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Fluorescent probe and raman spectroscopic investigation of the effects of pH, heating, and [kappa]-carrageenan… Alizadeh-Pasdar, Nooshin 2001

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FLUORESCENT PROBE AND RAMAN SPECTROSCOPIC INVESTIGATION OF THE EFFECTS OF pH, HEATING, AND K-CARRAGEENAN ON WHEY PROTEIN STRUCTURE B y NOOSHIN A L I Z A D E H - P A S D A R B . S c , The University o f Shahid Beheshti, Tehran, Iran, 1991 M . S c , M c G i l l University, Montreal, Canada, 1995 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F PHILOSOPHY In T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Food Science) We accept this thesis as conforming to thejequired standard T H E UNIVERSITY O F BRITISH C O L U M B I A A p r i l 2001 © Nooshin Alizadeh-Pasdar, 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of £Q* cf <j? o t e ^ cc The University of British Columbia Vancouver, Canada Date Apr\\ 6, &o<ol DE-6 (2/88) A b s t r a c t Surface hydrophobicity (S0) is an important structural parameter describing food proteins due to its correlation with functionality. A new method to measure S 0 was established based on the neutral fluorescent probe, 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN). S 0 was determined for whey protein isolate (WPI), bovine serum albumin (BSA) and B-lactoglobulin (BLG) at various pH (3.0, 5.0, 7.0 and 9.0) under heated (80°C for 30 min) or unheated conditions. S 0 values measured by P R O D A N and cis-parinaric acid (CPA) were lower at pH 3.0 than at other pH, while values measured by 1-anilinonaphthalene-8-sulfonic acid (ANS) were highest at pH 3.0. The anionic probes ANS and CPA yielded opposing results on the effects of pH and heating on protein hydrophobicity. Heating, pH and addition of K-carrageenan (KCG) at low (1.0:62.5), medium (1.0:6.0) or high (1.0:1.2) ratios of KCG:protein, as well as the interactions between these factors, significantly (P<0.05) affected S 0 measured by P R O D A N . Generally, proteins had higher S 0 and were more sensitive to heating and K C G addition at pH 9.0, than at other pH. Heating increased S 0 of high ratio KCG:protein mixtures at pH 3.0, but generally decreased SQ of mixtures at higher pH, especially pH 9.0. Raman spectroscopy was used to analyze structural changes at higher protein concentration (15% w/v) than those (0.002-0.01%) used in spectrofluorometry. Decreased SS and Trp band intensities, and lower helical and higher (3-sheet content, were generally observed after heating at pH 9.0 or K C G addition at pH 5.0. Decreased helical and increased (3-sheet contents obtained by heating B L G at pH 7.0 or 9.0 were not ii observed after heating K C G : B L G mixtures at these pH. Addition of K C G to BSA at pH 7.0 or 9.0 resulted in increased helical content. Heating KCG:WPI mixtures at pH 5.0 and 9.0 resulted in large increases in SS and Trp band intensities and helical content, and decreased ratio of the tyrosine doublet, indicating a more buried or hydrophobic environment around aromatic residues. Raman and fluorescent probe spectroscopy provided information on protein structural properties as a function of pH, heating and interactions with other macromolecules, which may be important in applications of these proteins in food systems. iii Table of Contents Abstract ii List of Tables vii List of Figures viii List of Abbreviations x Preface xii Acknowledgments xiii CHAPTER I Introduction and Objectives 1 CHAPTER II Literature Review 7 1 Protein Conformation 7 2 Hydrophobicity 9 2.1 Definition of hydrophobicity 9 2.2 Importance of hydrophobicity in protein function 11 2.3 Methods for determination of protein hydrophobicity 12 3 Fluorescence Spectroscopy 15 4 Fluorescent Probes 17 5 Whey Proteins 24 5.1 Whey protein composition and properties 24 5.2 (3-Lactoglobulin 26 5.3 Bovine serum albumin 29 6 Polysaccharides 31 6.1 Structure 31 6.2 Sources 32 6.3 Functional properties 32 6.4 Applications 37 7 Protein-Polysaccharide Interactions 37 8 Raman Spectroscopy 44 9 Raman Spectroscopy of Proteins 47 10 Raman Spectroscopy of Polysaccharides 51 CHAPTER III Comparison of Whey Protein Surface Hydrophobicity Measured at Various pH, Using Three Different Fluorescent Probes 53 1 Abstract 53 2 Introduction 54 3 Experimental Procedures 57 3.1 Materials 57 iv 3.2 Preparation of proteins for measurement of S0 58 3.3 Hydrophobicity determination 60 3.4 Statistical Analysis 63 4 Results and Discussion 64 4.1 Comparison of hydrophobicity measured using anionic probes, ANS and CPA 64 4.2 Comparison of hydrophobicity measured using a neutral probe, PRODAN to the anionic probes, ANS and CPA 70 5 Conclusions 73 CHAPTER IV Application of PRODAN Fluorescent Probe to Measure Surface Hydrophobicity of Whey Proteins Interacting with K-Carrageenan 74 1 Abstract 74 2 Introduction 76 3 Experimental Procedures 80 3.1 Materials 80 3.2 Preparation of samples for measurement of S0 80 3.3 Hydrophobicity measurement 82 3.4 Statistical analysis 82 4 Results 82 4.1 Effects of pH and heating 88 4.2 Effects of KCG on S c of proteins at various pH and KCGiprotein ratios 88 4.3 Comparison of S 0 of proteins heated at various pH and KCG:protein ratios 89 4.4 Effect of heating on proteins containing KCG 91 5 Discussion 91 6 Conclusion 96 CHAPTER V Raman Spectroscopic Study of the Effects of pH, Heating, and Presence of K-Carrageenan on Protein Structure 98 1 Abstract 98 2 Introduction 100 3 Experimental Procedures 105 3.1 Materials 105 3.2 Preparation of samples 105 3.3 Method 106 4 Results and Discussion 108 4.1 Effect of pH, heating, and K-carrageenan on the secondary structure of proteins 117 4.2 The effect of pH, heating, and addition of K-carrageenan on the disulfide bond 125 4.3 The effect of pH, heating, and addition of K-carrageenan on tryptophan band 129 4.4 The effect of pH, heating, and presence of K-carrageenan on the tyrosine doublet bands 132 4.5 The effect of pH, heating, and addition of K-carrageenan on the C H 2 bending band .135 4.6 The effect of pH, heating, and addition of K-carrageenan on the C=0 stretching band 138 4.7 The effect of pH, heating, and addition of K-carrageenan on the C-H stretching bands 140 4.8 The effect of pH, heating, and addition of K-carrageenan on the OH stretch (-3200 cm"1).... 146 5 General Discussion 149 CHAPTER VI Conclusions and Recommendations 158 v 1 Conclusions 158 2 Recommendations 160 References 163 Appendix 1 186 Appendix II. Colorimetric determination of carrageenans 195 Materials and Methods 195 Appendix III. SDS-PAGE profile of whey protein samples 196 L i s t o f T a b l e s Table 1: Classification and characteristics of methods for determining hydrophobicity 13 Table 2: The characteristics of some of the major proteins in whey 25 Table 3: Amino acid composition of some of the major proteins of whey from bovine milk 26 Table 4: Secondary structure of P -lactoglobulin 28 Table 5: Physical-chemical characteristics of different carrageenans 35 Table 6: Main Amide I Frequencies Characteristic of Protein Secondary Structure 48 Table 7: Typical assignment of side-chain vibrations and wavenumber in the Raman spectra of proteins.. 49 Table 8: Assignment of major bands in the Raman spectra of deuterated P-lactoglobulin 50 Table 9: The assignments of bands in the FT-Raman and FT-IR spectra of sulfated polysaccharides 52 Table 10: Specific experimental parameters for each fluorescent probe 62 Table 11: Results of the ANOVA (analysis of variance) of the effects of pH, heating, presence of K-carrageenan (KCG) and their interactions on the surface hydrophobicity values of whey protein isolate (WPI), P-lactoglobulin (BLG), and bovine serum albumin (BSA) 83 Table 12: Effects of heating (H), and presence of K-carrageenan (KCG) on changes in the Raman shift of the C=0 stretching band of the Raman spectrum of protein samples at pH 3.0 139 Table 13: Effects of pH, heating, and K-carrageenan on protein secondary structure content (%) estimated by the Raman Spectral Analysis Package for the amide I band 187 Table 14: Effects of pH, heating, and K-carrageenan on the normalized intensity and Raman shift of bands typical of cc-helical structure of the Raman spectrum of protein samples 188 Table 15: Effects of heating, pH, and K-carrageenan on the normalized intensity and Raman shift of bands typical of SS stretching band of the Raman spectrum of the protein samples 189 Table 16: Effects of heating, pH, and K-carrageenan on the normalized intensity and Raman shift of bands typical of tryptophan in two wavenumber regions of the Raman spectrum of the protein samples.. 190 Table 17: Effects of heating, pH, and K-carrageenan on the normalized intensity and Raman shift of bands typical of tyrosine doublet ratio (860/825 cm"1) of the Raman spectrum of protein samples 191 Table 18: Effects of pH, heating, and K-carrageenan on the normalized intensity and Raman shift of the C H 2 bending band of the Raman spectrum of protein samples 192 Table 19: Effects of pH, heating, and K-carrageenan on the normalized intensity and Raman shift of the CH stretching band of the Raman spectrum of protein samples 193 Table 20: Effects of pH, heating, and K-carrageenan on the normalized intensity and Raman shift of the OH band of the Raman spectrum of protein samples 194 VII L i s t o f F i g u r e s Figure 1: Absorption, fluorescence, and phosphorescence phenomenon .' 16 Figure 2: Structures of ANS, CPA, and PRODAN 19 Figure 3: The primary sequence of P-lactoglobulin 27 Figure 4: The primary sequence of bovine serum albumin 29 Figure 5: Comparison of three carrageenan types 36 Figure 6: Ionic interactions of carrageenans and protein 43 Figure 7: The relationship between infrared absorption, Rayleigh and Raman scattering 46 Figure 8: Flow chart of protein preparation for measurement of S0 59 Figure 9: Flow chart of fluorescence measurement 62 Figure 10: Surface hydrophobicity (S0) of whey protein isolate (WPI) measured at pH 3.0-9.0 with ANS, CPA, and PRODAN (top, middle and lower graphs, respectively) 65 Figure 11: Surface hydrophobicity (S0) of P-lactoglobulin (BLG) measured at pH 3.0-9.0 with ANS, CPA, and PRODAN (top, middle and lower graphs, respectively) 66 Figure 12: Surface hydrophobicity (S0) of bovine serum albumin (BSA) measured at pH 3.0-9.0 with ANS, CPA, and PRODAN (top, middle and lower graphs, respectively) 67 Figure 13: Flow chart of sample preparation for the K-carrageenan:protein interaction experiment 81 Figure 14: Effect of heating (H) at 80°C for 30 min at pH 3.0-9.0 on the surface hydrophobicity (S0) of whey protein isolate (WPI), P-lactoglobulin (BLG), and bovine serum albumin (BSA) 84 Figure 15: Effect of pH and presence of K-carrageenan (KCG) at KCG:protein ratios of 1.0:1.2, 1.0:6.0, or 1.0:62.5 on the the surface hydrophobicity (S0) of whey protein isolate (WPI), P-lactoglobulin (BLG), and bovine serum albumin (BSA) 85 Figure 16: Surface hydrophobicity (SQ) of proteins heated (H) at 80°C for 30 min at pH 3.0-9.0, in the absence or presence of K-carrageenan (KCG) at KCG:protein ratios of 1.0:1.2, 1.0:6.0, or 1.0:62.5. 86 Figure 17: Effect of heating (H) at 80°C for 30 min at pH 3.0-9.0, on the surface hydrophobicity (S0) of proteins in the presence of K-carrageenan (KCG) at KCG:protein ratios of 1.0:1.2, 1.0:6.0, or 1.0:62.5 87 Figure 18: Raman spectra in the 400-1800 cm"1 wavenumber regions of unheated BSA at various pH.... 109 Figure 19: Raman spectra in the 400-1800 cm"1 wavenumber regions of heated BSA at various pH 110 Figure 20: Raman spectra in the 400-1800 cm"1 wavenumber regions of unheated mixture of KCG:BSA at various pH I l l Figure 21: Raman spectra in the 400-1800 cm"1 wavenumber regions of heated mixture of KCG:BSA at various pH 112 Figure 22: Raman spectra in the 2500-3400 cm"1 wavenumber regions of unheated BSA at various pH.. 113 Figure 23: Raman spectra in the 2500-3400 cm"1 wavenumber regions of heated BSA at various pH 114 Figure 24: Raman spectra in the 2500-3400 cm"1 wavenumber regions of unheated mixture of KCG:BSA at various pH 115 Figure 25: Raman spectra in the 2500-3400 cm'1 wavenumber regions of heated mixture of KCG:BSA at various pH 116 Figure 26: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on whey protein isolate (WPI) secondary structure content (%) estimated by the Raman Spectral Analysis Package for the Amide I band 119 Figure 27 Effects of pH, heating (H), and presence of K-carrageenan (KCG) on P-lactoglobulin (BLG) secondary structure content (%) estimated by the Raman Spectral Analysis Package for the amide I band 120 Figure 28: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on bovine serum albumin (BSA) secondary structure content (%) estimated by the Raman Spectral Analysis Package for the Amide I band 121 Figure 29: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of bands typical of a-helical structure of the Raman spectrum of the protein samples 124 Figure 30: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of SS stretching band (-508 cm"1) of the Raman spectrum of the protein samples 128 Figure 31: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of band typical of the tryptophan (near 761 cm"1) of the Raman spectrum of the protein samples 131 Figure 32: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of bands typical of the tyrosine doublet ratio (860/825 cm"1) of the Raman spectrum of the protein samples 134 Figure 33: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of the C H 2 bending band of the Raman spectrum of the protein samples 137 Figure 34: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of the CH stretching band (2880 cm'1) of the Raman spectrum of protein samples 143 Figure 35: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of the CH stretching band (2930 cm"1) of the Raman spectrum of protein samples 144 Figure 36: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of the CH stretching band (3060 cm'1) of the Raman spectrum of protein samples 145 Figure 37: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of the OH band (3200 cm"1) of the Raman spectrum of protein samples 148 Figure 38: SDS-PAGE profile of whey protein samples 196 ix L i s t o f A b b r e v i a t i o n s °c Degree(s) Celsius uL Microliter(s) A N O V A Analysis of variance ANS 1 -anilinonaphthalene-8-sulfonic acid B L G P-Lactoglobulin B S A Bovine serum albumin cm Centimeter(s) CPA Cis-parinaric acid CD Circular dichroism CT Charge transfer DD Distilled deionized D P H 1,6-di phenyl-1,3,5-hexatriene DS Dextran sulfate DSC Differential scanning calorimetry D 2 0 Deuterium oxide E Extinction coefficient Fig. Figure FTIR Fourier transform infrared g Gram(s) H Heated H K C G : B L G Heated mixture of K-carrageenan and P-lactoglobulin H K C G : B S A Heated mixture of K-carrageenan and bovine serum albumin H KCG.WPI Heated mixture of K-carrageenan and whey protein isolate IR Infrared K a Association constant K C G K-Carrageenan K C G : B L G mixture of K-carrageenan and P-lactoglobulin K C G : B S A Mixture of K-carrageenan and bovine serum albumin KCG:WPI Mixture of K-carrageenan and whey protein isolate K D Dissociation constant L Liter(s) L E Locally excited L 0 Ligand concentration M Mole(s) mg Milligram(s) min Minute(s) mL Milliliter n Number of binding sites n.b. No band n.d. Not determined nm Nanometer(s) Po Protein concentration pD Deuterium ion exponent pH Hydrogen ion exponent pi Isoelectric point PROD A N 6-propionyl-2-(dimethylamino)naphthalene PSC Phenyl sepharose chromatograhy S 0 Protein surface hydrophobicity S/N Signal to noise ratio w/v Weight per volume WPC Whey protein concentrate WPI Whey protein isolate A c k n o w l e d g m e n t s dlearn andstudu j^rom cradle to ^raue. Prophet Mohammed I wish to thank God, the Compassionate and the Merciful who gave me health and strength throughout my life. My sincere gratitude is for my supervisor Professor E .C .Y. Li-Chan of the department of Food Science for her excellent and unique supervision, patience, and encouragement. She has always been an excellent model for her students due to her dedication in teaching and research, and also her moral support, which is greatly treasured. I also would like to appreciate the members of my supervisory committee, Dr. B.J. Skura, Dr. D.D. Kitts, and Dr. C H . Seaman for their caring criticism, encouragement, and advice. Many thanks to V . Skura, S. Yee, and Dr. E. Akita for their technical assistance and to J. Law and J. Tom for their efficient secretarial service. I wish to thank all staff and my fellow graduate students for their encouragement and care, especially Judy Chan for her comments on my thesis. I am sincerely grateful and dearly indebted to the members of my family, my husband, A . Ghanbari and our daughters, Mona and Sana, and our parents for their support and encouragement throughout this study. xii i C H A P T E R I I n t r o d u c t i o n a n d O b j e c t i v e s Protein functionality is a characteristic that relates to the utilization of the protein as a food ingredient. Some examples of protein functionality include solubility, water-and fat-binding capacity, emulsifying, foaming, gelation, and elasticity. Functional properties of proteins are closely related to surface hydrophobicity (S0) (Nakai et al., 1996). Several techniques, such as fluorescent probe methods, have been used to measure S 0 . Based on quantitative structure activity relationships (QSAR), functionality or activity of a protein is influenced by hydrophobic, steric, and electronic parameters (Nakai and Li-Chan, 1988b). Besides, extrinsic factors such as pH, salts, and temperature as well as presence of other molecules such as lipids, sugars, other proteins, and polysaccharides may all affect protein structure and consequently functional properties (Nakai et al., 1996). Due to the complexity of the large amounts of information that affect protein function, the extraction or "mining" of the useful or relevant data is necessary to understand the structure-function relationship, and data mining techniques are therefore proposed as a long-term approach for prediction of functional properties. For example, Bochereau et al. (1992) predicted apple quality by combining data analysis (principal component and regression analysis) and neural networks. Horimoto et al. (1995) predicted the volume of wheat flour loaf using neural networks and principal component regression. Lipp et al. (1998) applied neural networks for characterization of pyrolysis-mass spectrometry data. Najjar et al. (1997) used neural networks for predictive microbiology. 1 However, in the investigation of protein structure-function relationships, most of the attention has been given to hydrophobicity due to its correlation with protein stability. Furthermore, a review by Nakai and Li-Chan (1993) on QSAR of food proteins also revealed the importance of hydrophobicity in functions such as emulsifying, foaming and gelation properties. The value of hydrophobicity has thus been used commonly to predict functional properties of proteins. Over the years, scientists have been trying to find the best method for the quantification of protein surface hydrophobicity; however, they still have not reached any agreement to identify a method as a "gold standard". Many anionic fluorescent probes such as l-anilinonaphthalene-8-sulfonic acid (ANS) and cis-parinaric acid (CPA) are available for the measurement of S 0, However, interpretation of the relationship between functionality and hydrophobicity measured using S 0 values based on these probes is not always accurate, due to the possible contribution of both electrostatic and hydrophobic interactions to the binding of these anionic probes to proteins. Using a neutral probe may circumvent this problem. Because of the limitation of anionic fluorescent probes, a method was established by Tsutsui et al. (1986), using a neutral probe, diphenylhexatriene (DPH), for fat-binding capacity as an index of protein hydrophobicity. Due to the non-polar nature of this probe, and consequently low solubility in aqueous solution, they devised an approach to first dissolve the probe in a corn oil-heptane mixture and then use it for S 0 measurement, after heptane removal by evaporation. Although this probe is neutral, it actually measures fat binding capacity rather than hydrophobicity. This probe was not widely used later due to tediousness of the procedure (Nakai et al., 1996). 2 Royer (1995) reported that 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN) exhibits a very large excited-state dipole which causes sensitivity of this probe to the environmental polarity. Recently the neutral probe P R O D A N was used by Haskard and Li-Chan (1998) for quantitation of protein surface hydrophobicity. In that study, the influence of ionic interactions on quantitation of protein surface hydrophobicity was assessed by comparing the binding constants of P R O D A N versus ANS to bovine serum albumin and ovalbumin, and by comparing the S 0 values of these two proteins at various ionic strength. Due to the common naphthyl structure of the two probes, ANS and P R O D A N , their hydrophobic interactions with proteins are expected to be similar, and indeed the probes were reported to probably bind to a common region in the protein tubulin (Mazumdar et al., 1992). Nevertheless, comparison of binding constants indicated a 6-fold greater value for the BSA-ANS complex than the BSA-P R O D A N complex, suggesting enhanced binding of ANS to B S A through a positively charged residue adjacent to the hydrophobic binding site (Haskard and Li-Chan, 1998). Conversely, ovalbumin was suggested to possess a slight negative charge adjacent to the hydrophobic binding site, resulting in a binding constant for ANS which was about half that for PRODAN. By increasing ionic strength up to 1.0 M , the S 0 value of B S A measured by ANS decreased while that of ovalbumin increased. Using PRODAN, however, increase in the S 0 value of B S A and no change for ovalbumin was observed by increasing the ionic strength. These results therefore confirmed the importance of considering charge effects on the S 0 value measured by fluorescent probes. Unfortunately, due to low solubility (3.5 uM) of P R O D A N in water (the P R O D A N has to be stirred overnight to reach such solubility, as stated by Weber and 3 Farris, 1979), the protocol established by Haskard and Li-Chan (1998) yielded low fluorescence readings and measurements were difficult to reproduce due to batch-to-batch variability in the solubility of the P R O D A N aqueous stock solution (Alizadeh-Pasdar, 1998). The methodology of Haskard and Li-Chan (1998) was therefore modified as suggested by the supplier's handbook (Haughland, 1996), to dissolve the probe in methanol instead of water (Alizadeh-Pasdar, 1998). This thesis was thus initiated, in response to concerns about the possible effects of ionic interactions on the measurement of surface hydrophobicity using fluorescent probes, to modify the method proposed by Haskard and Li-Chan (1998), using a neutral fluorescent probe, 6-propionyl-2-(N,N-dimethylamino)naphthalene or PRODAN, prepared as a methanol stock solution for the measurement of S 0 . The effect of pH on the surface hydrophobicity of proteins determined by the fluorescent probe method was investigated by comparing the S 0 values measured with the neutral probe, P R O D A N , versus two anionic probes, ANS and CPA. Whey proteins, bovine serum albumin (BSA) and P-lactoglobulin (BLG) were chosen since their structure is well established, albeit mainly at neutral pH, while WPI was chosen as an example of a protein which is actually used in food systems as an ingredient and which contains B L G and BSA. In the second part of this study, P R O D A N fluorescent probe was used to monitor changes in surface hydrophobicity resulting from interactions of whey proteins with K-carrageenan (KCG) at various KCG:protein ratios, under varying pH and heat treatments. Polysaccharides have been widely used as stabilizing, thickening, and gelling agents in the food, agricultural, cosmetics, and pharmaceutical industries. They contribute to the physical stability and therefore quality as well as shelf life of various dairy products. 4 Polysaccharides and proteins are two major macromolecules in most food systems. The understanding and consequently, control of the interaction of proteins and polysaccharides is necessary in the development of new food products containing these two macromolecules. The study of this interaction can enhance the application of polysaccharides into foods and improve their functional properties. Raman spectroscopic investigation of the effect of various factors, including pH, heating, and presence of K-carrageenan on whey protein structure was performed in the third part of this research. Although fluorescent probe methods are simple to conduct and therefore may be applied easily by the food industry to monitor changes in protein surface hydrophobicity, they require very dilute protein solutions. Use of higher protein concentrations in visible laser Raman spectroscopy is advantageous to the study of food proteins, due to the relevance of such protein concentrations in real food systems. Furthermore, various aspects of protein structure may be investigated using Raman spectroscopy. It can be a useful analytical technique for the study of proteins in solid and liquid food systems, to obtain valuable information regarding the secondary structure and to monitor the environment around the amino acid side chains. To the best of our knowledge, Raman spectroscopy has not been reported for the study of the interaction of food proteins and polysaccharides. The purpose of this part of the thesis was therefore to use Raman spectroscopy to study possible effects of pH, heating, and presence of K-carrageenan on protein secondary structure (a-helix, P-sheet, and total random coil), various amino acid side chain vibrations (disulfide stretching, aromatic rings of tryptophan and tyrosine, C H stretching bands of aliphatic amino acids, C=0 stretch of the C O O H group of aspartic and glutamic acids) and protein-water interactions. 5 This brief introduction only outlines the rationale and main objectives of this study. More detailed information is provided in the introduction of each chapter (Chapters HI, IV, and V). 6 C H A P T E R II L i t e r a t u r e R e v i e w 1 Protein Conformation Proteins are polymers composed of 20 amino acids, which are linked together through peptide bonds. The unique structure and function of these biopolymers arises from the composition and distribution of amino acids, their sequence and the final three-dimensional structure of the protein. Formation of the three dimensional structure of any protein depends on the net effect of attractive and repulsive forces in the protein molecule and the interaction of the protein chain with its surrounding environment (Damodaran, 1989). Since the net thermodynamic stability of the native structure of most proteins is about 10-20 Kcal/mole, even a small decrease in the free energy of stabilization may affect protein stability and function. Factors such as heat, salts, pH, and the presence of other components contribute to such changes in free energy in food systems. Brownian or thermal energy is one of the factors that affect molecular interactions, since an increase in temperature increases mobility of molecules. At temperatures between 0 and 50°C, the influence of entropy is dominant. When temperature increases, hydrophobic association also increases. At higher temperatures, the effect of the increase in enthalpy is greater than the increase in entropy, so hydrophobicity decreases (Koning and Visser, 1992). Various forces that maintain the folded structure of proteins include electrostatic, van der Waals, hydrogen, and hydrophobic bonding. Electrostatic interactions are pH or ionic strength dependent. pH is a factor that influences the total charge on the protein, while the type of salt and its concentration determines the nature and extent of interaction 7 among those charges. Protein stability can be affected by electrostatic interactions through two ways, non-specific repulsion, or specific charge. Classical electrostatic effects (non-specific repulsion) occur at extremes of pH, when the protein is highly charged. Electrostatic interactions have no contribution to protein stability near the isoelectric point of protein. By increasing the net charge on the protein upon increasing acidity or basicity of the solution, however, the folded protein destabilizes because charge density on the folded molecule is greater than on the unfolded molecule. As a result of unfolding, a lower state of electrostatic free energy occurs. On the other hand, a specific charge interaction such as ion pairing (salt bridging) occurs when amino acid side chains with opposite charges are very close together (Damodaran, 1989; Phillips, 1992). Hydrogen bonding occurs when a hydrogen atom is shared between two electronegative atoms, while van der Waals attractions arise from interactions among fixed or induced dipoles (Cybulski and Scheiner, 1989; Koning and Visser, 1992). Hydrogen bonding is known to be a driving force for helix-coil transition. This transition is influenced by various factors including temperature, chain length, and pH (Phillips, 1992). Hydrophobic interactions are the dominant force responsible for protein folding in aqueous solutions. Globular proteins are known to have most of their charged groups outside (to aid contact with water) and the majority of their non-polar residues inside the molecule (to escape contact with adjacent water). However, uncharged polar groups are located both inside and outside and hydrogen bonded to water molecules or other parts of the protein (Bigelow, 1967). Due to the relevance of hydrophobicity to the objectives of this thesis, it has been elucidated in more detail as follows. 8 2 Hydrophobicity 2 . 1 Definition of hydrophobicity The term "hydrophobicity" describes the dislike of a molecule toward a water molecule (general abhorrence of water) (Zubay, 1983). Hydrophobicity has also been defined as the tendency of non-polar solutes to adhere to one another in aqueous environments (Cardamone and Puri, 1992). Hydrophobic effect, in an aqueous medium, may also be due to van der Waals interactions between macromolecules whose hydrophobic sites interact with each other (Nakai and Li-Chan, 1988b). Although the surface of a protein molecule has a hydrophilic nature, a significant number of hydrophobic amino acid groups are exposed at the molecular surface of proteins such as bovine serum albumin and P-lactoglobulin (Kato et al, 1983). The hydrophobicity of the side chains of some amino acid residues is due to their apolar chemical structure; thus side chains of this kind do not interact (except for van der Waals interactions) with polar molecules like water (Koning and Visser, 1992). These side chains prefer to avoid contact with water and therefore tend to associate (hydrophobic interactions) in internal hydrophobic regions of protein. Amino acids with hydrophobic side chains are alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine (Cheftel et al, 1985). An example of the hydrophobic effect is the existence of stable casein micelles in milk. Casein structure is composed of a hydrophobic and a charged polar domain. Sensitivity of the physicochemical properties of this protein to pH and ionic strength is mainly due to the anionic clusters in the polar domain. The associative characteristic of this protein is not only due to the interactions of the hydrophobic domain, but also, some H-bonding or the extension of the secondary structure at hydrophobic surface contact 9 sites which may cause self-association as well (Swaisgood, 1992). Besides, their peculiar charge distribution causes the association (Rollema, 1992). Various terms, used in relation to protein hydrophobicity, include total hydrophobicity and surface hydrophobicity. The term "total hydrophobicity" is the average hydrophobicity of a protein multiplied by the number of residues in the molecule and is a measure of molecule stabilization if all of its non-polar residues were buried. This method is based on the sum of each amino acid residue's free energy of transfer from aqueous to organic solvent. There are some limitations in using "total hydrophobicity" as a method of measuring hydrophobicity (Bigelow, 1967; Ponnuswamy, 1993). Based on Bigelow's study, proteins with higher charge density and lower average hydrophobicity have a higher solubility, which is true for most proteins. Bigelow's theory can not be applied, to some proteins, such as myoglobin and serum albumin, which have the same charge frequency. Since the average hydrophobicity of serum albumin is greater than that of myoglobin, on the basis of average hydrophobicity of these proteins, serum albumin should be less soluble than myoglobin. Myoglobin, however, is insoluble at its isoelectric pH, while serum albumin is extremely soluble at this pH. Based on this observation, it seems that functional behavior of a protein is more influenced by physical and chemical properties of the protein surface and the thermodynamics of its interaction with the surrounding solvent, rather than the average or total hydrophobicity. So, effective, exposed or surface hydrophobicity, which has a great influence in characterizing the protein structure and function, can give more accurate information regarding the hydrophobic nature of a protein. Surface hydrophobicity influences intermolecular interactions such as the binding of small ligands or the 10 association with other molecules, e.g., protein-protein, protein-polysaccharide, or protein-lipid interactions. 2.2 Importance of hydrophobicity in protein function Beside hydrophobic interactions (surface or exposed), electronic (net or surface charge) and steric (molecular size, molecular flexibility, and sulfhydryl disulfide) effects must be considered to elucidate both the functional and biological properties of protein molecules in foods (Li-Chan, 1999; Nakai and Li-Chan, 1989a). The most attention, however, has been given to the hydrophobic interaction due to its significance for prediction of functionality and role in protein stability and conformation (Gueguen, 1989; Kato et al, 1983; Li-Chan and Nakai, 1989; Voustinas et al, 1983a; Voustinas et al, 1983b). The development of equations that describe the quantitative structure-activity relationship (QSAR) is one important outcome of using protein hydrophobicity as a factor for predicting protein functionality (Nakai and Li-Chan, 1989a; Nakai et al, 1991). Some extrinsic factors such as pH, temperature, and ionic environment as well as - intrinsic protein properties such as disulfide bonds may affect molecular flexibility or stability (Harwalker and Ma, 1989; Koning and Visser, 1992). Heating may affect protein surface hydrophobicity in two ways, increased hydrophobicity due to protein unfolding and therefore more exposure of hydrophobic sites, and decreased hydrophobicity due to aggregation, loss of solubility, and less exposure of hydrophobic sites (Bonomi et al, 1988; Nakai and Li-Chan, 1989b). Li-Chan et al. (1984 and 1987), in a study of the effect of heating on the functional and physicochemical properties of salt-extractable muscle proteins, found that heating affects solubility and hydrophobicity of proteins and therefore their functionality. Kato and 11 Nakai (1980), who studied the relationship of surface hydrophobicity of proteins and their surface properties, reported a significant (p<0.01) correlation between the emulsifying capacity and surface tension with protein surface hydrophobicity. Kato et al. (1983), in a study of emulsifying and foaming properties of proteins during heat denaturation, showed that emulsifying activity and emulsion stability of proteins correlated linearly with surface hydrophobicity, while foaming power of proteins correlated curvilinearly with surface hydrophobicity. 2.3 Methods for determination of protein hydrophobicity The most common methods for evaluation of protein hydrophobicity are: fluorescence methods, partition coefficients and hydrophobic binding (Magdassi and Toledano, 1996). In fluorescence methods, either the extrinsic fluorescence of hydrophobic probes such as l-anilinonaphthalene-8-sulfonic acid (ANS) or the intrinsic fluorescence of aromatic amino acids is used. Partition coefficient methods are based on combination of polymers such as polyethylene glycol and dextran in aqueous solutions. Protein is partitioned in this two-phase system in which each phase has different polarity. Therefore, the relative concentration of protein partitioning in the two phases can be evaluated and utilized as a hydrophobic parameter (Magdassi and Toledano, 1996). In hydrophobic binding methods, the amount of hydrophobic markers (such as aliphatic or aromatic hydrocarbons, sodium dodecylsulfate, and simple triglycerides) adsorbed to the protein molecule is evaluated. Fluorescence probe techniques are quick 12 compared to hydrophobic partition and hydrophobic chromatography, which require 2 and 5 hours, respectively (Kato and Nakai, 1980). Nakai and Li-Chan (1988a) classified hydrophobicity methods as shown in Table 1. Table 1: Classification and characteristics of methods for determining hydrophobicity3 Method Characteristics Hydrophobicity scale Inconsistency among scientists about definitions and scales Protein intrinsic fluorescence Problem of measuring only aromatic amino acid residues, not all hydrophobic amino acids Fluorescence quenching Problem of measuring only aromatic amino acid residues, not all hydrophobic amino acids Hydrophobic probe methods Simple and quick Hydrophobic chromatography Problem of protein denaturation during chromatography Hydrophobic binding methods Good technique; caution in choosing a proper method Hydrophobic partition Tedious; problem of insolubility of some proteins in non-polar phase Adapted from Nakai and Li-Chan (1988a) Among these methods, fluorescent probe methods are still the most popular (Nakai et al, 1996). Hayakawa and Nakai (1985) classified hydrophobicity measured by these probes into two groups, aromatic and aliphatic. For example, ANS is composed of aromatic rings, while cis-parinaric acid (CPA) is composed of an aliphatic hydrocarbon chain. These probes could be used for the measurement of aromatic and aliphatic hydrophobicity, respectively (Hayakawa and Nakai, 1985). Hayakawa and Nakai (1985) studied the relationships of hydrophobicity and net charge to the solubility of milk and soy proteins using two fluorescent probes, ANS and 13 CPA. They compared the hydrophobicity values obtained with ANS and CPA with a chromatographic method using phenyl Sepharose (PSC). It has been reported (Kato and Nakai, 1980) that CPA hydrophobicity (S0, protein surface hydrophobicity, measured with CPA probe) was well correlated to the protein functionality, but was affected by pH (Kato et al., 1984). However, CPA hydrophobicity did not correlate well with insolubility, while ANS hydrophobicity (S 0 measured with ANS probe) and PSC showed a good correlation with insolubility and zero zeta potential (Hayakawa and Nakai, 1985). They also mentioned that among the 42 samples (under different conditions) that were studied, only CPA hydrophobicity of BSA and P-lactoglobulin, unlike others, was negatively correlated to insolubility. Those two proteins show strong interactions with aliphatic hydrocarbon chains. Some proteins preferentially bind the aliphatic adsorbents, while others aromatic ones; so the classification of protein hydrophobicity as aliphatic and aromatic seems reasonable due to aliphatic (alanine, valine, leucine, and isoleucine) and aromatic amino acid (phenylalanine, tryptophan, and tyrosine) residues, respectively. Although aromatic residues are more hydrophobic than aliphatic residues, their effective burial in the interior of the protein is hindered due to their bulkiness (Hayakawa and Nakai 1985). Greene (1984) noted that besides hydrophobic interaction, electrostatic forces are involved in the interaction between charged probes and proteins. In that study, he evaluated the effect of charge on the interaction of 2-p-toluidinylnaphthalene-6 sulfonate (TNS) and its neutral and cationic sulfonamide derivatives with apomyoglobin and bovine serum albumin. He suggested that since cations and anions do not have equal access to the same number of binding sites (since the number of binding sites for anionic 14 probes is more than for cationic probes), electrostatic forces increase protein binding by anions more than cations. So, the use of anionic probes for the measurement of protein surface hydrophobicity may cause overestimation, compared to cationic probes (Greene, 1984). Electrostatic interactions are expected to play a major role in the measurement of protein surface hydrophobicity. For example, ANS carries one unit of negative charge while B L G is highly positive below its isoelectric point (D'Alfonso et al, 1999). 3 Fluorescence Spectroscopy Fluorescence spectroscopy is an important and widely used method for studying the structure, dynamics, and interactions of proteins in solution (Eftink, 1991; Guilbault, 1989; Royer, 1995; Slavic, 1994a). The fluorescence phenomenon is shown in Figure 1. "Fluorescence" relates to the light or luminescence that is emitted by molecules, while excited by photons. Fluorescence represents the emission of a photon accompanying the return of the molecule from its singlet excited state to its ground state (Slavic, 1994b). Since this emitted light is from the singlet state, it discontinues when the exciting energy source is eliminated. The afterglow lasts less than 10"9 s and is not temperature dependent. On the contrary, in "phosphorescence" the emitted light originates from the triplet state, with longer than 10"6 s afterglow and it is temperature dependent (Guilbault, 1989). 15 A: Absorption F: Fluorescence P: Phosphorescence S0: Ground state Si: First excited singlet state S2: Second excited singlet state TV Lowest triplet state T 2 : Excited triplet state Figure 1: Absorp t ion , fluorescence, and phosphorescence phenomenon Adapted from: Eftink, (1991), Johnston (1996) and Slavic (1994b) 16 Fluorescent probes are defined as chromophores whose absorption characteristics are affected by environmental conditions. Fluorescence methods depend on the response of some fluorescent component, either intrinsic or extrinsic, to its environment following optical excitation (Damodaran, 1989; Hudson et al, 1986). "Intrinsic ultraviolet fluorescence" is due to the presence of aromatic amino acids, tyrosine, tryptophan, and phenylalanine in the protein, while proteins in combination with fluorescent molecules have "extrinsic fluorescence" (Chen, 1973). Extrinsic fluorescence itself can be due to two types of probes, covalently bound and non-covalently bound. The basis for using most non-covalently bound fluorescent probes, such as ANS and CPA, is that these dyes are only weakly fluorescent in aqueous solution, but strongly fluorescent when adsorbed onto proteins (Chen, 1973). ANS and P R O D A N are known for sensing the polarity of the environment in biological materials (Rettig, 1993). Most fluorescent dyes react covalently with proteins only if they are modified to contain a reactive group, which attaches the dye to a specific protein group. Based on this definition, these reagents can be classified into the following groups, dye reagents attaching to primary amino groups (e.g., dansyl chloride) and sulfhydryl group reagents (e.g., didansyl cystine) (Chen, 1990). 4 Fluorescent Probes Molecules exhibiting a strong dependence of their fluorescence on environment polarity are of special interest in various fields, especially in biochemistry/food chemistry. Three such molecules are l-anilinonaphthalene-8-sulfonic acid (ANS), cis-parinaric acid (CPA), and 6-propionyl-2-(dimethylamino)-naphthalene (PRODAN), the 17 structures of which are illustrated in Figure 2. A comprehensive knowledge of the nature of the dependence of the emissive characteristics of the probes on environmental polarity and probe-protein interactions is essential in understanding the spectral properties of the probes (Baiter, 1988). Fluorescent probes are commonly used for binding studies due to the easy experimental procedure and the fact that the separation of the free ligand from bound one is not required. However, determination of the number of binding sites (n) and the dissociation constant (K D ) is still common (Laligant et al, 1991). In this approach, for a given ligand (L 0) concentration (e.g., ANS), the concentration of protein (P0) is varied and the fluorescence intensity is measured, or P 0 is kept constant and L Q is varied. These binding data are then analyzed using Scatchard (1949) or Klotz (1947) plots. 18 NH S0 3 H (CH 2 ) 7COOH CPA PRODAN O Figure 2: Structures of ANS, CPA, and PRODAN l-Anilinonaphthalene-8-sulphonate or ANS, is the most popular fluorescent probe for the measurement of protein surface hydrophobicity or demonstration of conformational changes in proteins (Slavic, 1994a). This probe is hardly fluorescent in aqueous solution; however, it shows an enhanced fluorescence when added to proteins such as bovine serum albumin (BSA), due to a decrease in dielectric constant and a change in molecular rigidity of the probe (Cardamone and Puri, 1992; Chang et al, 1994; Gibrat and Grignon, 1982; Kraayenhof et al., 1975). X-ray analysis of bilayer membranes has shown that the ANS molecules locate at the polar/non-polar interface, and their charged groups are oriented toward the hydrophilic phase (Kraayenhof et al., 1975). Fluorescence emission of ANS depends on the environment (Penzer, 1972). For example, adding acids to a solution containing ANS forms a quaternary aniline derivative due to protonation of nitrogen in ANS. Also, ANS molecules quench due to loss of molecular rigidity of ANS in water (Penzer, 1972). Aromatic hydrophobicity can be measured using ANS probe (Aluko and Yada, 1995). Bonomi et al. (1988) studied the effect of heat on milk protein surface hydrophobicity and concluded that the determination of surface hydrophobicity is a useful means to elucidate changes in protein conformation due to heating. Cardamone and Puri (1992) used ANS binding as a method of measuring protein surface hydrophobicity. They established a method for assessing the average protein surface hydrophobicity by estimating the association constant (K a ) of ANS-protein binding. ANS has been used widely for the measurement of protein surface hydrophobicity (Ainsworth and Flanagan, 1969; D'Alfonso et al, 1999; Jeyarajah and Allen, 1994; Laligant et al, 1991; Lee etal, 1992; Miller etal, 1991; Wicker et al, 1986). 20 Although ANS has been widely used for measurement of protein surface hydrophobicity, it has some limitations as a fluorescent probe. For example, since ANS contains one unit of negative charge, electrostatic interactions will play a major role, especially when used at pH values below the protein isoleletric point where proteins carry positive charge (D'Alfonso et al, 1999). Overestimation of quantum yield of this probe due to electrostatic potential of adsorbed ANS has also been reported (Vanderkooi and McLaughlin, 1975). The fluorescence intensity of ANS in blood serum albumin increased when placed in acidic environment, due to the increase in the positive charge of the protein and increase in the binding constant of protein when interacting with a negatively charged probe (Miller et al, 1991). In another study by Guha and Bhattacharyya (1995), the pH dependence of protein (tubulin) binding by ANS was reported. They noted that the relative fluorescent intensity (RFI) of ANS, in presence of tubulin, increased upon lowering pH from 7.0 to 3.0. The RFI of ANS-tubulin at pH 3.0 was 6 times that of the RFI at pH 7.0. The quantum yield of ANS is not sensitive to pH from 2.0-8.0 (Gibrat and Grignon, 1982). Cis-parinaric acid or CPA was first applied by Sklar et al. (1977) as a probe of lipid-protein interactions. Later, Kato and Nakai (1980) introduced a simpler methodology, using CPA, for measurement of protein surface hydrophobicity. Since then, the CPA probe has been used by many researchers for the measurement of protein surface hydrophobicity (Ibrahim et al., 1993; Lee et al, 1992; Li-Chan et al, 1984; Li-Chan et al, 1985; Mattarella and Richardson, 1983). CPA is a naturally occurring fatty acid and is an aliphatic fluorophore (Nakai et al, 1996). CPA and its derivatives are the closest structural analogs of intrinsic membrane lipids among currently available fluorescent 21 probes. The optical properties of CPA include a very large fluorescence Stokes shift (-100 nm) and no fluorescence in water. Similar to many other fluorescent probes, however, CPA is photolabile. CPA undergoes photodimerization under intense illumination resulting in loss of fluorescence (Haugland, 1996). CPA is not soluble in aqueous acidic solutions (below pH 5.0) (Nakai et al, 1996). Variations between the results of ANS and CPA hydrophobicities have been reported before (Hayakawa and Nakai, 1985; Lee et al, 1992). Weber and Farris (1979) were the first researchers who studied 6-propionyl-2-(N,N-dimethyl)aminonaphthalene (PRODAN). Since then, many researchers have studied this interesting fluorescent probe (Baasov and Sheves, 1987; Baiter et al, 1988; Bruins & Epand, 1995; Bunker et al, 1993; Catalan et al, 1991; Chakrabarti and Basak, 1996; Chakrabarti, 1996; Chong, 1988; Harianawala and Bogner, 1998; Heisel et al, 1987; Krasnowska et al, 1998; Lasagna et al, 1996; Macgregor & Weber, 1986; Mazumdar et al, 1992; Rottenberg, 1992; Sun et al, 1997; Wald et al, 1990). However, it has not been used for the measurement of S 0 before. P R O D A N is a neutral species and highly sensitive to polarity; it shows a single broad fluorescence band with red shift upon increasing solvent polarity (Heisel et al, 1987). The neutral nature of P R O D A N will eliminate possible effects of electrostatic interaction contributions in the measurement of protein hydrophobicity (Hermetter et al., 1993). P R O D A N shows a large Stokes shift (emission maximum minus absorption maximum). The Stokes shift increases with increasing solvent polarity (Bunker et al, 1993). This shift is dependent on the increase in dipole moment in the excited state over the ground state. Due to the alignment of the solvent dipoles in a hydrophilic 22 environment, there is a greater transfer of energy from the excited P R O D A N to adjacent solvent molecules, causing decreases in the energy of the excited state and a shift in the maximum emission. In hydrophobic environment, however, lower transfer of energy to adjacent solvent molecules occurs and a much smaller shift in the maximum emission is observed (Harianawala and Bogner, 1998). Macgregor and Weber (1986) considered P R O D A N an ideal fluorescent probe. They noted that a suitable polar fluorescent probe shouldn't have any charge. It also must be soluble in a range of solvents with various polarities. It has been shown that the fluorescence of P R O D A N in non-polar solvent is due to a locally excited state (LE), while in polar solvents the fluorescence is attributed to a charge transfer (CT) state of energy, which can be influenced by the polarity of the solvent (Heisel et al, 1987). The maximum fluorescence emission of P R O D A N in a polar environment versus a hydrophobic environment is around 520 nm versus 420 nm, respectively (Bruins and Epand, 1995). Hydrogen bonding interactions are important in the photophysical behavior of P R O D A N in protic solvents such as water and methanol. Time-resolved fluorescence results have shown that an excited-state relaxation process which exists for P R O D A N in polar solvents, polarity and temperature (viscosity) dependent (Bunker et al, 1993). Catalan et al. (1991) studied the dependency of P R O D A N fluorescence on 38 protic, nonprotic and amphiprotic solvents. Their results suggest that specific interactions exist between P R O D A N and some solvents. P R O D A N has no acid group prone to change in the electron transition. On the other hand, basic sites (the tertiary amine group and the carbonyl group) can experience significant changes with the electron transition. Catalan 23 et al. (1991), concluded that the electron absorption and emission transitions of P R O D A N are sensitive to solvent acidity. 5 Whey Proteins 5.1 Whey protein composition and properties The significance of whey proteins as an additive or food ingredient is rapidly growing due to their excellent functional properties. However, improvement of their functional characteristics is essential, if they are to be used to meet the needs of the food industry (Pittia et al, 1996). Whey proteins can be obtained through acidification of skim milk at pH 4.6 and 20°C, for removal of casein (Pearce, 1989). Whey protein concentrate (WPC) and whey protein isolate (WPI) are manufactured from the same source, whey, but vary in protein concentration (25-95%), minerals, lipids and lactose. The composition of any whey product depends on the whey source and processing conditions (Bottomley et al, 1990; Dybing and Smith, 1991; Ziegler and Foegeding, 1990). Whey protein isolate is a highly purified protein product that contains about 90-95% protein (dry basis) and is obtained by ion exchange and ultrafiltration (Dybing and Smith, 1991; Mate and Krochta, 1996). Foaming (Phillips et al, 1990), emulsifying (Foley and Oconell, 1990), and gelation (Barbut and Foegeding, 1993) are some of functional properties of whey protein isolate. Mate and Krochta (1996) used WPI as a coating agent for roasted peanuts. Yildirim et al. (1996), who studied the properties of biopolymers from cross-linking whey protein isolate and soybean l i s globulin, showed that WPI is soluble over a wide range of pH 24 (2.0-9.0). They also noted that WPI has excellent foaming and emulsifying properties. Errington and Foegeding (1999), who compared the Theological properties of egg white and whey protein gels, showed that egg white and WPI gels, at the same protein concentrations, have similar shear stress at fracture. Mleko and Foegeding (2000) noted that gelation property of WPI was pH dependent. Foley and Oconell (1990), however, found that emulsion capacity of aqueous solutions of WPI was not pH dependent. Whey proteins retain good solubility and minimal heat sensitivity around pH 2.5-3.5 (de Wit, 1981). The structure and solubility of whey proteins are governed by factors such as pH and temperature (de Wit and Klarenebeek, 1984). Structural unfolding of whey proteins generally occurs around 60-70°C and involves molecular interactions such as hydrogen and hydrophobic bonding. Protein aggregation, which depends on compositional factors (such as total solids, and pH) and involves disulfide linkages, may occur at higher temperatures. The composition and characteristics of some of the major whey proteins are shown in Table 2, and their amino acid compositions are given in Table 3. Table 2: The characteristics of some of the major proteins in whey Protein Approximate Molecular Isoelectric Approximate concentration in weight (daltons) point percentage of total whey (g/L) whey protein P-lactoglobulin 3.0 18,363 5.35-5.49 50 a-lactalbumin 0.7 14,175 4.2-4.5 12 Immunoglobulins 0.6 (1.61 - 10) x 105 5.5-8.3 10 Bovine serum albumin 0.3 66,267 5.13 5 Source: Bottomley etal, 1990 25 Table 3: Amino acid composition of some of the major proteins of whey from bovine milk Amino acid a-Lactalbumin P-Lactoglobulin Bovine serum albumin Asp 9 11 41 Asn 12 5 13 Thr 7 8 34 Ser 7 7 28 Glu 8 16 59 Gin 5 9 20 Pro 2 8 28 Gly 6 3 15 Ala 3 14 46 ViCys 8 5 35 Val 6 10 36 Met 1 4 4 Ile 8 10 14 Leu 13 22 61 Tyr 4 4 19 Phe 4 4 27 Trp 4 2 2 Lys 12 15 59 His 3 2 17 Arg 1 3 23 Total 123 162 581 Source: Kinsella and Whitehead, 1989 5.2 (3-Lactoglobulin The principal whey protein is P-lactoglobulin (BLG). B L G is a globular protein that was isolated by sodium sulfate fractionation of acid whey by Palmer in 1934 (McKenzie, 1971). B L G A and B L G B are the two most common genetic variants of this molecule. These two variants were originally differentiated based on their mobility on paper electrophoresis at pH 8.6, where the mobility of A was higher than B (McKenzie, 1971). B L G has 162 amino acid residues in a single chain, with a monomer molecular weight of 18,363 for P-lactoglobulin variant A (Bottomley, 1990; de Wit and Klarenebeek, 1984; Ziegler and Foegeding, 1990). The primary sequence of P~ lactoglobulin A is shown in Figure 3. 26 MKCLLLALALTCGAQALIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDA QSAPLRVYVEELKPTPEGDLEILLQKWENGEC 6 6AQKKIIAEKTKIPAVFKI DALNENKVLVLDTDYKKYELFC , 0 6 MENSAEPEQSLAC 1 1 9 QC 1 2 I LVRTPEVDDEALE KFDKALKALPMHIRLSFNPTQLEEQC 1 6 0HI Figure 3: The primary sequence of p-lactoglobulin Source: http://pir.georgetown.edu/pirwww/search/textpsd.html Abbreviations: A=Alanine, C=Cysteine, D=Aspartic acid, E=Glutamic acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine (Cheftel et al, 1985). This protein has two disulfide bridges, between residues 106-119 and 66-160, and a free thiol group at residue 121, (shown in bold superscript in Figure 3) which provide possibility of intermolecular and intramolecular disulfide link interchange due to alterations in pH, temperature, and ionic strength (Bottomley, 1990). It has been known that P-lactoglobulin is soluble over a wide range of pH from 2.0-10.0 (Jouenne and Crouzet, 2000). P-Lactoglobulin exists as a monomer, containing two disulfide bonds, at extreme pH below pH 3.5 and above pH 8.0 (Harwalker and Ma, 1989). It forms a dimer at pH values between 3.5 and 5.2 at room temperature, while it forms an octamer at temperature close to 0°C. Some conformational changes, which are accompanied by the expansion of the molecule, occur at pH 7.5. Increase in the reactivity of the thiol group and dissociation of the dimer occurs above pH 7.0. Unfolding and aggregation of this protein occurs above 55°C, where cysteine and cystine groups and hydrophobic surfaces are exposed (Bottomley et al, 1990; Hambling et al, 1992; Jelen, 1989; Ziegler and Foegeding, 1990). 27 The secondary structure of proteins can be predicted based on the primary sequence of amino acids and their preferred conformation, hydrophobicity profile, influence of neighboring residues, and statistical computations (Sawyer and Holt, 1992). The reported range of a-sheet in the secondary structure predicted from the sequence of P-lactoglobulin is quite wide (10-50%). Estimates of the secondary structure of P-lactoglobulin are shown in Table 4. Based on CD spectra, P-lactoglobulin is composed predominantly of P-sheet, with a secondary structure that does not change over a wide range of pH (Matsuura and Manning, 1994), due to rigidity of B L G conformation around pH 3.0 to 7.0 (Das and Kinsella, 1989). Shimizu et al. (1985) investigated the emulsifying and structural properties of B L G at pH 3.0, 5.0, 7.0, and 9.0, using ANS fluorescent probe and circular dichroism (CD). The emulsifying activity index of B L G was lowest at pH 3.0 but increased with an increase in pH. The ANS hydrophobicity of B L G was the highest at pH 3.0 and lowest at pH 7.0. They also noted that pH had no effect on the secondary structure of B L G at pH 3.0 and 7.0. Table 4: Secondary structure of P -lactoglobulin Sequence Prediction (%a-helix: P-sheet: P-turn) CD Spectra (%cc-helix: P-sheet) (10-50):(20-30):(17-24) 10:43 Source: Swaisgood, 1989 P-Lactoglobulin is the most abundant protein in whey. It is suggested that P-lactoglobulin has a physiological function in retinol binding (Pearce, 1989), since a homology was found between retinol-binding protein and P-lactoglobulin. Some 28 experimental data suggest that the transport of retinol from mother to offspring could be the biological role of P-lactoglobulin (Kella and Kinsella, 1988; Narayan and Berliner, 1998). 5.3 Bovine serum albumin B S A consists of 582 amino acids in a single chain, with a molecular weight of 66,267 daltons. It contains 17 disulfide bonds with one free sulfhydryl group at position 34. The primary sequence of BSA is shown in Figure 4. This protein has 17 disulfide bridges, between residues (shown in bold superscript in Figure 4) 53-62, 75-91, 90-101, 123-167, 166-175, 198-244, 243-251, 263-277, 276-287, 314-359, 358-367, 390-436, 435-446, 459-475, 474-485, 512-557, and 556-565 and a free thiol group at residue 34 (Brown and Shockley, 1982). MKWVTFISLLLLFSSAYSRGVPPJRDTHKSEIAHRFKDLGEEQFKGLVLIA FSQYLQQC 3 4 PFDEHVKLVNELTEFAKTC 5 3 VADESHAGC 6 2 EKSLHTLFGDELC 7 5 K VASLRETYGDMADC 9 0 C 9 1 EKQEPERNEC 1 0 1 FLSHKDDSPDLPKLKPDPNTLC 1 2 3 DEF KADEKKFWGKYLYEIARRHPYFYAPELLYYANKYNGVFQDC 1 6 6 C 1 6 7 QAEDKGAC 1 7 5 LLPKIETMREKVLASSARQRLRC 1 9 8 ASIQKFGERALKAWSVARLSQKFPKAE FVEVTKLVTDLTKVHKEC 2 4 3 C 2 4 4 HGDLLEC 2 5 1 ADDRADLAKYIC 2 6 3 DNQDTISSKLKE C 2 7 6 C 2 7 7 DKPLLEKSHC 2 8 7 IAEVEKDATPENLPPLTADFAEDKDVC 3 1 4 KNYQEAKDAFL GSFLYEYSRRHPEYAVSVLLRLAKEYEATLEEC 3 5 8 C 3 S 9 AKDDPHAC 3 6 7 YSTVFDKL KHLVDEPQNTLIKQNC3 9 0 DQFEKLGEYGFQNALIVRYTRKVPQVSTPTLVEVS RSLGKVGTRC 4 3 5 C^'TKPESERMPC^'TEDYLSLE.lSrRLC 4 5 9 VLHEKTPVSEKVTKC 4 7 4 C 4 7 5 TESLVNRRPC 4 8 5 FS ALTPDETYVPKAFDEKLFTFHADIC 5 1 2 TLPDTEKQIKKQT ALVELLKHKPKATEEQLKT VMENFVAFVDKC 5 5 6 C 5 S 7 AADDKEAC S 6 5 FAVEGPKLVV STQTALA Figure 4: The primary sequence of bovine serum albumin Source: http://pir.georgetown.edu/pirwww/search/textpsd.html Abbreviations as Figure 3. \ 29 BSA is considered a monomelic globular protein, although small quantities of dimers and higher polymers can occur. BSA molecules undergo acid denaturation at pH 4.0, due to charge repulsion (Bottomley et al, 1990). When exposed to an acidic environment, B S A shows a two-step structural transition (Rainbow et al, 1987). It has been shown that upon heating, BSA maintains its native form up to 42°C. Between 42°C and 50°C, reversible conformational changes occur; the native structure is restored upon cooling. However, when heated above 50°C, the a-helical structure unfolds, causing only partial restoration of native structure after cooling. At 60°C, significant aggregation of molecules occur while gelation of aggregates occurs around 70°C. Both B S A and B L G have an inaccessible free thiol group at or below neutral pH. However, above pH 6.5, the hidden thiol of P-lactoglobulin activates and facilitates thiol-disulfide interchange reactions, which leads to formation of new structures upon heating. At pH 7.0, B S A is in monomeric form while P-lactoglobulin is in dimeric form. B S A contains 17 disulfide bridges, while P-lactoglobulin contains two. Although BSA is relatively large and contains a large number of disulfide bridges, it is more flexible than p-lactoglobulin with its dimeric structure and thiol-disulfide interchange reactions (Suttiprasit et al, 1992). At neutral pH, BSA and p-lactoglobujin both carry a net negative charge, but electrostatic interactions may take place between negative charges on anionic polysaccharides and positively charged regions on the protein to form soluble ionic complexes (Galazka et al, 1996). 30 6 Polysaccharides Several terms including gums, hydrophilic colloids, hydrocolloids, mucilages, etc. have been used to describe the materials that are capable of thickening or gelling aqueous systems. To facilitate categorization, gums have been divided into water-soluble and water insoluble materials. Rubber is an example of water-insoluble gums (resins). Hydrophilic colloid or hydrocolloid is the terminology that is used for the water-soluble gums such as carrageenans. Most hydrocolloids are polysaccharides, so the suffix "-an" is added to the end of the materials in this group to designate the substance as a polysaccharide (Glicksman, 1982). 6.1 Structure Generally, polysaccharides consist of polymers of varying length composed of a single monosaccharide or its derivative (e.g., D-glucose in starch), or of a very few monosaccharides or their derivatives (e.g., chondroitin which contains D-glucuronic acid and N-acetyl-D-galactosamine), which are called as homo- and heteropolysaccharides, respectively. The configuration of any polysaccharide depends on both the individual monosaccharide unit and its environment (Whitney, 1977). Both homo- and heteropolysaccharides are classified into linear and branched types (Sand, 1982). The viscosity of solutions containing branched polysaccharides is usually lower than those of linear molecules of equal molecular weight (Glicksman, 1982). Polysaccharides are classified into three types based on pendant or ring ionizable groups. For example, anionic polysaccharides may possess sulfate, carboxyl, or phosphate groups, while cationic polysaccharides, may possess monoacetylated amino 31 groups. Starch and cellulose are typical of the neutral group (Glicksman, 1982; Walter, 1998a). The name 'carrageenan' refers to a class of galactan polysaccharides. The carrageenans are classified, based on their sulfation patterns and the distribution of 3,6-anhydro-D-galactose residues, into three types: kappa (K), iota (t), and lambda (A.) carrageenan (Roberts and Quemener, 1999). K-Carrageenan, [l-4(D-a-galactose-4-sulphate)-l-3 (3,6 anhydro-D-galactose)]n, dissolves only when heated and forms strong and brittle gels, while t and A-carrageenans are cold-soluble (Bixler, 1994; Lockwood, 1985; Piculell, 1995). K-Carrageenan contains 25-30% sulfate (S0 4) (Lin, 1977). The ionisable groups of the carrageenans are sulfate ester groups (Gillberg and Tornell, 1976). 6.2 Sources The purest form of K-carrageenan is obtained from Eucheuma cottonii, of tropical waters, or Chondrus crispus from the colder waters of Maine, Nova Scotia, Ireland, and Brittany (Lockwood, 1985) or the cold water Gigartina stellata species (Thomas, 1992). Various sources of hydrocolloids, especially food grade gums, include 1) plant exudates, 2) seaweed extracts, 3) flours, 4) fermentation or biosynthesis, 5) chemical modification, and 6) chemical synthesis. Carrageenans are examples of seaweed extracts (Glicksman, 1982; Pedersen, 1978; Walter, 1998a). 6.3 Functional properties One can observe the unique water retention capability (80-90% water) of polysaccharides, naturally in fruits such as apples, oranges, melons, etc., or similarly in fruit jellies where they retain 35-50% water. The great hydration property of 32 carrageenans is due to the presence of large number of hydrogen bonds in their molecule in the solid state, which can hydrate easily (Walter, i998a). Typically, carrageenans form highly viscous solutions due to their unbranched, linear macromolecular structure and polyelectrolyte nature. The repulsion of negatively charged ester sulfate groups causes the rigidity of the carrageenan molecule. A sheath of immobilized water molecules surround the molecule, due to the hydrophilicity of carrageenan. A l l these factors cause carrageenan solutions to resist flow. Various factors affect viscosity including type, molecular weight, and concentration of carrageenan, temperature, presence and concentration of other solutes. For example, viscosity increases almost exponentially with concentration due to increased interaction between polymer chains. Both K- and i-carrageenans have the ability to form gels upon cooling of a hot solution. The gelling property of K-carrageenan depends upon various factors including its concentration, cooling procedure, ionic content, presence of ions, and salt concentration (Piculell, 1995; Tziboula and Home, 1998). K-Carrageenan forms strong crisp gels in the presence of potassium ions (Percival and McDowell, 1990). K-Carrageenan gels are subject to syneresis, which is the appearance of fluid on the surface of the gel. This fluid is produced as a result of increasing aggregation of junction zones and contains a proportionate part of the soluble substances of the gel such as salts, sugars, or other type of carrageenans. The syneresis will not occur if the concentration of gelling cations is properly chosen (Guiseley et al., 1980). Double helices have been implicated in carrageenan gelation. Under non-gelling conditions, K and i-carrageenans exist as a disordered coil at high temperatures (above 33 50°C), and as a double helix, which forms the junction zones, at low temperatures (Eceda and Kumagai, 1998; Walter, 1998c). Studies by X-ray diffraction have shown that K-carrageenan molecules form helices in the solid state (Piculell, 1995). Practically, the presence of monovalent cations, especially potassium, is essential for preparing gels from K-carrageenan. Divalent anions such as magnesium and calcium also can affect the strength and texture of the gel. K-Carrageenan gels are. known to be thermally reversible; the difference between the setting and the melting temperature is much less than that of gels made of agar (Lockwood, 1985). Upon gelation of polysaccharides, ordered regions or junction zones are formed through the association of certain length of polysaccharides. Intermolecular hydrogen bonding is responsible for this association (Lockwood, 1985). In the presence of milk proteins, gelation occurs at relatively low carrageenan concentrations and at temperatures below the carrageenan helix-to-coil transition temperature (Drohan et al, 1997). Table 5 and Figure 5 demonstrate the most important properties of carrageenans, and the structures of carrageenans, respectively. 34 c C3 B 01 CJ 05 u i~ a CJ C 03 C 0J a 01 03 VI C a c 0> M CS t< S* 03 CJ C a> i« s-C3 « "3 u <u Y CB vi >, 3 CJ H c es C a cj Of a] c C3 c CJ CJ ec a u u . « o . u c C o o + + + + H + + C o !-• <L> a. cj 'a, o i_ o >< 15 c c O o ca *—^  <L> + ca SW z cd en + + u + 1) C3 Z „ + + + w + c o _3 o o 3 ~o o .is ca ca o c a 03 ca u u u u 3 .a O 3 fa S o c a 3 ca o o oo <N o o o o OO CS "O <-H c o 5 3 c o •4—. ~ + + ca <D JS J3 . O £ ca j= j=: jg oa ca C o a o •e .S CD 2 5 8 oo O Pi oo PH 60 c ^ 3 (A w w oo -a o o O -a o o O o o a, cu CJ B 2 5> 3 3 c « 3 crt —' O •'•8 3 « + + o H - ' 3 -*-• o o oo Z 35 CH 2 OH -O3SO •O OH K-carrageenan CH 2 OH C H 2 -O3SO > O o o OH i-carrageenan CH 2 OH CH2OSO HO •O > o OH OSO3-X-carrageenan Figure 5: Comparison of three carrageenan types Source: Thomas, 1992 6.4 Applications Coatings that contain carrageenans have been used, as a major or only component, to carry antimicrobials and to decrease moisture loss, oxidation, or disintegration in a variety of foods (Krochta and De Mulder-Johnston, 1997). These polysaccharides have been widely used as stabilizing, thickening and gelling agents in the food, agricultural, cosmetics and pharmaceutical industries. They are also used as processing aids in the fining of beer and wine (Guiseley et al, 1980; Huffman and Shah, 1995; Tziboula and Home, 1997). A concentration of about 1% is required for applications in water, while the usual concentration in milk or other proteins varies from 0.005% in evaporated milk to 0.03% in chocolate milk, to 0.2 to 0.8% for milk puddings (Guiseley et al, 1980; Moirano, 1977). Since carrageenan absorbs water rapidly, clumping is a problem. It is recommended to mix the carrageenan with a diluent, such as sugar, before addition of water. Or, if no diluent is to be used, the carrageenan has to be slowly added to a cold liquid, while stirring (Guiseley et al, 1980). 7 Protein-Polysaccharide Interactions Proteins and polysaccharides are two of the major macromolecules found in various food products. The structure, stability and textural characteristics of foods mainly depend upon the individual biopolymers as well as mixed biopolymer interactions (Galazka et al, 1999b). Several variables including pH, ionic strength, temperature, and 37 variations in biopolymer structure can determine the type of polysaccharide-protein interactions that will occur. When aqueous solutions of polysaccharides and proteins are mixed, three situations may result. The first type is when the mixture contains a liquid two-phase system and these two components are mainly in different phases due to the limited thermodynamic compatibility of polysaccharides and proteins in that medium. The next condition, complex coacervation, occurs when both components are in the same concentrated phase due to the formation of an insoluble electrostatic protein-anionic polysaccharide complex. A homogeneous stable solution is obtained when the two components either do not interact or exist as soluble complexes (Tolstoguzov, 1986). Polysaccharides do not strongly react covalently with proteins since proteins have few free amino acids or guanidyl groups, while polysaccharides contain few free aldehyde groups. Because of these reasons, covalent bonds can not be easily formed. The interaction between a polysaccharide and protein is mainly due to ionic (coacervates) or hydrogen bonds, electrostatic, or van der Waals forces (Sand, 1982; Walter, 1998b). Laurent (1963) hypothesized that polysaccharides may affect the solubility of proteins. To examine this hypothesis, he used several proteins in combination with dextran (which is an uncharged polysaccharide). He found that the presence of dextran, in media of high ionic strength, decreased the solubility of albumin, fibrinogen, and y-globulin. Also, the size of protein and the concentration of dextran determines the extent of insolubility, i.e., the larger the protein, the less soluble it is in dextran, and the higher the protein concentration, the lower the solubility (Laurent, 1963). 38 Hidalgo and Hansen (1969) studied the interactions between carboxymethylcellulose (CMC) and P-lactoglobulin (1:3 ratio) at various pH. They found that, under specified conditions of pH, ionic strength, and the ratio of these components, B L G forms insoluble complexes with anionic polysaccharides. They showed that the amount of soluble protein was the lowest at pH 4.0, while no protein was precipitated at pH below 2.5 or above 7.0. Ionic strength was also found to affect the complex formation. This was confirmed by varying ionic strength of a mixture of C M C : B L G at pH 4.0. At constant pH, the higher the ionic strength (salt concentration), the lower the amount of precipitated protein. The effect of C M C concentration on precipitation of B L G was also studied at pH 4.0, with constant ionic strength of 0.05, which showed the highest amount of precipitated protein in the previous experiment. Increasing C M C concentration (to 0.3:1.0 ratio), initially caused a sharp decrease in the amount of soluble protein; most of the protein was precipitated. However, further increase (to 0.8:1.0 ratio and up) of C M C concentration increased the amount of soluble protein, for two out of three examined types of C M C . Two separate reactions occurred. First, a primary reaction where insoluble complexes were formed, and second, a reaction that stabilized the initial product. This primary reaction is pH sensitive, so it should be ionic in nature (Hidalgo and Hansen, 1969). Many researchers (e.g., Grinberg and Tolstoguzov, 1997; Lin, 1977) studied dependence of compatibility of polysaccharide.protein solution on conditions such as pH and ionic strength. Ganz (1974) also studied the effect of pH on the viscosity of CMCprotein mixtures and found that by decreasing pH to the isoelectric point (pi) of the protein, the viscosity increased because of the formation of a soluble complex. By 39 lowering pH to below the pi, the overall net charge decreased and the complexes precipitated. Imeson et al. (1977) also showed that the interactions between B S A or myoglobin and anionic polysaccharides (pectate, C M C , and alginate) were dependent upon the charge carried by both macromolecules. Spectral changes in myoglobin occurred when mixed with polysaccharides at pH 6.0 and the thermal stability of both B S A and myoglobin decreased. However, heat denaturation at pH 6.0 formed stronger interactions, causing the formation of high molecular weight complexes, which inhibited precipitation. The ratio of polysacchariderprotein in a solution also affects the stability of a protein (Hansen, 1968). Dickinson (1998) showed the importance of this ratio on the structural states of a binary system of spherical particles and adsorbing polymer molecules (such as polysaccharides and proteins). He mentioned that "bridging flocculation" phenomenon occurs when there are not enough polymers to cover the entire particle, and some of the polymer molecules attach to more than one particle. If the polymers saturate the particles, "steric stabilization" occurs. Finally, if there is an excess amount of polymer, a gel network is formed, which is called "immobilization of polymer covered particles in a polymer gel network". Lin and Hansen (1970), who studied the stabilization of casein micelles by carrageenans (pH 6.7), showed that the higher the ratio of the carrageenanxaseins, the higher the casein stability. They also found that the stability of various hydrocolloids:^ casein mixtures at 1.0:4.0 ratio (pH 6.7) depended on the type of hydrocolloid. For example, guar gum caused precipitation of fx-casein at this ratio, while i and K-carrageenans could stabilize 90-100% of the 0;-casein. However, Skura and Nakai (1981), who studied the stabilization of Osi-casein by K-carrageenan 40 (10.0:2.0, w/w ratio, pH 6.6) in the presence of calcium, argued that if the ratio of the constituents was kept constant, while the amount of 0Csi-casein was increased (so the amount of K-carrageenan would increase accordingly), the stability of the GCsi-casein decreased. They speculated that at certain concentrations of the oCsi-casein, the stabilizer was not effective. When using equal concentrations of the gum in both milk and in water, one can get higher viscosity in milk compared to water. This is due to the gum characteristic, "milk reactivity", which is an important criterion in choosing stabilizers in dairy products (Schmidt and Smith, 1992). The overall effect of adding gums to dairy-type products depends on the amount of both gum and protein, as well as pH and concentration of counterions such as Na + , K + , Ca 2 + , etc (Dickinson, 1998). Schmidt and Smith (1992) showed that both whey proteins and casein proteins have the ability to interact with hydrocolloids. Interaction of K-carrageenan with whey protein isolate at various pH was studied by Mleko et al. (1997). They showed that the highest shear stress values for K-carrageenan-10% WPI mixtures was observed at pH 6.0, while the mixtures around pH 7-11 had similar shear stress. Complex coacervation is the spontaneous phase separation of mixed biopolymer systems, into two phases, due to interaction of negatively charged polysaccharides (such as carrageenans) with protein. Ionic bonds are important in this phenomenon. On the acid side of the isoelectric point of the protein, the positively charged epsilon amino groups of lysine react strongly with the negatively charged groups (OSO3) of the carrageenan. After ionic bonding has brought the molecules close together, hydrogen bonds also form. 41 On the other hand, at the alkaline side of the isoelectric point of the protein, both macromolecules are negatively charged and divalent cations are required for coupling. At the isoelectric point, the protein and polysaccharide form ionic bonds with and without divalent cation (Dickinson, 1993 and 1998; Imeson et al, 1977; Lin, 1977; Samant et al, 1993). In other words, the pH of the solution of a mixture of protein and polysaccharide compared to the pi of the protein determines how these biopolymers interact. Most food proteins, with pi about 5.0, form complex coacervates with anionic polysaccharides, where they carry opposite charges (between pH 3.0 and 5.0). When both biopolymers carry a net negative charge (pH > protein pi), soluble protein-polysaccharide complexes are formed. In this situation, anionic polysaccharides interact with positively charged patches of the protein (Dickinson, 1998). Net repulsive protein-anionic polysaccharide interactions may occur at a pH>pI. Strong attractive interactions may occur between positively charged proteins (pH<pI) and anionic polysaccharides, especially at low ionic strength. In any system, however, the protein-polysaccharide interaction may change from net repulsive to net attractive, or vice versa, upon changing the temperature, pH or ionic strength (Dickinson, 1993). Ionic interactions of carrageenans and protein are shown in Figure 6. 4 2 Protein T~~\—i—i—i—i—i—i—r~ NH2 COf NH2 COf NH2 COf NH2 COf NH2 COf Ca SOA 2+ Ca SO* 2+ Ca 2+ SO* Ca' SO, Ca SO, .2+ Protein Carrageenan ' 2 NHf COf NH3 + COf NH3* II III IV so4' SOi so4 so4 so4 J L Carrageenan Protein i—i—i—r NH3* C02H NH3+ C02H NH3 + co2 NH i r + COf NH3+ CO, so4~ _L S04' SOf J L so4- so4~ Carrageenan Protein ~i—i—i—i—i—i—i i i r NH3+ C02H NH3+ C02H NH3* C02H NH3+ C02H NH3+ C02H S0£ J_ 50. S04 _L SOi S04' _L Carrageenan Figure 6 : Ionic interactions of carrageenans and protein I pH > pi Interaction mediated by divalent cation II pH = pi Protein/carrageenan net-charge ratio = 0. No direct interaction III pH < pi Protein/carrageenan net-charge ratio < 1. Partial interaction; no precipitation IV pH < pi Protein/carrageenan net-charge ratio = 1. Complete interaction; precipitation Source: Guiseley et al., 1980 43 8 Raman Spectroscopy Various spectroscopic techniques based on absorption, emission, and scattering processes, are being used as analytical techniques in science. Techniques such as visible laser Raman spectroscopy, a branch of vibrational spectroscopy, have proven to be a useful analytical tool for the study of the structure of biological materials. A l l these techniques are based on the interactions of electromagnetic radiation with electrons and nuclei of molecules (Li-Chan, 1996a). Vibrational spectroscopy involves two effects, the absorption of infrared radiation, and inelastic scattering of light in the visible, near infrared (NIR), and ultraviolet (UV) regions of the spectrum (Howell et al., 1999; L i -Chan, 1996a; Painter, 1984;). In infrared spectroscopy, the activity of a vibrational mode depends on a possible change in dipole moment during vibration. Change in the polarizability of the molecule, however, is the indicator of a Raman active vibrational mode (Yu, 1977). In 1928, Raman and Krishnan first observed that some of the light, scattered by a liquid, was changed in wavelength. Inelastic collisions between the molecules composing the liquid and photons (particles of light composing the light beam) form the basis for the physical origin of Raman scattering (Carey, 1982a). An inelastic collision can be defined as an exchange of energy between the photon and the molecule with a consequent change in energy and wavelength of the photon. Since the total energy is conserved during the scattering process, by measuring the energy lost or gained by the photon, changes in molecular energy can be determined. Since Raman spectroscopy is mainly concerned with the vibrational energy level transitions of the molecule, the Raman spectrum is a vibrational spectrum of a molecule. In other words, the Raman effect is considered a 44 scattering process and the interaction between the molecule and the photon happens in a short time. Also, Raman bands relate to photons that have inelastically bounced off the molecules. This is in contrast with an infrared spectrum that corresponds to energies where infrared photons are absorbed by the molecule (Carey, 1982a). Due to inelastic collision of the incident photons with molecules, a shift in wavelength of the exciting incident beam occurs; a phenomenon that is called "Raman scattering". In Raman scattering, the frequency of scattered radiation is shifted ± V j from the incident radiation, resulting in Stokes' (v 0 - V j ) lines and anti-Stokes' (v 0 + V i ) lines. Inelastic collisions can be divided into two types, resonance and non-resonance, which differ due to the energy of the incident or exciting beam. On the other hand, the elastic collision of photons and sample molecules is known as Rayleigh scattering (Li-Chan, 1996a; Li-Chan et al., 1994). The relationship between infrared absorption, Rayleigh and Raman scattering is shown in Figure 7. 45 Excited electronic states > J3 v 0 ' fa v 0 ' ^0 -Vj V 0 i VO +V; Vi Infrared Absorption Rayleigh scattering Stokes' Anti-Stokes I Raman scattering Stokes' Resonance Raman scattering Ground electronic states Figure 7: The relationship between infrared absorption, Rayleigh and Raman scattering v0: frequency of the exciting or incident light beam Vj=Av (Raman shift) for Raman scattering; the frequency at which incident electromagnetic radiation is absorbed in the infrared region. Source: Li-Chan (1996a) 4 6 9 Raman Spectroscopy of Proteins Raman spectroscopy is a valuable. technique for the study of the structure of molecules such as proteins in a solid or liquid form (Li-Chan and Qin, 1998). Various amino acid side chains and peptide backbone contribute to the Raman spectrum of a protein recorded under non-resonance conditions. Since the frequency and the relative intensity of vibrational motions of the amino acid side chains and polypeptide backbone of proteins are sensitive to chemical changes and environmental factors, Raman spectroscopy is a good technique to use in the study of proteins. Tables 6 and 7 show the principal amide I IR frequencies and typical assignment of side-chain vibrations and wavenumber of protein Raman spectra, respectively. Using Raman spectroscopy, one can obtain some information regarding the secondary structure of proteins based on amide I, amide m, and skeletal stretching modes of the polypeptide backbone. For example, amide I and amide rn modes are used to characterize the secondary structure of the peptide backbone (Carey, 1982b). There are nine normal modes for the amide bands of proteins, which are called A , B , and I to VII in order of decreasing frequency. The vibrational assignment of the amide I band, near 1650 cm"1, consists of primarily C=0 stretch, while amide II band, near 1550 cm' 1, contains primarily N - H bend and C-N stretch, and amide in band, near 1300 cm"1, contains mainly C-N stretch and N - H bend. A l l these bands are used to study protein structure, however, amide I and DI bands are commonly used for the purpose of assigning secondary structure of proteins (Pelton and McLean, 2000). 47 Table 6: Main Amide I Frequencies Characteristic of Protein Secondary Structure Conformation Wavenumber (cm'1) a-helix 1650-1657 Antiparallel P-sheet 1612-1640; 1670-1690 (weak) Parallel P-sheet 1626-1640 Turn 1655-1675; 1680-1696 Unordered 1640-1651 Source: Pelton and McLean, 2000 Raman spectroscopy can also detect vibrations due to various amino acid side chains. Examples are S-S (500-550 cm"1) and S-H (2550-2580 cm"1) groups of cystine and cysteine, the C-H (2800-3000 cm"1) groups of aliphatic amino acids, the aromatic rings of tyrosine (especially the tyrosine doublet at 830 cm"1 and 850 cm"1), tryptophan (mainly around 761, 880, 1360 cm"1), and phenylalanine (mainly around 1004 cm"1). Some other important amino acid side chains include the imidazole ring of histidine (1491 cm"1), and the COO" (1400-1430 cm"1) and C O O H (1700-1750 cm"1) groups of aspartic and glutamic acids (Li-Chan, 1996a). The band around 761 cm"1 of the Raman spectrum is assigned to tryptophan (an aromatic amino acid), which is useful for monitoring the polarity of the environment. A decrease in the intensity of the tryptophan band indicates that the tryptophan is exposed to a more polar environment. The intensity ratio of tyrosine doublet indicates the polarity of environment as well as the hydrogen bonding of the phenolic hydroxyl groups. However, the location and the intensity of the bands corresponding to phenylalanine are not sensitive to the environment and therefore can be used as an internal standard for normalization of the Raman spectra of proteins (Li-Chan, 1994 et al. and 1996a). 48 Table 7: Typical assignment of side-chain vibrations and wavenumber in the Raman spectra of proteins Assignment Wavenumber (cm" ) Aliphatic amino acid residues C-H stretch 2800-3000 C H 2 bend 1465, 1450 Polar or charged residues O O stretch of Asp, Glu COOH 1700-1750 C=0 stretch of Asp, Gly COO" 1400-1430 -CO-NH-ofAsn 1650 -CO-NH-ofGln 1615 N H 3 + of Lys 1640,1600 Imidazole of His 1491 OH of Ser, Thr (weak) 1339 Aromatic amino acid residues C-H stretch 3050-3100 Phe 1605, 1585, 1207, 1030, 1006, 622 Tyr 1610, 1590, 1263, 1210, 1180, 850, 830, 645 Trp 1622, 1582, 1553, 1363, 1014, 879, 761, 577, 544 Sulfur-containing residues Trans-gauche-trans (SS of cystine) 540 Gauche- gauche- trans (SS of cystine) 525 Gauche-gauche-gauche (SS of cystine) 510 Trans C-S of Met, Cys, Cys/2 745-700 Gauche C-S of Met, Cys, Cys/2 670-630 S-H of cysteine 2550-2580 Source: Li-Chan et al, 1994 Although general assignment of various bands of Raman spectrum of proteins is as mentioned in Table 7, detailed assignment may be slightly different for individual proteins. Table 8, for example, shows the assignment of bands for B L G in D2O. 49 Table 8: Assignment of major bands in the Raman spectra of deuterated (3-lactoglobulin Assignment Wavenumber (cm"1) vS-S 508 Trp 540 - 597 - 624 - 647 - 742 Trp 761 - 805 Tyr 831, 856 Trp 883 Amide III' (a-helix) 941 Amide IIP 960 Amide IIP O-sheet) 984 Phe 1005 Phe 1033 vC-N 1072, 1130, 1162 Tyr 1180 Tyr, Phe 1207 - 1241 Trp 1333 His 1406 5 C H 2 1453 Trp 1556 Amide F 1658 Notes: v, stretching mode; -, not assigned clearly; 8, deformation mode Source: Nonaka et al, 1993 Beside Raman spectroscopy, several other techniques are available for the study of protein structure. Examples include circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (NMR), and X-ray crystallography. C D requires very small amount and concentration of sample and is useful for measuring the temperature dependence of protein secondary structure. It can be used only for the analysis of clear samples. The three-dimensional structure of proteins can be obtained using X-ray crystallography, for protein crystals, and N M R , for protein 50 solutions (Venyaminov and Yang, 1996). X-ray crystallography and N M R methods have some drawbacks. For example, the former needs crystalline proteins, many of which are difficult or impossible to obtain, and for the latter, better resolution can be obtained for proteins with molecular weight higher than 15000 daltons. FTIR and Raman spectroscopy can provide complementary information (Pelton and McLean, 2000). Infrared absorption is due to the absorption of energy by vibrating chemical bonds. Although the same bonds are responsible for Raman scattering, a weak band in IR might be strong in the Raman spectrum and vice versa (Pelton and McLean, 2000). Due to the low intensity of O-H vibration in Raman spectra, Raman spectroscopy can be used for the study of conformation in aqueous solution; water is present in most biological systems (Tu, 1986). 10 Raman Spectroscopy of Polysaccharides Information on using Raman spectroscopy for studying the structure of polysaccharides is scarce. Infrared spectroscopy, however, has been widely used for this purpose (refer to references in the article by Caceres et al., 1997). Second derivative Fourier transform infrared spectroscopy (FT-IR) was used to identify various carrageenans (Caceres et al., 1997). Malfait et al. (1987) analyzed the sodium salt of K-carrageenan (KCG, 20 mg/mL H2O or D2O) in the 700-1500 cm"1 region using Raman spectroscopy. A strong band was observed around 730-740 cm"1 in both aqueous and deuterated solutions, which was assigned to complex ring vibrations. Bands near 1150, 1350, and 1460 cm"1 were assigned to CO stretching, COH, and C H 2 bending modes, respectively. In another study by Malfait et al. (1989), sodium (Na+), potassium (K + ) , and 51 rubidium (Rb+) salts of i- and K-carrageenan (4.5 and 2% w/v, respectively in H2O) were analyzed using Raman spectroscopy. The band around 730-740 cm"1 was observed at 730 cm"1 for K + and Rb + salts of K C G and at 734 cm"1 for Na + salt of K C G . They also observed some differences between ionic interaction of Na + salt compared to other salts using several other bands. Although sulfated polysaccharides could be analyzed using both Raman and infrared (IR) spectroscopy, the vibrations of the pyranose ring, however, can only be analyzed using Raman spectroscopy. The assignment of bands observed in FT-Raman and FT-IR spectra is shown in Table 9. Table 9: The assignments of bands in the FT-Raman and FT-IR spectra of sulfated polysaccharides Assignment Wavenumber (cm1) Polar OH group 3600-2800 C-H stretching vibration 2969.5 and 2905.6 C H 2 bending 1468.9, 1330.5, 1284.8 CO, COH, HCO, and HCC vibrational modes 1200-950 C-O stretching (with contributions of CC, CCO, and COH vibrations) 1079.6 CO stretching (with contributions of CC and CO modes) 965.5 Weak signal for 3,6-anhydro-galactopyranose 930.4 SO group 1089.8, 1075.5, 1072.5 CH vibration coupled with C-O-S vibration 850.7, 855.8 C-C and C-0 vibrations coupled with C-H mode of the anomeric carbon 890 of P-D-galactopyranose Adapted from: Matsuhiro (1996); Matsuhiro and Rivas (1993) 52 CHAPTER III Comparison of Whey Protein Surface Hydrophobicity Measured at Various pH, Using Three Different Fluorescent Probes 1 Abstract The influence of type of fluorescent probe on the surface hydrophobicity values determined for three protein samples was assessed using neutral (6-propionyl-2-(N,N-dimethylamino)naphthalene or PRODAN) versus anionic aliphatic (cis-parinaric acid or CPA) and aromatic (l-anilinonaphthalene-8-sulfonic acid or ANS) fluorescent probes. Surface hydrophobicity of whey protein isolate (WPI), beta-lactoglobulin (BLG), and bovine serum albumin (BSA) under heated (80 °C for 30 min) and unheated conditions and at varying pH (3.0, 5.0, 7.0, and 9.0) were measured using ANS, CPA, and PRODAN. ANS and CPA yielded opposing results for the effects of pH and heating on protein hydrophobicity. Hydrophobicity was lower at pH 3.0 than at other pH values for all proteins measured by P R O D A N , while the values measured by ANS and CPA at pH 3.0 were quite high compared to other pH values, suggesting the influence of electrostatic interactions on anionic probe-protein binding. These results suggest that the presence or absence of a permanent charge as well as the aromatic and aliphatic nature of fluorescent probes can affect protein surface hydrophobicity values measured under various pH conditions. 53 2 Introduction Due to their three-dimensional structures, food proteins are involved in many functional processes (Stryer, 1968). Over the years, food chemists have been trying to elucidate the mechanism of protein functionality. However, the food industry is still looking for ways to predict functional properties of proteins. Hydrophobic, steric and electrical parameters are the most important variables that affect the structure of proteins. Among these factors, hydrophobicity is known to be significantly related to the functional properties of proteins (Nakai, 1983). The tendency of non-polar solutes to adhere to one another in aqueous environment is called hydrophobicity (Cardamone and Puri, 1992). Due to high sensitivity, noninvasiveness, and availability of imaging techniques, fluorescence spectroscopy has been considered to be one of the most promising and widely used techniques in medicine, biology, biochemistry, and molecular biophysics for the 21st century (Slavic, 1994b and Royer, 1995). Fluorescent methods depend on the response of some fluorescent component, either intrinsic or extrinsic, to its environment following optical excitation (Damodaran, 1989; Hudson et al, 1986). One approach to quantify protein hydrophobicity is through fluorescent probe methods. The quantum yields of fluorescence and wavelength of maximum fluorescence emission of these compounds depend on the polarity of their environment (Li-Chan, 1999). Several fluorescent probes such as l-anilinonaphthalene-8-sulfonic acid (ANS) and c/s-parinaric acid (CPA) have been widely used to measure protein hydrophobicity. These probes have low quantum yield of fluorescence in aqueous solution. Upon binding of the probes to accessible hydrophobic regions of proteins, an increase in fluorescence is observed, 54 which is used as a measure of protein surface hydrophobicity. However, due to the possible contribution of both electrostatic and hydrophobic interactions to the binding of these anionic probes to proteins, interpretation of results based on these probes has not been easy. There has been a need for a neutral probe to circumvent this problem. Although ANS has been used widely for the measurement of protein hydrophobicity, its binding is greatly influenced by protein charge, due to influence of pH or ionic strength, as was shown by D'Alfonso et al. (1999). The contribution of electrostatic interaction to the binding of ANS and CPA to positively charged derivatives of B L G was also noted by Mattarella and Richardson (1983). ANS and 6-propionyl-2-(dimethylamino) naphthalene (PRODAN) are known for sensing the polarity of environment in biological materials (Rettig, 1993). The fluorescence emission of ANS and some close analogues depend on environment (Penzer, 1972). ANS is composed of aromatic rings, while C P A possesses an aliphatic hydrocarbon chain. The binding sites for CPA on protein molecules may therefore differ from the sites for ANS (Nakai and Li-Chan, 1988a). Both ANS and C P A are considered anionic probes, containing sulfonic acid and carboxylic acid groups, respectively. Depending on the pK of the groups and the pH of the environment, sulfonate and carboxylate groups may be formed. Yet, there is a need to conduct comprehensive studies of the structural and molecular properties of proteins such as P-lactoglobulin in the pH range 1 to 10, in order to predict the behavior of proteins in model systems in the presence of other variables (Phillips et al, 1994). Under these conditions of acidic and alkaline pH, contribution of charged interactions in the measurement of surface hydrophobicity using the anionic fluorescent probes may be encountered. 55 PRODAN, on the other hand, is a solvent-sensitive probe and has no charge. This will eliminate possible electrostatic interaction contributions in the measurement of protein hydrophobicity (Hermetter et al, 1993). The first reference on the synthesis and spectral properties of P R O D A N dates back to 1979 (Weber and Farris, 1979). Studies on the binding and spectral properties of P R O D A N with bacteriorhodopsin, membrane and protein interior, spectrin, tubulin and horseradish peroxidase have been cited (Baasov and Sheves, 1987; Baiter et al, 1988; Bruins and Epand, 1995; Catalan et al, 1991; Chakrabarti and Basak, 1996; Chakrabarti, 1996; Heisel et al, 1987; Krasnowska et al, 1998; Lasagna et al, 1996; Macgregor and Weber, 1986; Mazumdar et al, 1992). Wald et al (1990) investigated the lipid domains in high density lipoproteins, using PRODAN. Bunker et al (1993) studied photophysical properties of P R O D A N in solution. Royer (1995) stated that P R O D A N is an excellent example of solvent relaxation phenomena, since it shows a very large excited-state dipole that renders the emission spectrum quite sensitive to the relaxation of the solvent. Prendergast et al. (1983) pointed out that the sensitivity of P R O D A N to solvent polarity is due to the large dipole moment developed in the excited state as a consequence of facile charge derealization between the 2-dimethylamino moiety and the carbonyl group in the 6-position of the naphthalene. In response to a concern about the possible effect of anionic probes on the binding of proteins, the objectives of this study were to establish a fluorescent probe method using an neutral probe (PRODAN), prepared as a methanol stock solution, to compare the values of protein surface hydrophobicity measured using this probe with those measured by aliphatic (CPA) and aromatic (ANS) anionic probes. Surface hydrophobicity of three protein samples (whey protein isolate, P-lactoglobulin and bovine serum albumin) before 56 and after heating (80 °C for 30 min) at various pH (3.0, 5.0, 7.0 and 9.0) were measured using these three probes. 3 Experimental Procedures 3.1 Materials Whey protein isolate (WPI), (3-lactoglobulin (BLG) and bovine serum albumin (BSA) were from Foremost Farms USA (Waukon, Iowa; Daritek N V B 389, Lot #21-4080, containing 89.43% protein and 4.26 % moisture; obtained as a gift from Canadian Inovatech Inc. Abbotsford, B.C., Canada), Sigma (St. Louis, M O , L-2506, approximately 80% BLG) and Sigma (St. Louis, M O , A-4503, minimum 96% BSA), respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of WPI, B L G , BSA, whey powder (Canadian Inovatech Inc. Abbotsford, B.C., Canada), and high range (36,000-205,000 daltons) standard molecular weight markers (Sigma, St. Louis, M O , M -3788) was performed according to the method of Laemmli (1970), using 10-15 Gradient Phastgel, PhastSystem™ (Pharmacia L K B Biotechnology, Uppsala, Sweden). Whey powder was used as a comparison with WPI. The electrophoretic analysis of B L G and B S A samples (Fig. 38, Appendix LTI). Three fluorescent probes, l-anilinonaphthalene-8-sulfonic acid (ANS), cis-parinaric acid (CPA) and 6-propionyl-2-(dimethylamino) naphthalene (PRODAN) were obtained from Sigma (St. Louis, MO), Molecular Probes (Eugene, OR), and Molecular Probes (Eugene, OR), respectively. 57 Buffers were prepared according to the method of Dawson et al. (1969). For buffers at pH 3.0, 5.0, and 7.0, a mixture of 0.1 M citric acid (BDH, Toronto, ON) and 0.2 M Na2HP04 (Fisher Scientific, Fairlawn, NJ) in the following proportions (v/v) were used, respectively: 79.5/20.5, 48.5/51.5, 17.7/82.3 yielding final buffer composition of 0.079 M/0.041 M , 0.048 M/0.103 M , 0.017 M/0.165 M citric acid/sodium phosphate, respectively. For pH 9.0 buffer, 50 mL 0.025 M Na2B407 (Fisher Scientific, Fairlawn, NJ) was adjusted to pH 9.0 with 1 N NaOH (BDH, Toronto, ON) and was brought to 100 mL final volume (0.0125 M final concentration). In all buffers, 0.02% sodium azide (Sigma, St. Louis, MO) was included to prevent growth of microorganisms. 3.2 Preparation of proteins for measurement of S 0 Stock protein solutions containing 1.5% (w/v) protein sample in distilled deionized water, with 0.02% sodium azide, were prepared in duplicate. Protein concentrations of WPI, B L G and B S A were determined by absorbance at 280 nm using E1%icm of 11.7 (Kitabatake et al, 1994), 9.6 (Fasman, 1992) and 6.61 (Fasman, 1992), respectively. The stock protein solutions were diluted, with appropriate buffers, to an intermediate concentration (0.03%) and then either diluted to final concentrations as is, or after heat treatment for 30 minutes at 80 °C. A l l three proteins were soluble at the pH and ionic strength conditions studied; however, B S A showed slight turbidity after heating. Heat treatment of protein solutions in 50-mL volumetric flasks, which were covered with parafilm to avoid evaporation, was done in a water bath (Blue M Magni Whirl). After heating, samples were cooled immediately, under running water. For the fluorometric probe assay, the stock proteins were diluted with required buffers at pH 3.0, 5.0, 7.0, and 58 9.0 to typical concentration ranges of 0.005-0.025% w/v (5 concentrations) for measurements using ANS and CPA and 0.002-0.01% for measurements using PRODAN. A simple flowchart of the protein preparation procedure is shown in Figure 8. Stock: 1.5% WPI, 3-lg, and BSA in DD water Measure Absorbance @ 280 nm Intermediate: 0.03% in buffers (pH 3.0, 5.0, 7.0, 9.0) f Heating, 30 min at 80°C \ Cooling 0.005-0.025% in buffers for ANS and CPA 0.002-0.01% for P R O D A N Figure 8: Flow chart of protein preparation for measurement of S 0 59 3.3 Hydrophobicity determination Protein surface hydrophobicity using ANS and CPA probes was determined by modification of the method of Kato and Nakai (1980). A similar approach was used to develop a new method using PRODAN. Stock solutions of 8 x 10"3 M ANS, 3.6 x 10"3 M CPA and 1.41 x 10"3 M PRODAN were prepared in 0.1 M phosphate buffer (pH 7.4), ethanol and methanol, respectively. For CPA, 10 ug butylated hydroxyanisole (BHA) was added per mL of ethanol as an antioxidant. CPA and P R O D A N stock solutions were transferred to screw-capped vials, covered with aluminum foil, and the cap sealed with parafilm to prevent evaporation of ethanol or methanol. The CPA and P R O D A N stock solutions were stored in the freezer (< -10°C) until the day of experiment when they were held in ice throughout the experiment. ANS stock solution was stored in a screw-capped container at room temperature. A l l the probes were wrapped in aluminum foil to avoid exposure to light. Under these conditions, the stock solutions were stable for at least 6 months based on the relative fluorescence intensity (RFI) values of the probes in the solvents used for standardization (data not shown). Concentrations of the ANS, CPA and P R O D A N stock solutions were determined spectrophotometrically at 350, 303, and 360 nm, respectively, using molar absorption coefficients of 6350 (ANS)= 4.95 x 103 M " 1 cm"1 (Weber and Young, 1964), £303 (CPA)=7.6 x 104 M " 1 cm"1 (Haughland, 1996) and e36o (PRODAN) = 1.8 x 104 M " 1 cm"1 (Chakrabarti, 1996), respectively. A l l fluorescence measurements were made with a Shimadzu RF-540 (Shimadzu Corporation, Kyoto, Japan) spectrofluorometer. For hydrophobicity determination using P R O D A N , the excitation/emission slits and wavelengths were set at 5 nm/5 nm and 365 nm/465 nm, respectively. To successive 60 samples containing 4 mL of diluted proteins, 10 pL P R O D A N stock solution was added and mixed well by vortexing. After 15 minutes in the dark, the relative fluorescence intensity (RFI) of each solution was measured, starting from buffer blank (buffer + probe) and then the lowest to the highest protein concentration, and rinsing the fluorometer quartz cell between samples with a small volume of the solution to be measured. RFI of buffer and protein dilution blanks (no PRODAN) were also measured. The RFI of each protein dilution blank was subtracted from that of corresponding protein solution with P R O D A N to obtain net RFI. The initial slope (S0) of the net RFI versus protein concentration (percent) plot was calculated by linear regression analysis with Microsoft Excel for Windows 95 (Version 7.0), and used as an index of the protein surface hydrophobicity. To correct for day-to-day instrumental fluctuations in relative fluorescence intensity, standardization was performed by measuring the RFI of 4 mL methanol with 10 pL P R O D A N and correcting to a standard value of 50. ANS and C P A probe methods were performed essentially according to the method of Kato and Nakai (1980). The procedure was the same as for P R O D A N , with the following exceptions. Excitation and emission wavelengths were 390 and 470 nm, respectively for ANS, and 325 and 420 nm, respectively for CPA. The excitation and emission slit widths were 5 and 5 nm, respectively for ANS and 2 and 5 nm for CPA. The amount of probe stock solution for measuring hydrophobicity using these two probes was 20 pL, to be added to 4 mL diluted protein. For standardization of the ANS assay, the measured RFI for 10 mL methanol with 10 pL ANS was corrected to a value of 15. For the CPA assay, the measured RFI for 4 mL n-decane with 10 pL C P A was corrected to a value of 3. Specific experimental conditions for each probe are summarized in Table 10. 61 Table 10: Specific experimental parameters for each fluorescent probe Probe Stock solution in Standard RFI in Xcx nm Km nm ANS Methanol Methanol 365 465 CPA Buffer Methanol 390 470 PRODAN Ethanol Decane 325 420 Surface hydrophobicity values were determined using at least duplicate analyses. In all cases, R 2 values of 0.99 were noted for the linear regression analyses used to calculate surface hydrophobicity (S0) values. Quadruplicates of several samples were performed and the coefficients of variation (CV) of the replicates were found to be less than 3%. Figure 9 shows a simplified flow chart of fluorescence measurement. Diluted protein solutions, 5 concentrations ± Probe Measure RFT at specific emission and excitation wavelength Calculate slope of net RFI versus % protein Figure 9: Flow chart of fluorescence measurement 62 3.4 Statistical Analysis Data were analyzed by an analysis of variance (ANOVA) procedure applying the General Linear Model, with further analysis using Tukey's pairwise comparison test to determine significant differences (p<0.05) between treatment means (Minitab for Windows Version 12, Minitab Inc., State College, PA). Due to the range in magnitude of data obtained, logarithmic transformation of the S 0 values was performed prior to the Tukey's test (Arteaga, 1994; Zar, 1974).. 63 4 Results and Discussion 4.1 Comparison of hydrophobicity measured using anionic probes, ANS and CPA Protein surface hydrophobicity (S0) values measured using the two anionic fluorescent probes, ANS and CPA, are depicted in the top and middle panels, respectively, in Figures 10-12. Surface hydrophobicity values based on the ANS probe method ranged from 78 for heated B L G at pH 5.0, to 3020 for heated B S A at pH 3.0. Using the ANS method, a significant (p<0.05) increase in the S 0 value after heating was observed at pH 7.0 and 9.0 for WPI and for B L G . No significant effects of heating were observed for WPI or B L G at pH 3.0 or 5.0. On the other hand, for BSA, heating significantly decreased the S 0 value at all pH, except at pH 3.0. At pH 3.0, 5.0, and 7.0, the hydrophobicity determined by ANS was in the following order for the unheated proteins: BSA>WPI>BLG, while the reverse was seen using CPA. For WPI and B L G at pH 7.0 and 9.0, heating significantly affected the S 0 value, but ANS showed significant (p<0.05) increase while CPA showed significant (p<0.05) decrease. C P A hydrophobicity values ranged from 12 for both heated and unheated B S A at pH 3.0, to 380 for unheated B L G at pH 5.0. Based on the CPA method, heating generally decreased the value of S 0 of the proteins at pH 7.0 and 9.0, and also at pH 5.0 for BSA. 64 Figure 10: Surface hydrophobicity (S0) of whey protein isolate (WPI) measured at p H 3.0-9.0 with ANS, CPA, and P R O D A N (top, middle and lower graphs, respectively). Open and shaded bars show mean values of duplicate determinations for unheated and heated samples, respectively. b ' c ' d bars with different letters represent significant (p<0.05) differences in S0 values within heated or unheated samples as a function of pH. y bars with different letters represent significant (p<0.05) differences in S 0 values between heated and unheated samples at a given pH. 65 700 600 -500 -o 400 • z < 300 -200 -100 -0 -ax ax bx bx cy bx ay cx E 450 -t 400 -350 -a? o 300 -< 250 -a o 200 -150 -100 -50 -o -ax ax PH bx bx cx cy dx dy 1200 •P1000 W 800 z Q 600 o °- 400 200 0 ax ax P H bx bx ^ cx cx i ii dx p H Figure 11: Surface hydrophobicity (S0) of (3-lactoglobuIin (BLG) measured at p H 3.0-9.0 with ANS, CPA, and P R O D A N (top, middle and lower graphs, respectively). Open and shaded bars show mean values of duplicate determinations for unheated and heated samples, respectively. * b ' c ' d bars with different letters represent significant (p<0.05) differences in SG values within heated or unheated samples as a function of pH. x ' y bars with different letters represent significant (p<0.05) differences in S 0 values between heated and unheated samples at a given pH. 66 3 5 7 9 PH 140 120 -Figure 12: Surface hydrophobicity (S0) of bovine serum albumin (BSA) measured at pH 3.0-9.0 with ANS, CPA, and P R O D A N (top, middle and lower graphs, respectively). Open and shaded bars show mean values of duplicate determinations for unheated and heated samples, respectively. a ' b ' c ' d bars with different letters represent significant (p<0.05) differences in SQ values within heated or unheated samples as a function of pH. x ' y bars with different letters represent significant (p<0.05) differences in SQ values between heated and unheated samples at a given pH. 67 The effect of heat on hydrophobicity of WPI at pH 7.0 and 9.0, measured using ANS probe in the present study, are in agreement with those of Mleko et al. (1997) and Monahan et al. (1995). The results of heating at pH 3.0 and 5.0 are, however, inconsistent. Mleko et al. (1997) reported that heating WPI at pH 3.0, 5.0, 7.0, and 9.0 increased the SQ values at each pH studied, while Monahan et al. (1995) reported decreases in surface hydrophobicity of WPI samples after heating at 80 °C at pH 3.0 and 5.0, and increases in surface hydrophobicity after heating at pH 7.0 and 9.0. No significant changes by heating WPI at either pH 3.0 or 5.0 were observed in the present study. These inconsistencies could be due to differences in the WPI products studied. As reported in a review on the processing and functional properties of whey protein concentrates and isolates (Morr and Ha, 1993), different processing conditions of whey from different sources can result in products with varying compositions, degrees of protein denaturation, aggregation, physicochemical and functional properties. Das and Kinsella (1989) measured the fluorescence intensity of protein bound ANS at different pH (2.8, 4.3, 5.0, 7.6 and 9.7). Their results indicated that the highest hydrophobicity was observed at pH 2.8 and the hydrophobicity decreased drastically with increase in pH. They did not find any correlation, however, between hydrophobicity and surface area of the emulsions stabilized by B L G . They speculated that lack of correlation may be due to the fact that hydrophobicity is usually determined at very low concentrations, and the hydrophobicity values may change at the higher concentrations used for studying functionality. Shimizu et al. (1985), who measured structural properties of B L G at different pH, also reported that the highest value of surface hydrophobicity measured by ANS was 68 found at pH 3.0. On the other hand, Phillips et al. (1994) cited a number of studies indicating a compact structure for B L G and increased thermostability under low pH conditions, in contrast to enhanced susceptibility to surface denaturation at pH 9.0, suggesting a more open flexible molecular structure at alkaline pH compared to pH 3.0. The results from the present study with the ANS probe (Figure 11) also indicate that unheated B L G had higher S 0 at pH 3.0 and 9.0, compared to pH 5.0 and 7.0. The S 0 values measured with CPA probe, however, showed the opposite trend, with hydrophobicity in the following order: pH 5.0 > pH 7.0 > pH 9.0 > pH 3.0. According to Laligant et al. (1991), B L G at pH 7.0 contains a high proportion of hydrophobic amino acid side chains which are turned, preferably, toward the inside of the molecule. These authors also stated that ANS binding can be influenced by the interactions between charged amino acids of B L G and the sulfonate groups of ANS. Ibrahim et al. (1993), using the CPA method, showed a decrease in S 0 value when B L G samples (pH 7.4) were heated at 80 °C in a dry state. The decrease in S 0 could be due to burial of effective hydrophobic regions as a result of the interaction of partially denatured molecules by dry-heat denaturation (Ibrahim et al., 1993). Kato et al. (1983) also showed the decrease of S 0 value of B L G and B S A solutions (pH 7.4) when heated from 20-80 °C at the rate of 1 °C/min, using CPA method. Those results are similar to the ones observed in the present study after heating WPI and B L G at pH 7.0 and 9.0 (Figures 10 and 11). Similarly, decreases in S 0 measured by both ANS and C P A probes were observed after heating of BSA solutions at pH 5.0, 7.0, and 9.0 (Figure 12). However, no change in S 0 measured by either probe was observed in B S A solutions at pH 3.0 after heating. 69 In summary, different observations were noted using ANS and CPA probes for the effect of pH and heating on surface hydrophobicity of these three protein samples. Similar discrepancies between results obtained by these two fluorescent probes have been noted previously (Hayakawa and Nakai, 1985). These discrepancies may be partly attributed to differences in probe chemistry arising from the aromatic versus aliphatic nature of the probes. Moreover, the difference could be due to interference of electrostatic interactions during the measurement of hydrophobicity (Li-Chan et al, 1985). CPA also has a poor solubility in acidic aqueous solutions, which may explain the lower CPA hydrophobicity values at pH 3.0, compared to pH 5.0 and 7.0. 4.2 Comparison of hydrophobicity measured using a neutral probe, PRODAN to the anionic probes, ANS and CPA Surface hydrophobicity values of the three proteins measured using P R O D A N are shown in the bottom panels in Figures 10-12. The pH significantly affected S 0 values for all three proteins (p<0.05), with the lowest S 0 values being found at pH 3.0. Heating had no effect on SG value of WPI or B L G at pH 3.0, 5.0, or 7.0, nor for B S A at pH 3.0. Heating WPI and B L G at pH 9.0 significantly decreased S 0 (p<0.05). Heating of B S A at pH 5.0, 7.0, and 9.0 caused significant decreases in S 0 (p<0.05). P R O D A N is an aromatic hydrophobic probe, similar to ANS but without an ionizable group. The general trends of protein hydrophobicity measured using P R O D A N were more similar to ANS than to CPA. For example, at pH 5.0 and 7.0, both ANS and P R O D A N showed higher S 0 for BSA than either WPI or B L G . However, major differences between the P R O D A N and ANS results were observed, especially at acidic 70 and alkaline pH. For example, using either the CPA or P R O D A N methods, BSA had higher hydrophobicity at pH 5.0, 7.0 and 9.0 than at pH 3.0. However, using ANS, hydrophobicity of BSA was higher at pH 3.0 and 5.0 than at pH 7.0 or 9.0. Furthermore, heating decreased hydrophobicity measured by P R O D A N for all three proteins at pH 9.0, similar to the results observed using CPA. In contrast, hydrophobicity measured by ANS increased after heating of B L G and WPI but decreased after heating of BSA. Generally, heating (80°C, 30 min) affected S 0 of WPI and B L G in a similar way, except in the case of B L G heated at pH 9.0 which showed a large decrease in S 0, when using P R O D A N and CPA methods, while a smaller decrease in S 0 was observed for WPI under similar conditions. At pH 8.0 and above, B L G can be regarded as unstable, forming aggregates of denatured protein (Bottomley et al., 1990). B L G is the major (-50%) protein constituent of whey (Marshall, 1982), thus similar changes in S 0 due to environmental conditions are expected. It has been shown that the thermal behavior of whey proteins is mainly governed by the properties of B L G (de Wit, 1981). However, differences between WPI and B L G can be attributed to different processing conditions, such as method of concentration and isolation which may cause protein denaturation (Kinsella, 1976). It has been shown that the least heat-sensitive pH range for whey proteins lies between pH 2.5 and 3.5, where proteins retain good solubility (de Wit, 1981). This is in agreement with the results of the present study using P R O D A N , ANS and CPA, where little or no significant change in surface hydrophobicity was observed after heating any of the three proteins at pH 3.0. 71 It is often expected that surface hydrophobicity should increase when the protein molecule unfolds during heating. However, unfolding may be followed by protein aggregation, through hydrophobic interactions or through SH/SS interchange reactions (Laligant et al 1991). These intermolecular interactions could lead to decreases in surface hydrophobicity. In other words, heating may have two different effects on the protein hydrophobicity, including unfolding of molecules, thus exposing hydrophobic sites, and heat-induced aggregation with decrease in the exposure of hydrophobic sites and then loss of solubility (Nakai and Li-Chan, 1989b). Of course food proteins also may differ in their response to heat treatment, as is the case for the proteins in the present study. At pH 3.0 and 5.0, hydrophobicity determined by P R O D A N was in the following order: B S A > WPI > B L G . At pH 7.0, the order was B S A > B L G > WPI. At pH 9.0, the hydrophobicity of B L G was greatly increased, and the order was B L G > B S A > WPI. For all three proteins, the lowest hydrophobicity values using the P R O D A N method were found at pH 3.0. The low hydrophobicity of B L G at pH 3.0 and marked increase in hydrophobicity at pH 9.0 observed using PRODAN are consistent with observations reported for this protein indicating the higher thermostability at acidic conditions and greater susceptibility to surface denaturation at alkaline conditions (Phillips et al., 1994). It has been speculated that the B L G molecule undergoes specific structural transitions characterized by a tighter conformation at acidic pH, compared to a more hydrophobic and flexible molecule at pH values above pH 7.5 (Phillips et al, 1994). In contrast, using the anionic probes ANS and CPA, hydrophobicity values were generally higher at acidic pH compared to neutral or alkaline pH. The anionic probes may 72 interact with positively charged sites on the proteins at low pH, thus overestimating hydrophobicity. This supports the advantage of using an neutral probe (PRODAN) for measurement of protein surface hydrophobicity especially under conditions of varying pH. The non-dissociable nature of the P R O D A N probe is an advantage in enabling investigation of the effects of changes in protein surface hydrophobicity over a broad range of pH. Nevertheless, the electron absoiption and emission transitions of P R O D A N are highly sensitive to the solvent acidity (Catalan et al, 1991). Use of an appropriate blank consisting of buffer and probe (without protein) is necessary to correct for the effect of the buffer on the probe and the subsequent estimation of the protein hydrophobicity values in different buffers. 5 Conclusions The neutral aromatic fluorescent probe P R O D A N may be used to determine surface hydrophobicity of proteins over a wide range of pH. Differences obtained in this study for the relative surface hydrophobicity values for three proteins, when measured by this probe compared to two anionic probes, ANS and CPA, confirm the importance of considering not only the aromatic or aliphatic nature, but also the presence or absence of a permanent charge when using fluorescent probes for measurement of protein hydrophobicity. 73 CHAPTER IV Application of PRODAN Fluorescent Probe to Measure Surface Hydrophobicity of Whey Proteins Interacting with K-Carrageenan 1 Abstract The fluorescent probe, 6-propionyl-2-(N-N-dimethylamino)-naphthalene or PRODAN, was applied to measure surface hydrophobicity (S0) of proteins interacting with polysaccharides. Whey protein isolate (WPI), P-lactoglobulin (BLG), and bovine serum albumin (BSA) were used as model systems in combination with K-carrageenan (KCG) at KCG:protein ratios of 1.0:1.2 (high ratio), 1.0:6.0 (medium ratio), and 1.0:62.5 (low ratio). Heating (80°C for 30 minutes), pH (3.0, 5.0, 7.0, and 9.0) and addition of K C G at low, medium or high ratio had significant effects (p<0.05) on S 0 value of each protein. Interactions between pH, heating and K C G addition also had significant effects on S 0. In general, the proteins had higher S 0 at pH 9.0 and were more sensitive to the effects of heating and K C G addition at pH 9.0 than at other pH. For WPI, K C G increased S 0 only when added at high ratio at pH 9.0 or at high or medium ratio at pH 5.0. In contrast, decreases in S 0 of B L G and BSA at pH 9.0 were more pronounced at the lower K C G ratio. Heating KCG:protein mixtures generally increased S 0 at pH 3.0 for high ratio mixtures, and decreased S 0 at higher pH, especially pH 9.0. The S 0 values of heated mixtures differed from proteins heated alone under some conditions; however, the nature of these changes depended on the protein, pH and KCG:protein ratio. This study demonstrates the usefulness of P R O D A N to monitor changes in surface hydrophobicity 74 resulting from interactions of proteins with K-carrageenan under varying pH and heat treatments. 75 2 Introduction Polysaccharides and proteins are two major macromolecules in most food products. These biopolymers contribute to the structure, stability, and textural properties of foods through their gelling and aggregation behavior (Galazka et al., 1999a). The control or manipulation of the protein-polysaccharide interaction is a key factor in the development of novel foods (Tolstoguzov, 1997). The overall structure-function relationship of a food containing protein and polysaccharide depends on both the individual biopolymers and also on the strength and interaction of the mixed biopolymers (Galazka et al., 1999b). The study of the interaction of milk proteins and polysaccharides may not only enhance the application of polysaccharides in dairy products, but also may improve their functional properties. Polysaccharides have been widely used as stabilizing, thickening and gelling agents in the food, agricultural, cosmetics, and pharmaceutical industries (Guiseley et al., 1980; Hidalgo and Hansen, 1969; Huffman and Shah, 1995; Moirano, 1977; Piculell, 1995; Thomas, 1992; Tziboula and Home, 1997). In dairy products, polysaccharides may be used at levels of 0.005-3.000% to confer these functional properties (Thomas, 1992). Some polysaccharides provide stability by modifying the rheological properties of the continuous aqueous phase while others, such as K-carrageenans, interact directly with the milk proteins (Drohan et al., 1997). Physical stability, an important factor in the quality and shelf life of various dairy products including puddings, ice cream, low fat frozen desserts, chocolate milk, egg nog, infant formula, and yogurt, mainly depends on the presence of stabilizers such as polysaccharides (Hidalgo and Hansen, 1969; Moirano, 1977). For example, the majority 76 of frozen desserts are stabilized to control ice crystal formation, and to prevent whey separation during storage prior to freezing and melt-down (Moirano, 1977). In chocolate milk, carrageenan keeps the cocoa in suspension, while in infant formula, carrageenan is required for fat and protein stabilization. Polysaccharides blends are also frequently used as fat replacers, especially in the production of low fat spreads, soft cheeses and milk desserts (Chronakis et al., 1996). Due to their special characteristics, which depend on the aqueous environmental conditions such as pH, temperature, and ionic strength (Dickinson, 1998), polysaccharides have been used to precipitate proteins (Gilberg and Tornel, 1976; Gurov et al, 1981). For example, the anionic polysaccharide K-carrageenan was used for precipitation of lysozyme, resulting in its separation from egg white (Yang et al, 1998). Under other conditions, polysaccharides have been used to prevent protein precipitation (Chakraborty and Randolph, 1972; Glahn, 1982; Hansen, 1968), for example, in fruit-flavored milk drinks (Imeson et al., 1977). The significance of whey proteins as a functional ingredient is rapidly growing due to their very good functional properties. However, improvement of their functional characteristics is essential, if they are to be used to meet the needs of food industry (Pittia et al, 1996). Extrinsic factors such as pH, temperature, and ionic environment as well as intrinsic protein properties such as disulfide bonds may affect molecular flexibility or stability (Harwalker and Ma, 1989; Koning and Visser, 1992) and therefore protein hydrophobicity. Adding polysaccharides to proteins may also alter the functionality of both biopolymers. 77 Galazka et al., (1999a) studied the effect of high pressure on P-lactoglobulin (BLG) or bovine serum albumin (BSA) complexes with dextran sulfate (DS, an anionic polysaccharide), by measuring surface hydrophobicity (S0) of the proteins using the ANS probe method, thermal stability using differential scanning calorimetry (DSC), and size exclusion chromatography. They found that adding DS to B L G (1.0:1.0 ratio, pH 7.0) did not affect the value of protein surface hydrophobicity, while adding DS to BSA (1.0:2.0 ratio, pH 7.0) significantly decreased the S 0 value. Galazka, et al. (1999b), who investigated the effect of high pressure treatment on BSA:sulfated polysaccharide complexes, showed that adding K-carrageenan or t-carrageenan to B S A (1.0:2.5, and 1.0:5.0 ratio, respectively, at pH 7.0), decreased surface hydrophobicity of the protein. They assumed that the decrease in S 0 was mainly due to electrostatic repulsion between the two negatively charged molecules (KCG and BSA) and blocking of the ANS binding sites by the complexation of polysaccharide:BSA. However, this decrease in S 0 may also be due to repulsion between the negatively charged probe, ANS, and macromolecules. Due to the importance of incorporation of polysaccharides into foods, especially foods containing proteins, studying the polysaccharide-protein interaction has been an area of interest among food scientists. This can be especially so when foods containing mixtures of these substances are heat-treated (Kelly et al., 1994). Hydrophobic, steric and electric parameters are important variables that affect the structure of proteins and their interactions with other molecules (Nakai, 1983). Fluorescent probe methods are simple techniques to evaluate protein surface hydrophobicity (Slavic, 1994a; Royer, 1995). However, anionic probes, such as ANS, which have been used widely, may not give accurate information in this regard due to ionic as well as hydrophobic interactions 78 between probe and protein. Using a neutral probe may circumvent this problem (Alizadeh-Pasdar and Li-Chan, 2000). The measurement of surface hydrophobicity as an approach for studying polysaccharide:protein interactions is not common in the area of food science. However, the importance of hydrophobicity is illustrated in a study on the interaction between drugs and polysaccharides, in which hydrophobicity of the drug and charge density and structure of the polysaccharide were demonstrated to influence the extent of their interactions (Persson et al., 1996). The application of a simple and reliable fluorescent probe method to monitor changes in protein surface hydrophobicity that may result from the interaction of polysaccharides with proteins under various conditions, can provide information regarding the impact on protein surface properties of the parameters that control these interactions. The purpose of this study was to use a neutral fluorescent probe, 6-propionyl-2-(N,N-dimethylamino)naphthalene or PRODAN, to measure surface hydrophobicity of whey protein isolate (WPI), P-lactoglobulin (BLG), and bovine serum albumin (BSA) as affected by their interactions with K-carrageenan (KCG, at KCG:protein ratios of 1:1.2, 1.0:6.0, and 1:62.5) at various pH (3.0, 5.0, 7.0, and 9.0) and with or without heat treatment (80°C for 30 min). In other words, P R O D A N was used as a probe to study the changes in surface hydrophobicity that may result from interaction of whey proteins and polysaccharides under varying conditions of pH and heat treatment. 79 3 Experimental Procedures 3.1 Materials The same materials as Chapter m, Section 3.1 were used, with the addition of K-carrageenan (C-1263), which was purchased from Sigma (St. Louis, MO). 3.2 Preparation of samples for measurement of S 0 The same procedure as Chapter HI, section 3.2 was used for preparation of the samples, with the addition of K C G . K C G stock solutions were prepared by dispersing the powder in distilled deionized water and continuously stirring for 20 minutes at 70°C. The concentration of the stock K C G solutions were 1.250%, 0.025%, and 0.0024% (w/v). K C G concentration was determined according to the methylene blue method of Soedjak (1994) (refer to Appendix 13 for more details). To prepare the intermediate solutions of mixtures, 1 mL stock protein solutions plus 1 mL stock K C G solutions were diluted, with the appropriate pH buffers. The concentration of protein in the intermediate solution was 0.030%, and that of K C G solutions were 0.025%, 0.005%, and 0.00048% K C G , respectively (1.0:1.2, high ratio; 1.0:6.0, medium ratio; and 1.0:62.5, low ratio of K C G to protein, respectively). The intermediate solutions were then either diluted to final concentrations as is, or after heat treatment for 30 minutes at 80 °C. Heat treatment of 20 mL protein solutions or KCG:protein mixtures in 50 mL volumetric flasks, which were covered with parafilm to avoid evaporation, was done in a water bath (Blue M Magni Whirl). After heating, the flasks were cooled under running 80 water for 15 minutes. Preparation of diluted samples (either heated or unheated, with or without KCG) were performed 24 hours after preparation of stock solutions. Except for the KCG:protein ratio of 1.0:6.0 and 1.0:62.5 at pH 3.0, and the heated mixture of K C G : B S A (1:6 ratio) at pH 5.0, which were coagulated, the other mixtures did not contain any coagulated particles. The flow chart for sample preparation is shown in Figure 13. Stock: 1.5% WPI, (3-lg, and BSA in DD water Measure Absorbance @ 280 nm Intermediate: 0.03% in buffers (pH 3.0, 5.0, 7.0, 9.0) With K-carrageenan (3 ratios) Without K-carrageenan With heating 30 min at 80°C Without heating With heating 30 min at 80°C Without heating Diluting to 0.002-0.01% for S 0 measurement Figure 13: Flow chart of sample preparation for the K-carrageenan:protein interaction experiment 81 3.3 Hydrophobicity measurement Surface hydrophobicity (S0) was determined essentially according to the method of Alizadeh-Pasdar and Li-Chan (2000), which was explained in Chapter in, Section 3.3. The intermediate samples with 0.030% protein in the absence or presence of K C G were diluted with the appropriate buffers at pH 3.0, 5.0, 7.0, and 9.0 to typical final protein concentration ranges of 0.002-0.010% (5 concentrations). These dilutions and fluorescence measurements were done the same day that the stock solution was diluted. Hydrophobicity was determined on dilutions from duplicate protein stock solutions and the mean values are shown in the Figures. Addition of K C G to buffers containing P R O D A N (blanks) did not change the relative fluorescent intensity (RFI); in other words, K C G alone did not have any fluorescence nor affect that of PRODAN. 3.4 Statistical analysis The analysis of the data was performed as described in Chapter in, Section 3.4. 4 Results Table 11 shows the results of A N O V A of the three factors (pH, heating, and presence of K-carrageenan) and their interactions on the value of surface hydrophobicity (S0) of whey protein isolate (WPI), P-lactoglobulin (BLG), and bovine serum albumin (BSA). The results indicate that protein surface hydrophobicity (S0) was significantly affected by pH, heating, and K C G concentration. A l l three factors and their interaction terms were significant at p<0.001, with the exception of the effect of heating on WPI which was significant at p=0.033. 82 Table 11: Results of the ANOVA (analysis of variance) of the effects of pH, heating, presence of K-carrageenan (KCG) and their interactions on the surface hydrophobicity values of whey protein isolate (WPI), P-lactoglobulin (BLG), and bovine serum albumin (BSA). p-values Source Df WPI B L G BSA pH 3 0.000 0.000 0.000 Heating 1 0.033 0.000 0.000 KCG 3 0.000 0.000 0.000 pH * heating 3 0.000 0.000 0.000 pH * KCG 9 0.000 0.000 0.000 Heating * KCG 3 0.000 0.000 0.000 pH * heating * KCG 9 0.000 0.000 0.000 Error 32 Total 63 The detailed effects of these treatments on the surface hydrophobicity of WPI, B L G , and B S A are shown in Figures 14-17, and described below. Within each series of 4 bars (representing S 0 of each sample at 4 pH), those not sharing common lower case letters a, b, c, or d are significantly (p<0.05) different in S 0 as a function of pH. Between samples at a given pH, those not sharing common upper case letters v, w, x, y, z have significantly (p<0.05) different S 0. 83 250 200 f " 150 wo 1 0 0 50 0 cy by axtf WPI WPI HWPI 800 600 °S 400 o w 200 BLG cz by bz az BSA B L G H BLG bx by B S A H BSA pH 3.0 [] pH 5.01] pH 7.0 | pH 9.0 Figure 14: Effect of heating (H) at 80°C for 30 min at pH 3.0-9.0 on the surface hydrophobicity (S„)* of whey protein isolate (WPI), P-lactoglobulin (BLG), and bovine serum albumin (BSA). a, b. c, d: bars with different letters represent significant differences (p<0.05) in S0 values within samples as a function of pH. w, x, y, z: bars with different letters represent significant differences (/?<0.05) in S c values between samples at a given pH. * Values are means of duplicate determinations. 8 4 BLG KCG: BLG 1.0:1.2 ratio KCG:BLG 1.0:6.0 ratio KCG: BLG 1.0:62.5 ratio BSA KCG:BSA 1.0:1.2 ratio KCG: BSA 1.0:6.0 ratio KCG:BSA 1.0:62.5 ratio pH 3.01 pH 5.01 pH 7.01 pH 9.0 Figure 15: Effect of pH and presence of K-carrageenan (KCG) at KCG:protein ratios of 1.0:1.2, 1.0:6.0, or 1.0:62.5 on the the surface hydrophobicity (S0)* of whey protein isolate (WPI), P-lactoglobulin (BLG), and bovine serum albumin (BSA). a, b. c, d: bars with different letters represent significant differences (p<0.05) in S0 values within samples as a function of pH. w, x, y, z: bars with different letters represent significant differences (p<0.05) in S 0 values between samples at a given pH. * Values are means of duplicate determinations. 85 350 -r 300 H WPI H KCG:WPI H KCG:WPI H KCG:WPI 1.0:1.2 ratio 1.0:6.0 ratio 1.0:62.5 ratio H BLG cy cw bz ay H KCG:BLG 1.0:1.2 ratio H KCG:BLG 1.0:6.0 ratio cxy cw J 2 3 H KCG:BLG 1.0:62.5 ratio pH 3.0 []pH 5.0 [ ] PH7.0[ pH 9.0 Figure 16: Surface hydrophobicity (S0)* of proteins heated (H) at 80°C for 30 min at pH 3.0-9.0, in the absence or presence of K-carrageenan (KCG) at KCG:protein ratios of 1.0:1.2,1.0:6.0, or 1.0:62.5. a, b. c, d: bars with different letters represent significant differences (p<0.05) in S 0 values within samples as a function of pH. v, w, x, y, z: bars with different letters represent significant differences (p<0.05) in S0 values between samples at a given pH. * Values are means of duplicate determinations on heated whey protein isolate (WPI), P-lactoglobulin (BLG), and bovine serum albumin (BSA) and the heated mixtures of KCG and proteins. 86 KCG:WPI H KCG:WPI 1.0:1.2 ratio 1.0:1.2 Ratio 1.0:6.0 Ratio 1.0:6.0 Ratio 1.0:62.5 Ratio 1.0:62.5 KCG:BLG H KCG:BLG o 1.0:1.2 Ratio 1.0:1.2 Ratio o ° 5 KCG:BLG H KCG:BLG KCG:BLG H KCG:BLG 1.0:6.0 Ratio 1.0:6.0 Ratio 1.0:62.5 Ratio 1.0:62.5 Ratio KCG:BSA, 1.0:1.2 Ratio H KCG:BSA, 1.0:1.2 Ratio KCG:BSA, 1.0:6.0 Ratio < < BBS . bx | H KCG:BSA, 1.0:6.0 Ratio KCG:BSA, 1.0:62.5 Ratio H KCG: 1.0:62.5 I pH 3.0 [] pH 5.0 g pH 7.0 | pH 9.0 Figure 17: Effect of heating (H) at 80°C for 30 min at pH 3.0-9.0, on the surface hydrophobicity (S0)* of proteins in the presence of K-carrageenan (KCG) at KCG:protein ratios of 1.0:1.2,1.0:6.0, or 1.0:62.5. a, b. c, d: bars with different letters represent significant differences (/?<0.05) in S0 values within samples as a function of pH. v, w, x, y, z: bars with different letters represent significant differences (p<0.05) in S0 values between samples at a given pH. * Values are means of duplicate determinations on mixtures and heated mixtures of KCG with whey protein isolate (WPI), P-lactoglobulin (BLG), and bovine serum albumin (BSA). 87 4.1 Effects of pH and heating Figure 14 shows the effect of pH and heating on S 0 of whey proteins. The pH significantly affected S 0 values of all three proteins (p<0.001), with the lowest S 0 being seen at pH 3.0 for each protein. For WPI and B L G , heating only significantly affected S 0 at pH 9.0, causing decreases. Heating had no effect on the S 0 value of WPI or B L G at pH 3.0, 5.0, and 7.0. However, significant decrease of S 0 was observed upon heating of BSA at all pH values studied. Differences were observed between the three proteins in their S 0 values as well as their variation as a function of pH and heating. At pH 3.0, 5.0 and 7.0, unheated BSA had higher S 0 than either WPI or B L G , while at pH 9.0, B L G had the highest S 0 . The range of S 0 values measured as a function of pH from 3.0 to 9.0 varied only ~4-fold for WPI and BSA, whereas B L G showed > 40-fold change. Similarly, although a significant decrease in S 0 was observed at pH 9.0 after heating for all three proteins, this change was rather small for WPI compared to the marked decreases observed for the other two proteins. Results of A N O V A also demonstrated a higher level of significance of the effect of heating for B L G and B S A (p<0.001) than for WPI (p=0.033). 4.2 Effects of KCG on S 0 of proteins at various pH and KCG:protein ratios Significant effects on S 0 were observed by the addition of K C G to unheated proteins (Figure 15), and these effects depended on the protein, pH, as well as the ratio of 88 K C G to protein (KCG:protein ratio by weight of 1.0:1.2, 1.0:6.0, or 1.0:62.5, designated as high, medium or low ratio, respectively). Significant increases in S 0 were observed in the KCG:WPI mixtures obtained by addition of a high ratio of K C G at pH 5.0 and 9.0, or a medium ratio of K C G at pH 5.0, compared to S 0 of WPI alone. Addition of K C G at the low ratio did not change S 0 of WPI under any of the pH conditions studied. In the case of B L G , significant decrease was seen when K C G was added at pH 9.0 and high ratio, at pH 7.0 with medium ratio and at pH 5.0, 7.0, and 9.0 with low ratio. The addition of a high ratio of K C G to B S A at pH 5.0 increased S 0 . However, significantly lower S 0 values were observed by addition of K C G to B S A at all other ratios and pH conditions investigated in this study, with the exception of the high ratio mixture at pH 7.0, which was not different from B S A alone. In summary, the effects of K C G addition were dependent on the specific protein, pH, and ratio of K C G added. For WPI, K C G increased SO J but only when added at a high ratio at pH 9.0, or at a high or medium ratio at pH 5.0. In contrast, the decrease in S 0 of B L G in the presence of K C G was most pronounced at the lowest ratio of K C G : B L G , and no effects of K C G were observed upon addition of a high ratio of K C G to B L G at pH 3.0, 5.0, or 7.0. The addition of K C G to B S A also generally caused a decrease in S 0, with the effects again being more pronounced at the low and medium ratios of K C G addition. 4 . 3 Comparison of S 0 of proteins heated at various pH and KCG:protein ratios The S 0 values of proteins heated in the absence or presence of K C G over the pH range from 3.0-9.0 are shown in Figure 16. 89 For WPI, heating the high ratio mixture of KCG:WPI at pH 3.0, 5.0 and 9.0 resulted in higher S 0 values than WPI heated under these pH conditions in the absence of K C G . For the other two ratios, however, the only significant change was an increased S 0 in the heated low ratio KCG:WPI mixture at pH 9.0. In the case of B L G , heating in the presence of a high ratio of K C G : B L G at pH 3.0 resulted in significantly higher S 0 than for B L G heated alone. In contrast, S 0 decreased when heating of B L G was conducted in the presence of a low ratio of K C G : B L G at pH 5.0 and 7.0, or medium ratio at pH 7.0. Heating low or high ratio K C G : B S A mixtures at pH 5.0 resulted in higher S 0 than heating B S A alone. Heating medium ratio K C G : B S A mixtures at 7.0 and 9.0, however, resulted in lower S 0 than for BSA heated alone. Lower S 0 values were also observed for the heated medium or low ratio K C G : B S A mixtures at pH 3.0 than for B S A heated alone. In summary, the presence of K C G during heating of the proteins resulted in S 0 values that were in some cases different from the S 0 of the proteins heated in the absence of K C G . Higher S 0 values resulted by heating WPI in the presence of a high ratio of K C G at pH 3.0, 5.0, or 9.0, or with a low ratio of K C G at pH 9.0. Higher S 0 values also resulted by heating B L G with a high ratio of K C G at pH 3.0, or by heating B S A with a high or low ratio of K C G at pH 5.0. However, lower S 0 values were observed by heating B L G at pH 7.0 with the low or medium ratio of K C G , or at pH 5.0 with the low ratio. Lower S 0 values were also observed for B S A when heated in the presence of a medium ratio of K C G at pH 3.0, 7.0 or 9.0, or with a low ratio at pH 3.0. 90 4 . 4 Effect of heating on proteins containing KCG Figure 17 shows the effect of heating on the S 0 values of KCG:protein mixtures. Heating the high ratio mixture of KCG:WPI increased S 0 at pH 3.0 while heating the medium ratio mixture decreased S 0 at pH 5.0 and 9.0. On the other hand, heating the low ratio mixture of KCG:WPI had no effect on the value of hydrophobicity. For B L G , a significant increase at pH 3.0 and a significant decrease at pH 9.0 were observed in S 0 values after heating of the high ratio mixture of K C G : B L G . In the case of medium and low ratio mixtures, the only significant changes upon heating were decreases in S 0 at pH 9.0. Heating the high ratio mixture of K C G : B S A increased the S 0 at pH 3.0, while significant decrease was observed at the other pH. Heating the medium ratio mixture of K C G : B S A also decreased SG at pH 7.0 and 9.0. Heating K C G : B S A mixture with low ratio increased the S 0 value at pH 5.0 and 7.0, but decreased S 0 at pH 9.0. In summary, heating of the high ratio KCG:protein mixtures at pH 3.0 resulted in increased S 0 values. With a few exceptions, heating decreased S 0 values of KCG:protein mixtures at higher pH, especially at pH 9.0. 5 Discussion Protein-polysaccharide interactions are electrostatic in nature. Temperature, pH, and ionic strength are important experimental variables controlling the strength of complexation (Dickinson and Pawlowsky, 1998). Carrageenan products are more stable at neutral and alkaline pH, even at elevated temperatures. By lowering pH, hydrolysis of glycosidic linkages occurs. Hydrolysis is greatly accelerated by heat when pH is low. 91 Hydrolysis at pH 3.0-4.0 is sufficient enough to limit the application of K C G , when the primary function is thickening (Moirano, 1977). In this study, the surface hydrophobicity S 0 of whey protein isolate, (3-lactoglobulin and bovine serum albumin was noted to be significantly affected by pH, heating and addition of K-carrageenan, as well as the interactions between these factors. The lowest S 0 values were observed at pH 3.0 for all three proteins studied. Highest S 0 values were observed at pH 9.0, and in general, greater changes in S 0 were observed as a function of heating and K C G addition at this alkaline pH than at neutral or acidic pH. These results are in agreement with a number of studies cited by Phillips et al. (1994), which reported a compact structure and increased thermostability of (3-lactoglobulin under low pH conditions, and enhanced susceptibility to surface denaturation and a more open flexible structure at pH 9.0. In contrast, using an anionic fluorescent probe (ANS), the highest S 0 for WPI as a function of pH was observed at pH 3.0 (Mleko et al., 1997). The discrepancies between that study and our present results could be attributed to the use of different fluorescent probes for measurement of surface hydrophobicity (Alizadeh-Pasdar and Li-Chan, 2000). Electrostatic interactions between the negatively charged anionic fluorescent probe and the positively charged protein molecules may occur at pH 3.0, resulting in overestimation of the hydrophobicity measured by the fluorescent probe method at acidic pH. It has been reported that the least heat-sensitive pH range for whey proteins is between pH 2.0 and 3.5, where proteins have good solubility (de Wit, 1981). Based on the results from the present study, no significant change in S 0 value was observed after heating either WPI or B L G at pH 3.0, 5.0 or 7.0. In contrast, all three proteins studied 92 showed significant decreases in S 0 value after heating at pH 9.0, and decreases were also observed after heating of BSA at pH 3.0, 5.0 and 7.0. It is often expected that surface hydrophobicity should increase when the molecule unfolds during heating. However, Laligant et al. (1991) suggested that unfolding may cause protein aggregation, through hydrophobic interactions or through SH/SS interchange reactions, leading to decreases in surface hydrophobicity. In other words, hydrophobicity could be affected by heating in two opposing ways. If heating only causes unfolding of molecules, hydrophobic sites will be exposed and hydrophobicity will be increased. However, if heating causes aggregation of the molecules, solubility will decrease and this will decrease the exposure of the hydrophobic sites (Nakai and Li-Chan 1989). Dickinson and Pawlowsky (1998) reported that the surface tension of K C G : B S A complex (at 1:4 ratio) was higher at pH 7.0 than at pH 5.0. They also noted that the strength of electrostatic interaction between K C G and B S A increased as pH was decreased from neutral towards the protein's isoelectric point. According to Kato and Nakai (1980), the higher the protein hydrophobicity, the lower the surface tension. Our results show that the hydrophobicity of K C G : B S A mixture for all three ratios was higher at pH 5.0 than that at pH 7.0 (Figure 15). Based on the observations of Kato and Nakai (1980) showing an inverse relationship between hydrophobicity and surface or interfacial tension, the hydrophobicity results from the present study are in agreement with the surface tension results reported by Dickinson and Pawlowsky (1998). It has been shown that K C G may precipitate milk and other proteins below their isoelectric point (Walter, 1998a). Solubility of polysaccharide:protein mixtures depends 93 on several factors including pH (Dickinson, 1998). Since carrageenan is negatively charged at low pH, it is possible that it may interact with positive sites in the proteins (Dalgleish and Hollocou, 1996). The results of the present study are in agreement with that of Goncalves et al. (1986), who studied the precipitation of plasma proteins by anionic polysaccharides (6:1 ratio). They showed that the coprecipitates, which were obtained at pH 3.0-4.0, correspond to the formation of complexes stabilized by electrostatic interactions between the positively charged groups of the proteins and the ionic groups of the polysaccharides. The results of the present study indicate significant effects of the KCG:protein ratios on protein surface hydrophobicity values. The three KCG:protein ratios studied, namely 1.0:1.2, 1.0:6.0, and 1.0:62.5, fall within the range of typical applications using 0.005-3.00% polysaccharides in dairy products (Thomas, 1992), assuming approximately 3.5% protein in dairy products such as milk. Lin (1977), who studied the stabilization of casein by carrageenan at pH 6.7, showed that casein stability increased with increasing carrageenan-casein ratio; in other words, the higher the amount of carrageenan (compared to constant casein), the greater the stability. Biopolymers, such as K C G , may show a protective action as a result of the adsorption of the macromolecules on their surfaces. This protective action of the biopolymer increases with increasing concentration (Napper, 1983). Dickinson (1998) classified binary systems of spherical particles and adsorbing polymer molecules into three structural states based on the polymer/particle ratio -bridging flocculation at low polymer concentration, steric stabilization of particles by intermediate concentration, and immobilization of polymer-covered particles in a gel network at high polymer concentration. 94 In our study, the formation of soluble complexes of KCG:proteins at high ratio (1.0:1.2) and pH 3.0 is probably due to steric stabilization. Bridging flocculation, however, likely occurred at pH 3.0, for both medium (1.0:6.0) and low ratios (1.0:62.5) of KCG:proteins and at pH 5.0 for the 1.0:6.0 ratio mixture of K C G : B S A ; in these instances, some coagulation was observed. Increases in S 0 of proteins in the presence of K C G that were observed in this study (e.g. upon addition of a high ratio of K C G to WPI at pH 9.0 or to B S A at pH 5.0) could be attributed to increased exposure of hydrophobic sites resulting from the unfolding of the protein molecule in the presence of K C G . Decreases in the values of S 0 by adding K C G to proteins (e.g. upon addition of a low ratio of K C G to B L G at pH 5.0, 7.0 or 9.0, or upon addition of low or medium ratios of K C G to B S A at pH 3.0, 5.0, 7.0 or 9.0) could be due to electrostatic repulsion between the negatively charged K C G molecules and/or possible blocking of the P R O D A N binding site by KCG:protein complexation, since P R O D A N does not bind to K C G alone. Effects of K C G on S 0 of heated mixtures compared to the heated proteins alone could be due to the effects of the negatively charged K C G molecules on both intramolecular folding as well as intermolecular aggregation of the complexes. It is interesting to note that there was little change in the S 0 values upon addition of K C G to either WPI or B L G at pH 3.0. This observation indicates that although electrostatic interactions between the negatively charged polysaccharide and positively charged protein molecules may have occurred at this acidic pH, these complexes did not exhibit any changes in surface hydrophobicity as measured by the fluorescent probe. 95 Although complex formation between milk proteins and charged polysaccharides arise primarily from electrostatic interactions, strong soluble complexes can form even at neutral or alkaline pH when both biopolymers carry net negative charge (Dickinson, 1998). Evidence for interactions between K C G and proteins was indicated in this study by changes in S 0 of proteins in the presence of K C G even at pH 9.0 for particular KCG:protein ratios. For example, SG was significantly higher for the high ratio mixture of KCG:WPI than WPI alone at pH 9.0, while SG values were significantly lower for K C G : B L G and K C G : B S A mixtures than B L G and BSA alone at pH 9.0. In some cases, these interactions also resulted in significant differences in SQ of the heated mixtures from the proteins heated alone. 6 Conclusion The present study of protein surface hydrophobicity using the fluorescent probe P R O D A N showed that whey proteins and K-carrageenan interacted over a broad range of pH, with or without heat treatment. Generally, the hydrophobicity of proteins alone, heated or mixed with K C G , was the lowest at pH 3.0 compared to the other pHs studied (5.0, 7.0, 9.0). Coagulates were formed upon addition of K C G to proteins at low or medium ratio at pH 3.0. The S 0 of the KCG:protein mixtures at pH 3.0 were not different from the S 0 of proteins alone for WPI and B L G , but were lower in the case of BSA. S 0 values of proteins were found to be generally higher at pH 9.0 compared to other pH, and were more sensitive to the effects of heating and K C G at pH 9.0 than at pH 7.0. The S 0 of KCG:protein mixtures was also dependent on the ratio of these constituents. Generally, for the unheated mixtures, the higher the ratio of KCG:protein, the higher the S 0 value. 96 Heating the mixtures decreased S 0 at pH 3.0 but increased S 0 at higher pH, especially at pH 9.0. This study confirmed that P R O D A N may be useful as a neutral fluorescent probe for the determination of surface hydrophobicity of proteins in the presence or absence of K-carrageenan and as a function of heating at different pH. K-Carrageenan was used as an example of anionic polysaccharide, and whey proteins as examples of globular proteins. The application of P R O D A N to study the interactions of other (charged or nonionic) polysaccharides and proteins should be explored in future studies. 97 CHAPTER V Raman Spectroscopic Study of the Effects of pH, Heating, and Presence of K-Carrageenan on Protein Structure 1 Abstract Raman spectroscopy was used to elucidate structural changes of whey protein isolate (WPI), P-lactoglobulin (BLG), and bovine serum albumin (BSA), at 15% concentration, as a function of pH (3.0, 5.0, 7.0, and 9.0), heating (80°C for 30 min), and presence of 0.24% K-carrageenan. Examination of the spectra showed bands characteristic of proteins at 508 cm"1 (cys S-S), 761 cm"1 (tryptophan), 860/820 cm"1 (tyrosine doublet), 938 cm"1 (a-helix), 1453 cm"1 (CH 2 bending), 1730 cm"1 (COOH), 2880 cm"1, 2930 cm"1, and 3060 cm"1 (CH stretching), and 3220 cm"1 (OH). At a concentration of 0.24%, the spectrum of K C G alone at these regions did not show any bands. Heating resulted in decreased intensity of the C H and O H stretching bands especially at pH 9.0, suggesting structural changes involving hydrophobic interactions and the protein-solvent (water) interactions, respectively. These changes were less marked for proteins heated in the presence of K C G . Decreases in SS and Trp band intensities, lower helical content and higher P-sheet content were also generally observed after heating (particularly at pH 9.0) or in the presence of K C G without heating (particularly at pH 5.0). At pH 7.0 and 9.0, however, presence of K C G increased the helical content of BSA. The decreased helical content and increased P-sheet content 98 obtained by heating B L G at pH 7.0 and pH 9.0 were not observed after heating K C G : B L G mixtures at these pH. On the other hand, heating KCG:WPI mixtures at pH 5.0 and 9.0 resulted in large increases in SS and Tip band intensities as well as helical content, and decrease in the ratio of the tyrosine doublet, indicating a more buried or hydrophobic environment of the aromatic residues. 99 2 Introduction Lysozyme was the first protein that was studied using Raman spectroscopy (Lord and Yu, 1970). Changes in chemical structure and environment around an atom can be monitored using Raman spectroscopy, a vibrational spectroscopic technique. More specifically, using this technique, one can obtain some information regarding the secondary structure of proteins based on amide I, amide HI, and skeletal stretching modes of the polypeptide backbone. The amide I band (around 1650 cm"1) and amide III band (near 1200-1300 cm _ 1) are two important vibrational modes that have been used for both quantitative and qualitative evaluation of the secondary structure of the polypeptide backbone (Li-Chan et al., 1994). Vibrational transitions assigned to various amino acid side chains such as SS and SH groups of cystine and cysteine, the aromatic rings of tryptophan, tyrosine and phenylalanine, the C-H groups of aliphatic amino acids, the COO" and C O O H groups of aspartic and glutamic acids, and imidazole ring of histidine can also be studied using Raman spectroscopy (Li-Chan, 1996a; Li-Chan et al., 1994). The great potential of Raman spectroscopy in analysis of liquids, gels, non-aqueous liquids, solids such as fibers, films, powders, and crystals makes it a useful analytical technique in the investigation of the structure of food systems (Painter, 1984). Raman spectroscopy can also be used to monitor structural changes in food proteins caused by processing conditions (Nonaka et al., 1993). Although both infrared (TR) and Raman spectroscopy can be used for the study of the secondary structure of proteins, the IR spectrum provides mainly information on |3-sheet and little can be determined about a-helix and unordered structure (Clark et al., 1981). In Raman spectroscopy, however, the nature of secondary structure changes can be fully obtained (Clark et al., 1981). 100 Another advantage of Raman spectroscopy over IR is the fact that it can be used to study aqueous solutions, without interference from water; water exhibits weak Raman scattering but strong infrared absorption (Tu, 1986). Without the interference of water, therefore, side chain and peptide skeletal frequencies can be monitored (Clark et al., 1981). One important advantage of Raman spectroscopy over other available methods (such as ultraviolet, fluorescence, near U V circular dichroism, resonance Raman spectroscopy, etc.), for studying the changes in protein structure, is its capability to analyze proteins in concentrated aqueous solutions, even solids, precipitates, and gels (Howell et al., 1999) without destruction of samples (Nonaka et al., 1993). The use of high protein concentrations is especially critical for investigations involving structural changes of protein due to formation of coagulum or gels as a consequence of processes such as heating and drying. Raman spectroscopy, therefore, can be used to study the changes in both intramolecular and intermolecular interactions (Li-Chan et al., 1994). Appearance of artificial signals associated with turbid samples, is a drawback of using circular dichroism (CD), beside the requirement of low concentration (Matsuura and Manning, 1994). X-ray crystallography can provide more precise parameters, at atomic level, than the other mentioned methods, however, it is limited to the analysis of samples in crystal form and is quite time consuming (Byler and Susi, 1988). Raman spectroscopy has been applied to study structure of various proteins. For example, Frushour and Koenig (1975) investigated the crystalline, solution, and alkaline denatured states of B L G and reported a shift in amide in band from 1242 to 1246 cm"1, when pH was increased from 6.0 to 11.0. This is an indication of conversion of (3-sheet to disordered conformation. Increase of pH from 6.0 to 11.0 was accompanied by a decrease 101 in the intensity ratio of the tyrosine doublet, 855 cm"'/830 cm"1, from 1.0:0.9 to 1.0:1.3, which indicates that the average tyrosine, in the denatured state, may be in a more hydrophobic environment at pH 11.0 than at pH 6.0. The tryptophan band (around 833 cm"1) also became weaker with increasing pH to 11.0. In a laser Raman spectroscopic study of BSA (4% w/w in 0.1 M NaCl, pH 6.0) by Chen and Lord (1976), the polypeptide backbone was found to be mainly (-60%) a-helical. The authors reported a strong band at 941 cm"1 (a-helix), tyrosine doublet ratio (827-852 cm"1) of 10:4, and S-S frequency at 503 cm"1. Bellocq et al. (1972) studied Raman spectra of B S A (20 mg/mL, pH 7.0) and B L G (48 mg/mL, pH 5.8). They identified thirty Raman bands for each protein in the 500-1600 cm"1 region, half of which belonged to functional groups of the constituent amino acids. Location, as well as intensity, of the amide in band near 1250 cm"1 indicated a predominance of random coil in B L G . For BSA, however, this band indicated the presence of higher proportion of ordered structure. The SS stretching band in B L G was found as a broad band around 507 cm"1. The band near 1450 cm"1, in both proteins, was assigned to C H 2 group. In a study by Byler and Purcell (1989), thermal denaturation (at 30°C, 60°C, 70°C, and 80°C, for 1 hour) and gel formation in the whey proteins, B L G and BSA (3.0 to 3.5% w/v, pD 6.2 or 7.8) was investigated using FTIR. They showed that heating B L G and B S A at 80°C or above for 1 hour induced new P-strands, which were intermolecularly hydrogen-bonded. An increase in the proportion of disordered structure was also observed due to heating. Conformational changes of globular proteins may occur due to heating, which depend on the type of protein and the temperature (Byler and 102 Purcell, 1989). Byler et al. (1983) studied sulfhydryl groups of the cysteine linkages of B L G using laser Raman spectroscopy. They reported the great advantage of Raman spectroscopy to monitor and estimate the sulfhydryl content of B L G with only one cysteine residues (one out of 162). Hydrophobic interactions can be studied using Raman spectroscopy as was performed by Howell et al. (1999). They investigated the involvement of aromatic (band near 3065 cm"1) and aliphatic (bands near 2880 cm"1 and 2900 cm"1) amino acids in the C-H stretching region (including C H , C H 2 , and C H 3 groups) in several proteins (15% w/v, solutions in D 2 0 , apparent pD 6.8) including B L G . Heating (90°C, 30 min) caused an increase in the band area near 3065 cm"1 for B L G . Raman spectroscopic investigation of B L G compared to lysozyme (both 15% w/v in D 2 0 , pD 6.8) by Howell and Li-Chan (1996) showed a weaker band at 508 cm"1 (S-S stretching) in B L G , due to the lower number of cystine residues (two) in B L G (compared to four in lysozyme). Also a weaker band at 761 cm"1 showed lower tryptophan content (two in B L G and six in lysozyme). They observed that heating (90°C, 30 min) caused changes in the disulfide bonds and SS-stretching band (500-535 cm"1). For heated B L G , this band was observed around 507 cm"1 with some extra bands at 494 and 525 cm"1. Some factors, including pH, heating, chemical agents, etc., can denature proteins, which may cause protein unfolding and changes in the peptide backbone and/or side chains, and sometimes cleavage of disulfide bonds. Raman spectroscopy can be used as a suitable and powerful technique to detect transitions from an ordered to a disordered structure due to protein denaturation (Tu, 1986). 103 Lin and Koenig (1976) studied the effect of pH (from 1.72 to 10.9 in 0.1 N NaCl) and heating (25°C to 70°C at pH 8.0 for 3 hours) on BSA (5% by weight in deionized water). They reported that heating to 70°C caused an increase of disordered structure and a-helical content. No conformational change was observed below 42°C, while reversible changes occurred between 42°C and 50°C. They found a transition of helical to disordered structure near pH 5.0, when lowering the pH. The onset of alkaline denaturation occured between pH 9.0-10.0, but was less extensive than the aggregation due to acidic conditions. The effect of heating (50, 70, or 90°C for 30, 60, or 90 min) on the conformation of whey proteins (BLG and cc-lactalbumin, 15% w/v in D 2 0 , pD 6.8, 20 m M NaCl) was studied using Raman spectroscopy (Nonaka et al., 1993). In heated (70 and 90°C) B L G , a simultaneous decrease of turn structure and increase of P-sheet was observed. Some effects of heating on amino acid side chains that were observed include the exposure of tryptophan, increase of hydrogen bonding of tyrosine, and loss of native form of disulfide bonds. The effects of pH (3.0, 5.0, 7.0, 9.0, and 11.0), salts, and protein perturbants such as sodium dodecyl sulfate, mercaptoethanol, and ethylene glycol, on oat globulin (10% in distilled water) conformation was studied using Raman spectroscopy (Ma et al., 2000). They reported a transition from P-sheet at neutral pH, to a random coil at pH 3.0 and at alkaline pH (9.0-11.0). At extreme pH the intensity of tyrosine doublet band decreased, which is the indicator of buriedness of tyrosine residues. Raman spectroscopic study of the interaction of proteins and polysaccharides has not been reported before. Aoki et al. (1982), in a Raman spectroscopic study of 104 complexes of bovine serum albumin and ionic detergents, reported some changes in the protein secondary structure as well as environment change in the tyrosine residue, due to complexation. Raman and infrared spectroscopy has been used to investigate conformational features of lipids and proteins in myelin membrane (Carmona et al., 1986). Carey et al. (1972) investigated ligand (methyl orange)-protein interactions, using Raman spectroscopy. Raman spectroscopy was used to investigate structural changes in cod myosin after frozen storage or modification with formaldehyde by Careche and Li-Chan (1997). The objective of this research was to use Raman spectroscopy to study possible changes in protein secondary structure (a-helix, f3-sheet, and total random coil), various amino acid side chain vibrations (disulfide stretching, aromatic rings of tryptophan and tyrosine, C H stretching bands of aliphatic amino acids, C=0 stretch of the COOH group of aspartic and glutamic acids) and protein-water interactions due to pH, heating, and presence of K-carrageenan. 3 Experimental Procedures 3.1 Materials List of materials was previously mentioned in Chapter TV, section 3.1. 3.2 Preparation of samples Stock protein solutions containing 25% (w/v) protein were prepared in various buffers as described in Chapter IV, section 3.2. The stock protein solutions were diluted 105 to 15% (w/v) with the appropriate buffer or by mixing with K-carrageenan, for Raman spectroscopic analysis as is (unheated), or after heating (80°C, 30 min). To prepare KCG:protein mixtures, 0.6 mL 25% protein and 0.4 mL 0.6% K-carrageenan were mixed to yield 15% and 0.24% final concentrations of protein and K-carrageenan, respectively. This corresponds to a KCG:protein ratio of 1.0:62.5. Since K C G gels at room temperature even at this low concentration, both protein and K C G stock solutions were warmed to 35°C before mixing, for preparation of KCG:protein mixtures. The heated samples were prepared by placing parafilm sealed capillary tubes (Nichiden-Rika Glass Co. Ltd., Japan) containing the sample in a glass petri-dish floating in a water bath at 80°C for 30 minutes. A come-up time of 4 seconds has been reported for protein solutions heated in a similar type of capillary tube, as determined by using a T-type thermocouple attached to a data logger and a computer (Fukumoto, 1992). The heated samples were allowed to cool for an hour at room temperature and then stored in a refrigerator (4°C), overnight, before analysis. A l l samples of protein:KCG mixtures, at pH 3.0, were coagulated, while gels were formed at other pH of these mixtures. 3.3 Method An argon ion laser (Coherent Innova 70C series, Coherent Laser group, Santa Clara, CA) cooled with the Coherent Laser Pure heat exchanger system was used to excite samples at 488 nm. Samples were placed in hematocrit capillary tubes (Nichiden-Rika Glass Co. Ltd., Japan), and both ends were sealed with parafilm to avoid leaking. The capillary tubes were held horizontally, and the incident laser beam was vertical to the 106 capillary axis. The Raman scattering of the samples was measured at ambient temperature on a JASCO model NR-1100 laser Raman spectrophotometer (Japan Spectroscopic Co. Ltd., Tokyo, Japan). Conditions of the instrument were as follows: laser power, 200 mW, slit height 1 mm (for calibration with standard) or 4 mm (for the samples), sampling speed 120 cm"1 with data collected every cm"1, resolution at 19,000 cm"1, and spectral resolution 5 cm"1. The frequency (wavenumber calibration) of the instrument was checked daily using a standard solution of 1 M potassium nitrate, at 1050 ± 2 cm"1 band. To increase signal to noise ratio, at least 10 scans of each sample were run to obtain averaged spectral data. Duplicate spectra, of two independent samples, (each an average of 10 scans) were measured for 8 samples, and showed similar trends for replicates. A coefficient of variation below 10% has been reported previously in this laboratory for a variety of protein and amino acid samples using the same instrument (Howell and Li-Chan, 1996, Howell et al, 1999; Li-Chan, 1996b; Nonaka et al, 1993). The collected averaged data from the scans of samples in the Raman spectrophotometer were baseline corrected and smoothed using the 5-point Savitsky Golay function, and normalized against the phenylalanine band at 1004 cm"1 (i.e., adjusted to 1.0 for band 1004 cm"1 for all spectra) using Grams 386 (Galactic Industries Corp., Salem, NH). The intensity and location of the phenylalanine band at 1004 cm"1 band is not sensitive to conformation or microenvironment, and therefore can be used as an internal standard to normalize the Raman spectrum of proteins (Li-Chan, 1996a). The algorithm of Williams (1983) was used for detailed analysis of the secondary structure composition of samples, based on the averaged scans of the "raw" (not baseline corrected, smoothed or normalized) Raman spectra in the amide I region, using the RSAP 107 program (version 2.1) of Przybycien and Bailey (1989). Based on this program, the secondary structure composition of proteins has been divided into three parts: 1) total oc-helix, which includes ordered and unordered a-helix, 2) total P-sheet, which includes parallel and anti-parallel p-sheet, and 3) total random coil, which includes unordered structure and P-reverse turn. Although this program tends to overestimate P-sheet content of proteins, it is useful for observing comparative changes in the secondary structure composition of samples as a function of different variables. Assignments of bands in the Raman spectrum were performed according to available literature (Howell and Li-Chan, 1996; Nonaka et al., 1993), based on specific vibrational modes of amino acids side chains or the polypeptide backbone. 4 Results and Discussion Raman spectra of three protein samples, WPI, B L G , and BSA, at various pH, before and after heating, with or without K-carrageenan were compared over two different wavenumber regions of 400-1800 cm"1 and 2500-3400 cm"1. The spectral data of samples which had signal to noise (S/N) ratio of less than 5, which includes most of the spectra for samples at pH 3.0, should be interpreted with caution. Figures 18-25 show the Raman spectra in the 400-1800 cm"1 and 2500-3400 cm"1 regions of B S A under various conditions of pH, heating, and presence of K-carrageenan. Similar spectra were collected for B L G and WPI. K C G alone in the various buffers and concentration (0.24%) examined here did not show any bands in the Raman spectrum. 108 S < -o e u X ) U H 8 00 I 00 1600 1400 1200 1000 800 600 Wavenumber cm" Figure 18: Raman spectra in the 400-1800 cm"1 wavenumber regions of unheated BSA at various pH*. * Assignment of bands is shown on top of the Figure. 109 1600 1400 1200 1000 800 600 Wavenumber cm"1 Figure 19: Raman spectra in the 400-1800 cm"1 wavenumber regions of heated BSA at various pH*. * Assignment of bands is shown on top of the Figure. 110 - " i ; — i 1 : 1 f r~ 1600 1400 1200 1000 800 600 Wavenumber cm" Figure 20: Raman spectra in the 400-1800 cm' 1 wavenumber regions of unheated mixture of K C G : B S A at various pH*. * Assignment of bands is shown on top of the Figure. I l l 1600 1400 1200 1000 Wavenumber cm"1 8 0 0 6 0 0 Figure 21: Raman spectra in the 400-1800 cm'1 wavenumber regions of heated mixture of KCG:BSA at various pH*. * Assignment of bands is shown on top of the Figure. 1 1 2 1 T I i I \ I ~ 1 3 3 0 0 3 2 0 0 3 1 0 0 3 0 0 0 2 9 0 0 2 8 0 0 2 7 0 0 2 6 0 0 Wavenumber cm'1 Figure 22: Raman spectra in the 2500-3400 cm" wavenumber regions of unheated BSA at various pH*. * Assignment of bands is shown on top of the Figure. 1 1 3 1 l { I r 3300 3200 3100 3000 2900 2800 2700 2600 Wavenumber cm" Figure 23: Raman spectra in the 2500-3400 cm'1 wavenumber regions of heated BSA at various pH*. * Assignment of bands is shown on top of the Figure. 114 3300 3200 3100 3000 2900 2800 2700 2600 Wavenumber cm Figure 24: Raman spectra in the 2500-3400 cm'1 wavenumber regions of unheated mixture of KCG:BSA at various pH*. * Assignment of bands is shown on top of the Figure. 115 is h oo oo o u "1 1 1 1 1 1 1 " """I 3300 3200 3100 3000 2900 2800 2700 2600 Wavenumber cm"1 Figure 25: Raman spectra in the 2500-3400 cm'1 wavenumber regions of heated mixture of KCG:BSA at various pH*. * Assignment of bands is shown on top of the Figure. 116 4.1 Effect of pH, heating, and K-carrageenan on the secondary structure of proteins 4.1.1 Secondary structure based on amide I band The results of examination of the secondary structure of the three protein samples under various conditions of pH, heating, and addition of K-carrageenan (KCG) are shown in Figures 26-28 (and Appendix 1, Table 13). The total a-helix:(3-sheet:random coil content of protein samples at pH 7.0 were 30:40:30%, 25:40:35%, and 60:15:25% for WPI, B L G , and BSA, respectively. These results are consistent with literature values. For example, the ratio of the components of secondary structure of BSA was reported as 55:16:29% (Suttiprasit et al, 1992). CD spectra of B L G by Swaisgood (1989) showed the a-helix:P-sheet ratio of 10:43%. Kinsella et al. (1989) noted the a-helix:p-sheet ratio of 15:50% and 54:18% for B L G and BSA, respectively. Generally pH did not have a very large effect on the secondary structure of whey proteins, but higher P-sheet and lower a-helix were observed at pH 7.0 and 9.0 than at acidic pH for WPI, at pH 7.0 than either pH 5.0 or 9.0 for B L G , and at pH 7.0 and 9.0 than at pH 5.0 for BSA. In terms of random coil, the most noticeable change occurred when B L G was heated at pH 5.0 or 9.0 or in heated K C G : B S A mixture at pH 3.0, compared to unheated B S A alone, where the random coil disappeared. Heating resulted in increased P-sheet and decreased helix content of the proteins under the conditions investigated in this study, not considering those spectra with low signal to noise ratio. 117 Adding K C G to WPI generally decreased helix content, but helical content increased upon heating the KCG:WPI mixture at pH 5.0. Adding K C G to B L G also decreased helix content and increased p-sheet content at pH 5.0, 7.0, and 9.0. Heating the K C G : B L G mixture at pH 5.0 increased helix content and decreased P-sheet content, whereas no further change in secondary structure was observed after heating mixtures at pH 7.0 or 9.0, compared to unheated K C G : B L G mixture. Adding K C G to BSA increased p-sheet and decreased helix content at pH 5.0, while the reverse was seen at other pHs. 118 X I a TO +-O 1-WPI H WPI KCG:WPI H KCG:WPI 100 80 60 40 20 0 0 ¥ * Ma, a' 5f WPI H WPI rvy< * 1 Wz . •>< l V J , T • " KCG:WPI H KCG:WPI WPI H WPI KCG:WPI H KCG:WPI pH 3.0 0 PH 5.0 0 pH 7.0 0 pH 9.0 Figure 26: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on whey protein isolate (WPI) secondary structure content (%) estimated by the Raman Spectral Analysis Package for the Amide I band3. * Due to low signal/noise ratio (<5), the results should be considered with caution. a The secondary structure estimation was performed using spectral data averaged over 10 scans. 119 100 ~ 80 = 60 0) X a o 40 1 20 0 & -vv BLG H BLG KCG: BLG H KCG:BLG 100 80 SZ re o 60 40 20 1 w> m F--J w> 1 BLG H BLG KCG: BLG H KCG:BLG 100 - 80 O O E o T3 c re rx re o l -60 40 20 0 BLG 222 H BLG KCG: BLG H KCG: BLG pH 3.0 • pH 5.0 0 pH 7.0 0 pH 9.0 Figure 27 Effects of pH, heating (H), and presence of K-carrageenan (KCG) on (3-lactoglobulin (BLG) secondary structure content (%) estimated by the Raman Spectral Analysis Package for the amide I band3. * Due to low signal/noise ratio (<5), the results should be considered with caution. a The secondary structure estimation was performed using spectral data averaged over 10 scans. 120 100 _ 80 | 60 1 40 CO o H 20 'A' * I BSA H BSA K C G : B S A H KCG:BSA 100 0) to +-» o BSA H BSA KCG: BSA H KCG:BSA 100 BSA H BSA KCG: BSA H KCG:BSA | | pH 3.0 0 pH 5.0 0 pH 7.0 Q pH 9.0 Figure 28: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on bovine serum albumin (BSA) secondary structure content (%) estimated by the Raman Spectral Analysis Package for the Amide I band 3. * Due to low signal/noise ratio (<5), the results should be considered with caution. The secondary structure estimation was performed using spectral data averaged over 10 scans. 4.1.2 Secondary structure based on the 938 cm"1 band assigned to a-helix The effects of pH, heating, and presence of K C G on the 938 cm"1 band, which arises from skeletal C-C stretching vibrations of the polypeptide in an a-helical conformation (Li-Chan et al, 1994), is shown in Figure 29 (and Appendix 1, Table 14). For example, the intensity of the band varied from 0.1 for heated BSA at pH 7.0 to 1.1 for heated B L G at pH 7.0. The range in which the bands were found in various proteins and treatments ranged from 935-948 cm"1; no pattern in the shifts was observed. The intensity of the a-helix 938 cm"1 band at pH 7.0 for WPI, B L G , and BSA was 0.3, 0.5, and 0.6, respectively. In most samples, the intensity of this band was the lowest at pH 7.0, compared to other pHs. Howell and Li-Chan (1996), who used Raman spectroscopy to study the interactions of lysozyme with whey proteins (15% protein in D2O, pD 6.8), reported the normalized band intensity at 938 cm"1 of 0.4 for both unheated and heated (90°C for 30 min) B L G . Nonaka et al. (1993) reported the band intensity of 0.58 and 0.53, respectively for unheated and heated (90°C for 30 min, pD 6.8) B L G . Bellocq et al. (1972), who studied the laser-excited Raman spectroscopy of B S A (2% solution, pH 7.0) and B L G (4.8% solution, pH 5.8) reported the band intensity of 0.8 (940 cm"1) and 0.2 (945 cm"1), respectively. The normalized band intensity assigned to a-helix (around 938 cm"1) was highest in B L G and B S A at pH 5.0 compared to other pHs, while the band intensity of WPI was highest at pH 9.0. The band intensity was similar at pH 7.0 and 9.0 for both B L G and BSA. Heating caused a noticeable increase in the band intensity of WPI and B L G at pH 7.0, while that of B S A decreased. Heating decreased the band intensity at pH 9.0 for WPI and BSA, but that of B L G did not change. Adding K C G to protein, under unheated 122 conditions, caused a decrease in the band intensity for WPI at pH 3.0, B L G at pH 5.0 and 7.0, and BSA at pH5.0. Adding K C G to proteins, followed by heating, caused decrease in the band intensity in B L G at pH 7.0, and BSA at pH 3.0. Increase in intensity of the band was observed only for WPI at pH 9.0. In summary, heating in the presence of K C G increased the 938 cm"1 band intensity for WPI and B L G , while no change or slight decrease was observed for BSA. Adding K C G to proteins, however, generally decreased the band intensity. 123 WPI H WPI KCG:WPI H KCG:WPI BLG H BLG KCG:BLG H KCG:BLG V) c o • D (U , N To E i _ o z BSA H BSA KCG:BSA H KCG:BSA U p H 3.0 Q pH 5.0 [j pH 7.0 [j pH 9.0 Figure 29: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of bands typical of a-helical structure of the Raman spectrum of the protein samples. * Due to low signal/noise ratio (<5), the results should be considered with caution. Notes: WPI: whey protein isolate, B L G : P-lactoglobulin, BSA: bovine serum albumin. Intensity values normalized to the phenylalanine band at 1004±2 cm"1 124 4.2 The effect of pH, heating, and addition of K-carrageenan on the disulfide bond The band around 508 cm"1 in the spectra is assigned as disulfide (v S-S) bonds in the all-gauche conformation (Nonaka et al, 1993). The intensity and location of this band in the Raman spectrum for WPI, B L G , and BSA under various conditions of pH, heating, and addition of K C G is shown in Figure 30 (and Appendix I, Table 15). For example, the normalized intensity of this band varied from 0.2 for heated B S A at pH 9.0 to 1.1 in the heated KCG:WPI mixture at pH 3.0. For unheated BSA and B L G , highest intensity of the SS band was observed for samples at pH 5.0, whereas WPI showed similar intensity at pH 3.0, 5.0, and 7.0, which were all higher than the intensity at pH 9.0. The range in which the bands were found for various proteins and treatments were from 505 to 515 cm"1. The normalized intensity of SS stretching band around 508 cm"1 varied among proteins at various pH. At pH 7.0, the band intensity of WPI, B L G , and B S A were 0.6, 0.4, and 0.4, respectively. Howell and Li-Chan (1996) reported the band intensity of 0.26 (508 cm"1) and 0.22 (507 cm"1) for unheated and heated (90°C for 30 min) B L G (15% in D 2 0 , pD 6.8), respectively. Nonaka et al. (1993) reported the band intensity of 0.24 and 0.17, respectively for unheated and heated (90°C for 30 min, pD 6.8) B L G . Bellocq et al. (1972) reported the band intensity of 0.25 (510 cm"1) for B L G (4.8% solution, pH 5.8). Generally, heated or unheated proteins, with or without K C G had the lowest band intensity at pH 9.0 compared to other pH, with the exception of the heated KCG:WPI mixture at pH 9.0. 125 When comparing unheated samples, those at pH 9.0 had the lowest SS band intensity, while the highest intensity was observed at pH 5.0 for B L G and BSA. Heating caused poor signal to noise ratio in the spectra of samples at acidic pH, especially at pH 3.0. Although it is difficult to assess overall trends in this case, some observations are as follows. Heating WPI caused a slight increase in SS band intensity at pH 5.0 and slight decrease at pH 9.0. Heating also caused slight decreases in the band intensity at pH 9.0 for B L G and at pH 7.0 and 9.0 for BSA. These results may be correlated with reports that proteins are more prone to SS/SH reactions and disulfide bond cleavage at high pH (Boye et al, 1996; Monahan et al. 1995; Relkin et al, 1993). Nonaka et al. (1993), who studied the thermally induced gelation of whey proteins, found a slight decrease in SS band intensity of the Raman spectrum of B L G after heating at 90°C in D 2 0 , pD 6.8 (90°C, 30 min), with little or no change at 70°C. Two hypotheses were suggested for the decrease of the intensity of the SS band by Nonaka et al. (1993). They hypothesized that the decrease could be attributed to losing the native conformation of disulfide bond due to heat induced gelation, or burying of SS bonds inside the gel matrix. Due to low signal/noise ratio of the Raman spectrum for heated B L G at pH 7.0, the reported intensity values of SS band can not be compared with those of the present study. Adding K C G to proteins generally decreased SS band intensity at pH 5.0 and 9.0 for WPI and at pH 5.0 for B L G and BSA. Heating the KCG:WPI mixture, however, caused significant increases in the band intensity. Heating the K C G : B L G mixture slightly decreased the band intensity at pH 7.0, while heating the K C G . B S A mixture caused no further change in the intensity of SS stretching band. 126 In summary, overall trends include lowest SS band intensity at alkaline pH and highest intensity at pH 5.0 for B L G and BSA. The SS band intensity decreased by the addition of K C G , especially at pH 5.0. 127 in c Q) "D d) N 75 E i _ o z WPI H WPI KCG:WPI H KCG:WPI in c Q) O N 75 E o z BLG H BLG KCG:BLG H KCG:BLG in c a) TJ 0) N 75 E i _ o z BSA H BSA KCG:BSA H K C G : BSA | | pH 3.0 [] pH 5.0 0 pH 7.0 Q pH 9.0 Figure 30: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of SS stretching band (-508 cm"1) of the Raman spectrum of the protein samples. * Due to low signal/noise ratio (<5), the results should be considered with caution. Notes: WPI: whey protein isolate, B L G : P-lactoglobulin, BSA: bovine serum albumin. Intensity values normalized to the phenylalanine band at 1004±2 cm"1. 128 4.3 The effect of pH, heating, and addition of K-carrageenan on tryptophan band The bands around 761 cm"1 (Figure 31, and Appendix 1, Table 16) and 1333 cm"1, 1340 cm"1, and 1360 cm"1 (Appendix 1, Table 16), which are derived from indole-ring vibrations, are assigned to tryptophan. Generally, indole rings in hydrophobic or buried environments cause sharp bands in protein Raman spectra in the vicinity of these wavenumbers (Li-Chan et al, 1994). Decrease in the band intensity at 761 cm"1 is due to exposure of buried tryptophan residues to the aqueous environment (Nonaka et al., 1993; Li-Chan and Nakai, 1991). The normalized intensity of 761 cm"1 band varied from 0.1 in heated B S A at pH 7.0 to 1.1 for unheated B L G at pH 5.0. It is more difficult to compare the other three wavenumbers for the tryptophan band, since all three bands were not observed for all samples. The intensity of the 1345 cm' 1 band for B L G was 0.4, similar to 0.36, which was observed by Howell and Li-Chan (1996). The intensity of the band around 761 cm"1 was 0.6, 0.7, and 0.3 for WPI, B L G , and B S A at pH 7.0, respectively. Howell and Li-Chan (1996) reported the band intensity of 0.43 (761 cm"1) and 0.45 (757 cm"1) for the unheated and heated (90°C for 30 min) B L G . Nonaka et al. (1993) reported the band intensity of 0.48 and 0.31, respectively for unheated and heated (90°C for 30 min, pD 6.8) B L G . The band intensity at 761 cm"1 of unheated proteins in this study was pH dependent. For all three protein samples, the highest band intensity was observed at pH 5.0, compared to pH 7.0 and 9.0. Ma et al. (1999), who studied the conformation of oat globulin at various pH, also found that the intensity of the tryptophan band was highest at pH 5.0, compared to other pHs. The higher the pH (from 5.0 to 7.0 to 9.0), the lower the 129 band intensity, which may indicate more exposure. Heating resulted in decreases in the 761 cm"1 band intensity for WPI at pH 5.0, B L G at pH 9.0, and B S A at both pH 7.0 and 9.0. Adding K C G to proteins resulted in decreases in the 761 cm"1 band intensity, therefore more exposure of tryptophan residues, for WPI at pH 5.0, B L G at pH 5.0, 7.0, and 9.0, and B S A at pH 5.0 and 7.0. Adding K C G to WPI followed by heating caused increases in the band intensity at pH 5.0, 7.0, and 9.0. Adding K C G to B L G followed by heating decreased the band intensity of mixtures at pH 7.0. Heating K C G : B S A mixture decreased the band intensity at pH 9.0, compared to unheated mixture, but no change was observed for pH 7.0 mixture. In summary, some overall trends based on the 761 cm"1 band intensity include greater exposure of tryptophan at neutral and alkaline pH than at pH 5.0, and after heating of B L G or BSA at pH 9.0. Adding K C G also caused greater exposure, except for B S A at pH 9.0. Heating WPI or BSA in the presence of K C G resulted in more buried tryptophan than heated samples without K C G . 130 •</> c WPI H WPI KCG:WPI) H KCG:WPI 1.5 v. 1 •a <u N E i _ o z 0.5 BLG H BLG KCG: BLG H KCG:BLG BSA H BSA KCG:BSA H KCG:BLG pH 3.0 Q pH 5.0 0 pH 7.0 [j pH 9.0 Figure 31: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of band typical of the tryptophan (near 761 cm"1) of the Raman spectrum of the protein samples. * Due to low signal/noise ratio (<5), the results should be considered with caution. Notes: WPI: whey protein isolate, BLG: P-lactoglobulin, BSA: bovine serum albumin. Intensity values normalized to the phenylalanine band at 1004±2 cm"1. 131 4.4 The effect of pH, heating, and presence of K-carrageenan on the tyrosine doublet bands The intensity ratio of bands around 850/830 cm"1 is reported to be an indicator of hydrogen bonding of the phenolic hydroxyl group (Bouraoui et al., 1997, Siamwiza et al., 1975). Bellocq et al. (1972), who studied laser-excited Raman spectroscopy of BSA, B L G , and lysozyme, identified tyrosine doublets at 820 and 852 cm"1 for BSA and 832 and 860 cm"1 for B L G . Tu (1986) reported that a decrease in the intensity ratios of 850/830 bands is due to an increase in "buriedness" because of involvement of tyrosyl residues in intermolecular interactions. Tyrosine residues are considered exposed when the doublet ratio is >1 (Careche and Li-Chan, 1997). According to Tu (1986), if the intensity ratio of the tyrosine doublet is between 0.7-1.0, the tyrosine is considered to be buried. However, a ratio around 0.3 shows a strong H-bonding to a negative acceptor. The effect of pH, heating, and K-carrageenan on the ratio of these bands is shown in Figure 32 (and Appendix I, Table 17). For example, the intensity of these tyrosine doublet bands varied from 0.4 for heated K C G : B L G mixture at pH 7.0 to 1.75 for heated K C G : B S A mixture at pH 9.0. The Raman shifts of the bands vary from 820-834 cm"1 and from 852-861 cm"1. The ratio of tyrosine doublet of the Raman spectrum of protein samples at pH 7.0 for WPI, B L G , and B S A was 1.5, 1.0, and 0.9, respectively. These results are similar to those reported in the literature. Nonaka et al. (1993) and Howell and Li-Chan (1996) reported tyrosine doublet ratio of B L G , at pH 6.8, of 1.02 and 0.86, respectively, while Bellocq al. (1972) noted tyrosine doublet ratio of 0.8 for B S A (2% solution, pH 7.0) and 1.0 for B L G (4.8% solution, pH 5.8). Higher tyrosine doublet ratios for WPI and 132 B L G at pH 7.0 than pH 5.0 and 9.0, and higher ratio for B S A at pH 9.0 than 5.0 and 7.0 were observed. Heating WPI caused an increase in the tyrosine doublet ratio at pH 5.0, and a decrease at pH 9.0. Heating caused a large increase in the ratio for B L G at pH 9.0. For BSA, heating caused a decrease at pH 9.0. Adding K C G to proteins resulted in increased tyrosine doublet ratio at pH 9.0 for WPI, and at pH 7.0 and 9.0 for B L G and BSA; while decrease in the ratio was observed when K C G was added to BSA at pH 5.0. Heating KCG:protein mixtures resulted in decreased tyrosine doublet ratio at pH 5.0 and 9.0 for WPI, at pH 9.0 for B L G , and pH 7.0 for BSA, compared to proteins heated alone or unheated mixtures. In summary, high (>1) intensity ratio of the 850/830 cm"1 band was observed in the unheated mixture of KCG:proteins at pH 7.0 and 9.0 and most of the samples at pH 9.0. Adding K C G to proteins at pH 7.0 and 9.0 increased the band intensity ratio, compared to unheated mixtures. 133 (/) c X) N E WPI H WPI KCG:WPI H KCG:WPI c TJ 0) _N E o BLG H BLG KCG:BLG H KCG:BLG 2 BSA H BSA KCG:BSA H KCG:BSA @pH 3.0 [] pH 5.0 0 pH 7.0 Q pH 9.0 Figure 32: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of bands typical of the tyrosine doublet ratio (860/825 cm"1) of the Raman spectrum of the protein samples. * Due to low signal/noise ratio (<5), the results should be considered with caution. Note: WPI: whey protein isolate, BLG: P-lactoglobulin, BSA: bovine serum albumin. Intensity values normalized to the phenylalanine band at 1004±2 cm'1. 134 4.5 The effect of pH, heating, and addition of K-carrageenan on the CH 2 bending band The band near .1453 cm"1 is assigned to CH2 group (Bellocq et al., 1972). The results for the CH2 bending band around 1453 cm"1 are shown in Figure 33 (and Appendix I, Table 18). For example, the normalized intensity of these bands varied from 0.3 for the heated K C G : B S A mixture at pH 7.0 to 1.1 for heated KCG:WPI at pH 7.0. The shifts in bands varied between 1450 to 1459 cm"1. The normalized intensity of CH2 bending band of the Raman spectrum of unheated proteins at pH 7.0 were as follows: 0.8, 0.6, and 0.6 for WPI, B L G , and BSA, respectively. Howell and Li-Chan (1996) reported the band intensity of 0.92 for B L G (1454 cm"1) at pH 6.8 (in D 2 0) , which increased to 1.31 (1454 cm"1) after heating (90°C, 30 min). Bellocq et al. (1972) noted the band intensity of 1.4 (1450 cm"1) and 1.2 (1455 cm"1), respectively for B S A (2% solution, pH 7.0) and B L G (4.8% solution, pH 5.8). Generally, the intensity of this band decreased as the pH increased from pH 5.0 to 7.0 to 9.0 for WPI and BSA. For B L G , however, the lowest band intensity was observed at pH 7.0. Heating increased 1453 cm"1 band intensity for KCG:WPI mixtures at pH 5.0, 7.0, and 9.0, while decreased band intensity was observed by heating B L G at pH 9.0, B S A at pH 7.0 and 9.0, and K C G : B S A mixture at pH 9.0. Adding K C G to WPI or B S A at pH 5.0 and 7.0 caused decreases in the intensity of the C H 2 band. No change in the band intensity was observed when K C G was added to B L G . The intensity of the C H 2 band did not change when K C G was added to B L G or when this mixture was heated. Adding K C G followed by heating, generally, caused an 135 increase in the band intensity for mixtures of KCG:WPI at pH 7.0 and 9.0, and decrease in the band intensity for K C G : B S A mixtures at pH 7.0 and 9.0. Some overall trends include decrease in the band intensity as pH increased to alkaline (in unheated samples), decrease in the band intensity after heating B L G at pH 9.0 or BSA at neutral/alkaline pH, marked increase in the band intensity when B L G was heated at pH 7.0, and decrease in the band intensity when K C G was added to WPI or B S A at pH 5.0 and 7.0. 136 WPI H WPI KCG:WPI H KCG:WPI ID C Q> N TO E o BLG H BLG KCG: BLG H KCG:BLG BSA H BSA KCG:BSA H KCG:BSA pH 3.0 [] pH 5.0 0 pH 7.0 | pH 9.0 Figure 33: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of the C H 2 bending band of the Raman spectrum of the protein samples. * Due to low signal/noise ratio (<5), the results should be considered with caution. Notes: WPI: whey protein isolate, BLG: P-lactoglobulin, BSA: bovine serum albumin. Intensity values normalized to the phenylalanine band at 100412 cm"1. 137 4.6 The effect of pH, heating, and addition of K-carrageenan on the C=0 stretching band The band around 1700-1750 cm"1 has been assigned to the C=0 stretch of the COOH group of aspartic and glutamic acids (Li-Chan et al, 1994). The effect of heating and addition of K C G on the Raman shift of the C=0 stretching band of the proteins samples at pH 3.0 is shown in Table 12. No band was detected at pH 5.0, 7.0, and 9.0, due to ionization of the COOH groups to COO" at these pH. The shift in the band varied from 1720 cm"1 in WPI to 1745 cm"1 in heated BSA. Heating or presence of K C G slightly increased the intensity of the band around 1750 cm"1, however, some shift in the band was observed. Such shifts have been attributed to changes in the environment around the bands (Bouraoui et al, 1997), which corresponds to changes in the energy of the scattered photons and consequently to the differences in rotational and vibrational levels of the molecule (Freeman, 1974). Slight increase in the band intensity was observed when WPI, B L G , BSA, KCG:WPI or K C G : B L G mixture were heated, while slight decrease resulted when K C G : B S A mixture was heated. 138 Table 12: Effects of heating (H), and presence of K-carrageenan (KCG) on changes in the Raman shift of the C=0 stretching band of the Raman spectrum of protein samples at pH 3.0. Sample Raman Shift (cm1) Normalized Intensity WPI 1720 0.5 HWPI 1743 0.7 KCG:WPI 1732 0.5 H KCG:WPI 1730 0.6 BLG 1740 0.4 HBLG 1720 0.5 K C G : B L G 1732 0.5 H K C G : B L G 1731 0.6 BSA 1732 0.4 HBSA 1716 0.5 K C G : BSA 1732 0.5 H KCG:BSA 1741 0.4 Notes: WPI: whey protein isolate, BLG: P-lactoglobulin, BSA: bovine serum albumin. Intensity values normalized to the phenylalanine band at 1004+2 cm _ 1 . 1 3 9 4.7 The effect of pH, heating, and addition of K-carrageenan on the C-H stretching bands Bands near 2874-2897 cm"1 have been assigned to C H 3 symmetrical stretching and R 3 C -H stretching bands of aliphatic amino acids, while =C-H stretching bands of aromatic amino acids can be found around 3061-3068 cm"1. Both aromatic and aliphatic amino acids as well as charged amino acids, proline, threonine and histidine, have C-H stretching bands near 2935-2955 cm"1 (Howell et al., 1999). The intensity of the C H stretching bands around 2880, 2930, and 3060 cm"1 of the Raman spectra of proteins are shown in Figures 34-36 (and Appendix I, Table 19). For example, the intensity of 2880 cm"1 band varied from 0.3 for heated K C G : B S A mixture at pH 3.0 to 1.6 for unheated WPI at pH 5.0. The shift in the band varied from 2871 cm"1 for B L G at pH 7.0 to 2885 for heated WPI at pH 9.0. The normalized intensity of C H stretching band around 2880 cm"1 for unheated proteins at pH 7.0 were 1.1, 1.2, and 1.0 for WPI, B L G , and BSA, respectively. The intensity of 2880 cm"1 band was the highest at pH 5.0 for WPI, while pH had no effect on the intensity for B L G and was slightly higher at pH 7.0 for BSA, compared to other pHs. Generally, heating decreased the intensity of 2880 cm"1 band in all three proteins at various pH with or without K C G . Howell et al. (1999) similarly noted a decrease in the band intensity of lysozyme but observed increases in the band intensity of B L G and a-lactalbumin (15% w/v solution in D 2 0 , pD 6.8) after heating (90°C for 30 min). Adding K C G to all the proteins at pH 5.0 and 7.0, generally, decreased the 2880 cm"1 band intensity. Adding K C G to the proteins, followed by heating, resulted in decreased band intensity for WPI at pH 5.0 (compared to unheated mixture or unheated 140 WPI alone) and decreased band intensity at pH 7.0 and 9.0 for B L G and B S A (compared to unheated mixtures). The intensity of 2930 cm"1 band varied from 0.4 in K C G : B S A mixture at pH 3.0 to 3.0 for WPI at pH 5.0. The shifts in bands varied from 2928 cm"1 for BSA at pH 7.0 to 2943 cm"1 for KCG:WPI at pH 5.0. The normalized intensity of this band at pH 7.0 were 2.1, 2.0, arid 1.9 for WPI, B L G , and BSA, respectively. For WPI the highest band intensity was noted at pH 5.0, while for B L G the intensity at pH 5.0 was the lowest, compared to other pHs. Heating generally decreased the band intensity. Adding K C G to WPI decreased the band intensity at pH 5.0 and 9.0, but increased it at pH 7.0. Adding K C G to the proteins resulted in decreased band intensity at pH 5.0 and 9.0 for WPI and at pH 5.0 for BSA, and increased band intensity at pH 5.0 for B L G . Heating KCG:WPI at pH and 7.0, or K C G : B L G and K C G : B S A at pH 7.0 and 9.0 decreased the band intensity, compared to unheated mixtures or unheated proteins alone. The 2930 cm"1 band intensity of heated K C G : B L G or K C G : B S A was higher than B L G or B S A heated alone. The intensity of band around 3060 cm"1, which is assigned to C - H stretching of aromatic amino acids varied from 0.1 for heated B L G at pH 9.0, heated B S A at pH 7.0, heated K C G : B S A at pH 3.0, and K C G : B L G at pH 7.0 to 0.9 for heated B L G at pH 7.0. The shift in the band varied from 3040 cm"1 in K C G : B L G at pH 3.0 to 3073 cm"1 in heated B L G at pH 7.0. The normalized intensity of this stretching band at pH 7.0 in various proteins were 0.2, 0.2, and 0.3 for WPI, B L G , and BSA, respectively. The effects of pH on the - C H stretching band intensity at 3060 cm"1 are as follows. For WPI, the highest band intensity was observed at pH 5.0, while pH had little effect on the band intensity for B L G and BSA. Heating WPI increased the band intensity 141 at pH 5.0). Heating B L G increased the band intensity at pH 7.0 and decreased it at pH 9.0. Heating BSA decreased the band intensity at pH 7.0 and 9.0. Adding K C G to WPI caused a slight decrease in the band intensity at pH 5.0 and 9.0, and a slight increase at pH 7.0. The 3060 cm"1 band intensity of K C G : B L G mixture at pH 7.0 and 9.0 was slightly lower than B L G alone, while little effect was observed when K C G was added to BSA. Adding K C G followed by heating did not appreciably change the band intensity, except for increase in the band intensity for B L G at pH 5.0 and 7.0, and decrease in the band intensity at pH 7.0 and 9.0 for BSA, compared to B L G or BSA, respectively. In summary, some overall trends based on 2880 and 2930 cm"1 bands include decreased band intensity due to heating. While little change in intensity of these two bands was observed due to the presence of K C G in unheated proteins, the heated KCG:protein mixtures showed less decrease in band intensity compared to proteins heated in the absence of K C G . The 3060 cm"1 band intensity was increased by heating at pH 5.0 and 7.0 for WPI and B L G and at pH 5.0 for BSA, but decreased by heating at pH 7.0 and 9.0 for B S A and at pH 9.0 for B L G . 142 </) c TJ d) N TO E *_ o WPI H WPI KCG:WPI H KCG:WPI 1.5 •£ 1 TJ N "TO E 0.5 1 i 1 1 nt. I I vv>< * * • 1 r ..•» " Si (.."••> r','i> 1 % : v BLG H BLG KCG:BLG H KCG:BLG BSA H BSA KCG:BSA H KCG.BSA @pH 3.0 0 pH 5.0 0 pH 7.0 Q pH 9.0 Figure 34: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of the C H stretching band (2880 cm'1) of the Raman spectrum of protein samples. * Due to low signal/noise ration (<5), the results should be considered with caution. Notes: WPI: whey protein isolate, B L G : P-lactoglobulin, BSA: bovine serum albumin. Intensity values normalized to the phenylalanine band at 1004±2 cm' 1, n.b., no band. 1 4 3 WPI H WPI KCG:WPI H KCG:WPI BLG H BLG KCG: BLG H KCG:BLG in c < D +-* c -a 0) N To E 1.5 0.5 T 7 3 i • + J. BSA H BSA KCG: BSA b-'v ,i'J. H KCG:BSA pH 3.0 [] pH 5.0 pH 7.0 Q pH 9.0 Figure 35: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of the CH stretching band (2930 cm'1) of the Raman spectrum of protein samples. * Note: Due to low signal/noise ratio, the results should be considered with caution. WPI: whey protein isolate, B L G : P-lactoglobulin, BSA: bovine serum albumin. Intensity values normalized to the phenylalanine band at 1004±2 cm"1, n.b., no band. 144 1 BSA H BSA KCG:BSA H KCG:BSA gpH 3.0 [] pH 5.0 g pH 7.0 Q pH 9.0 Figure 36: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of the C H stretching band (3060 cm"1) of the Raman spectrum of protein samples. * Due to low signal/noise ratio (<5), the results should be considered with caution. Notes: WPI: whey protein isolate, BLG: P-lactoglobulin, BSA: bovine serum albumin. Intensity values normalized to the phenylalanine band at 1004±2 cm"1, n.b., no band. 145 4.8 The effect of pH, heating, and addition of K-carrageenan on the OH stretch (-3200 cm"1) The band around 3200 cm"1 has been assigned to O H stretch (Careche and L i -Chan, 1997). The effects of pH, heating, and K-carrageenan on changes in the Raman shift of the O H band of the Raman spectrum of protein samples are shown in Figure 37 (and Appendix I, Table 20). The normalized intensity of OH stretching band, of the Raman spectrum of the protein samples, around 3200 cm"1 varied from 2.8 for WPI at pH 5.0 to 0.4 for heated B L G at pH 9.0. The shift in the location of the band varied from 3203 cm"1 for B L G at pH 5.0 to 3236 cm"1 for K C G : B S A at pH 3.0. The intensity of this band for the protein solutions at pH 7.0 was: 2.0, 1.9, and 1.4 for WPI, B L G , and BSA, respectively. This O H band primarily arises from stretching vibrations of water in the solution. Since all these protein samples contained 15% (w/v) protein, it is expected to observe similar intensity for the O H band. This discrepancy can be attributed to the normalization procedure. Normalization of the spectral intensities was done based on the intensity of the phenylalanine band around 1004±2 cm"1. The number of phenylalanine residues differ between proteins; 27 out of 581 total residues and 4 out of 162 total residues for B S A and B L G , respectively. This means that the number of phenylalanine residues per total is higher in B S A than B L G (-4 out of 100 and -2 out of 100 residues, respectively). So, the intensity of bands based on different number of phenylalanine residues will also be about 2 fold different. This may explain why the intensity of the O H band was lower in BSA compared to B L G (1.4 and 1.9, respectively). 146 The highest band intensity for WPI was observed at pH 5.0, while for B L G pH 5.0 had the lowest intensity. Generally, heating decreased the intensity of water band in the absence or presence of K C G . Adding K C G to WPI or B S A at pH 5.0 and 7.0 decreased the band intensity. However, adding K C G increased the band intensity of B L G at pH 5.0 and of BSA at pH 9.0. Adding K C G to proteins followed by heating decreased the band intensity compared to unheated mixtures. Decrease in the intensity of OH stretch can be attributed to evaporation or immobilization of water molecules due to heat treatment. 147 W P I H W P I K C G : W P I H K C G : W P I 2.5 W 2 c 0) 7 3 <U N 75 E i — o 1.5 1 0.5 i jk •, 7 ™ * i -:'/• B L G H B L G K C G : B L G H K C G : B L G B S A H B S A K C G : B S A H K C G : B S A pH 3.0 1 pH 5.0 F] pH 7.0 0 pH 9.0 Figure 37: Effects of pH, heating (H), and presence of K-carrageenan (KCG) on the normalized intensity of the O H band (3200 cm'1) of the Raman spectrum of protein samples. * Due to low signal/noise ratio (<5), the results should be considered with caution. Notes: WPI: whey protein isolate, BLG: P-lactoglobulin, BSA: bovine serum albumin. Intensity values normalized to the phenylalanine band at 1004±2 cm"1. 148 5 General Discussion Raman spectroscopy is a powerful technique for elucidation of structural changes due to the effects of pH, heating and presence of K-carrageenan on whey proteins. The great flexibility of Raman spectroscopy allowed not only the study of solutions (such as individual proteins at various pH), but also the study of coagulated samples of protein:KCG mixtures (at pH 3.0) and gelled samples (many of the heated or unheated protein:KCG mixtures at pH 5.0, 7.0, and 9.0). However, poor signal/noise ratio was observed for most samples at pH 3.0, which consisted of white coagulum. The results of the present study showed that secondary structure of B L G consisted of 25% a-helix, 40% P-sheet, and 35% random coil. a-Helix:P-sheet ratios of 10:43 and 15:50 were reported in the literature (Swaisgood, 1989; Kinsella et al., 1989). Matsuura and Manning (1994) mentioned that native B L G is composed predominantly of P-sheet, with a secondary structure that does not change over a wide range of pH. Also, Das and Kinsella (1989) explained that the conformation of B L G is rigid around pH 3.0 to 7.0, therefore, pH has no effect on its secondary structure. The results of the present study also show that there was not much change in the secondary structure between unheated B L G at all pH studied, except slight increase in a-helix at pH 5.0. Casal et al. (1988), who used FTER to study structural changes of B L G due to effect of pH (2.0 to 13.0) and temperature (-100°C to 90°C), reported a P-sheet content of 57% and 59% at pH 3.0 and 7.0, respectively. They also noted no changes in the secondary structure of B L G up to about 58-60°C. 149 In the study of effects of pH (3.0-9.0) and heating (26-97°C) on the secondary structure of P-lactoglobulin (BLG, 200 mg/mL in D 2 0) , using FTER and differential scanning calorimetry (DSC), Boye et al. (1996) suggested that increasing pH from 3.0 to 9.0 increased the formation of P-sheet structure (based on a qualitative observation), which is not in agreement with the results of the present study nor those reported by others (Casal et al. 1988; Das and Kinsella, 1989; Matsuura and Manning, 1994). B L G showed maximum heat stability at pH 3.0 and most susceptibility to heat denaturation at pH 9.0. The P-sheet structure broke down at about 72°C for pH 3.0 sample, but at about 59°C and 55°C for pH 5.0 and 9.0, respectively. They suggested that at initial unfolding stage of B L G , due to heating, random coil structures are formed and then a-helical structures unfolded, resulting in the formation of intermolecular P-sheet structures and aggregates. They also reported that heating B L G increased the intensity of the bands at 1684 and 1618 cm"1, which are attributed to intermolecular hydrogen-bonded P-sheet structures (Li-Chan et al., 2001). Other findings included a slight decrease in the number of turns at 1673 cm"1 and loss of cc-helical and p-sheet structures at 1648, 1636, and 1629 cm"1. Lin and Koenig (1976) reported that B S A contains 55% a-helix and 45% disordered structure. They also noted that above 70°C, P-conformation is formed due to intermolecular hydrogen bonding subsequent to intermolecular disulfide exchanges. These authors reported reversible acid and alkaline denaturation of B S A between pH 1.72 and 10.9. The secondary structure of BSA reported by Suttiprasit et al. (1992) (a-helix, 55%; P-sheet, 16%, and unordered 29%) is very similar to the findings at pH 7.0 (60%: 15%: 25%, respectively) in the present study. Lin and Koenig (1976), who studied bovine 150 serum albumin using Raman spectroscopy, found that heat (70°C) denaturation of BSA (5% w/v, in H 2 0 , pH 8.0) caused a decrease in band intensity around 938 cm"1, which is in agreement with the results of the present study. This decrease is attributed to the loss of cc-helical content. In another study by Boye et al. (1995), the effects of pH (3.0-9.0), protein concentration, NaCl, time and temperature on the gelation of whey protein concentrate (WPC) were studied using various techniques including Fourier transform infrared spectroscopy (FTIR) in the 1700-1600 cm"' region to monitor changes in the secondary structure of whey proteins. Two new bands appeared at 1618 and 1684 cm"1 when WPC or B L G were heated to about 67-72°C, which were not observed while heating a-lactalbumin. This suggests interactions involving B L G , but not oc-lactalbumin, are responsible for aggregation of WPC. Comparison of IR spectra of heated WPC at acidic and alkaline pH showed that the thermal stability of protein secondary structure was greater at acidic than at- alkaline pH. They suggested that extent of denaturation was higher at pH 9.0 than at pH 3.0. The comparison of FTIR spectra of B L G , a-lactalbumin, and WPC also indicated that molecular transitions involving only B L G are responsible for aggregation of WPC at pH 7.0 and 9.0 (Boye et al., 1995). Some similar trends were observed between the results of the present study and that of Boye et al. (1995) including increased fi-sheet content (aggregation band) due to heating and the tendency for greater sensitivity to denaturation at pH 9.0. The band around 508 cm"1 is assigned to S-S stretching of cystinyl (cysteine-S-S cysteine) residues. Nonaka et al. (1993) reported a slight decrease in the 508 cm"1 band intensity after heating B L G (pH 6.8) which was time-temperature related. Heating B L G 151 at pH 9.0 decreased intensity of this band. Similarly heating WPI and B S A (pH 7.0) decreased the 508 cm"1 band intensity, which is in agreement with Nonaka et al. (1993). The lowest intensity of this band observed in samples at pH 9.0 may indicate either a decrease in the number of S-S bonds (through reduction or reaction of the cystinyl residues at high pH or heating), or changes in the environment around the S-S bonds leading to reduced vibrational motions and consequently decreased signal of the Raman band at 508 cm"1. The band near 761 cm"1 is assigned to tryptophan. Decrease in the intensity of this band is due to exposure of buried tryptophan residues to the aqueous environment, which occurred at neutral and alkaline pH either with or without heating or in the presence of K C G . However, slight increase in the intensity of this band in heated mixture of KCG:WPI or K C G : B S A , compared to heated samples without K C G , is an indicator of more buried tryptophan residues. The exposed tryptophan plays an important role in hydrophobic interactions (Nonaka et al, 1993). General trends of the effect of pH on the hydrophobicity values of proteins determined by the P R O D A N fluorescent probe method (Chapters lTf and IV) are similar to what was found from the intensity of the band around 761 cm"1; at pH where the intensity was higher, the hydrophobicity (S 0 value) was lower, indicating less exposed or more buried tryptophan residues. However, the effect of heating on the intensity of this band for B L G and B S A was opposite to the hydrophobicity values, while similar results were observed for WPI. In of KCG:protein mixtures, generally, the trends of the effects of pH on the hydrophobicity values of WPI and B L G (both heated and unheated), were similar to the intensity of the band around 761 cm"1, however, opposite results were obtained for BSA. This discrepancy between the 152 results of Raman spectroscopy and P R O D A N fluorescence probe method can be attributed to the fact that different concentrations of proteins may affect the results At low protein concentrations, molecules are unlikely to associate/aggregate even if unfolded, so hydrophobic groups are exposed and this causes increase in S 0. However, with high protein concentrations, aggregation may occur, so the hydrophobic groups come together, causing increase in the intensity of the tryptophan band. The bands around 850 and 830 cm"1 of the protein Raman spectrum are assigned to tyrosine residues which can be used for monitoring the microenvironment around these residues. The intensity ratio of 850/830 cm"1 of >1 indicates that the tyrosine residues are exposed on the protein surface, and may interact with water molecules as a hydrogen acceptor or donor. The intensity ratio between 0.7 and 1.0 suggests the buriedness of the tyrosine residues. Noticeably high (>1) intensity ratio of the 850/830 cm"1 band in the unheated mixture of KCG:proteins at pH 7.0 and 9.0 and most of the samples at pH 9.0 is indicative of exposure of tyrosine residues. The band near 1453 cm"1 is assigned to CH> group. A decrease in the 1453 cm"1 band intensity, which was observed by increasing pH to the alkaline region and heating B L G at pH 9.0 or B S A at neutral/alkaline pH, could be due to hydrophobic interactions of aliphatic residues. A marked increase in the intensity of this band occurred when B L G was heated at pH 7.0. This is in agreement with the study of Howell and Li-Chan (1996) who studied the interactions of lysozyme with whey proteins, using Raman spectroscopy. This increase could be attributed to changes in the environment around aliphatic or hydrocarbon side chains after heating. 153 The band around 1700-1750 cm"1 is assigned to the C=0 stretch of the COOH group of aspartic and glutamic acids. Absence of this band for samples at pH 5.0, 7.0, and 9.0 could be attributed to ionization of the COOH groups to COO". The shift in the wavenumber associated with this band could be the result of changes in the environment around the band. Hydrophobic groups of amino acids, peptides, and proteins exhibit C-H stretching vibrational bands in the 2800-3100 cm"1. Some overall trends based on 2880 cm"1 and 2930 cm"1 (CH stretching) bands include decreased band intensity due to heating, little change in the band intensity due to presence of K C G , and higher band intensity in heated KCG:protein mixtures compared to heated proteins without K C G . Howell et al. (1999) showed that heating (90°C, 30 min, pH 6.8) increased the C-H stretching envelope of B L G . This is in agreement with the result of the present study on B L G where heating increased the 3060 cm"1 band intensity from 0.2 to 0.9. A decrease was observed in the 2880 cm"1 band intensity of the Raman spectrum of B L G at pH 7.0 and 9.0, which is not in agreement with the study by Howell et al. (1999), who reported increases due to heating. Bands around 2874-2897 cm"1 are assigned to C H 3 symmetrical stretching and R3C -H stretching bands of aliphatic amino acids. C-H stretching bands of aromatic amino acids can be found near 3061-3068 cm"1. However, both aromatic and aliphatic amino acids as well as charged amino acids have C - H stretching bands around 2935-2955 cm"1. Decrease in the intensity of C H stretching, which was observed due to heating, indicates changes in the vibrational motions of the hydrophobic side chains of the amino acid residues. The C H stretching of these amino acid side chains was not influenced by the 154 presence of K C G . On the other hand, higher intensity of the C H stretching bands was observed in heated KCG:protein mixtures compared to proteins heated without K C G . Heating proteins without K C G caused a larger decrease in the band intensity than the decrease caused by heating proteins with K C G , suggesting protective effect of K C G on the extent of changes in the band intensity due to heating. Increase in the intensity of the band around 2930 cm"1 relative to the broad water band at 3230 cm"1 suggests increased polarity of the environment around hydrocarbon chains (Li-Chan and Nakai, 1991). Slight shift to higher wavenumbers was observed when water or D 2 0 was added to simple organic solvents such as alcohols (Arteaga, 1994). In other words, both the changes in location and decrease in intensity of the vibrations of the C - H stretching band represent changes in the environment of aliphatic C-H groups which may be related to hydrophobic interactions (Bouraoui et al., 1997). The band intensity at 3060 cm"1 was increased by heating at pH 5.0 and 7.0 for WPI and B L G but decreased at pH 9.0 for B L G and BSA. Adding K C G , however, caused little or no change in the band intensity around 3060 cm"1. Adding K C G followed by heating generally increased the band intensity. Changes in the intensity of the band at 3060 cm"1 indicates changes in the environment of the aromatic C - H groups which may be related to hydrophobic interactions (Arteaga, 1994; Li-Chan, 1996b). The band around 3200 cm"1 has been assigned to O H stretch, which is an indicator of amount of water in the sample. Generally, heating decreased the intensity of this band, except for WPI at pH 9.0. Decrease in the intensity of O H stretch can be attributed to evaporation or immobilization of water molecules due to heat treatment. 155 Some discrepancies between this study and the reported literature may be attributed to different experimental conditions, such as temperature and its duration, which was 80°C for 30 min in the present study, versus 70°C or 90°C for 30, 60, or 90 min in the study of thermally induced changes in whey proteins by Nonaka et al. (1993) or 90°C for 30 min in the study on interactions of lysozyme with whey proteins by Howell and Li-Chan (1996). Another difference may be the manner in which protein solutions were prepared. In some studies, the proteins were dissolved in D2O, while in this study various aqueous buffers were used. The neutral pH that was used in the present study was obtained using buffers adjusted to pH 7.0, while in the literature samples were dissolved in D 2 0 adjusted to apparent pD 6.8. Boye et al. (1996), in an FTIR study of the effects of physicochemical factors on the secondary structure of B L G , indicated a shift in the amide I and II bands of B L G when D 2 0 was used as solvent instead of H 2 G\ Differences may also result when using water versus aqueous buffers due to possible interactions of protein molecules with buffer components or dependence of structural properties on ionic strength. Researchers have also used different concentrations of proteins, which may affect the extent of intermolecular interactions. The approach used for mathematical processing and presentation of the spectral data may contribute to differences in the intensity values reported here and the literature. Some researchers have used the width or the area under a band as a measure of intensity, while in the present study the height of the bands was used. The values would also depend on the specific band selected for internal normalization of spectral intensities. For example, Nonaka et al. (1993) and Bellocq et al. (1972) normalized the spectra to the intensity of the H-C-H deformation mode at 1453-1458 cm"1 and 1450 cm"1, respectively. 156 However, Li-Chan and Qin (1998) and Howell and Li-Chan (1996) used the intensity of the phenylalanine band near 1004-1005 cm"1 for normalization similar to the present study. Despite the above mentioned differences, overall trends in the effects of pH, heating and presence of K C G on protein structure are expected to be similar. However, many factors may affect the trends as well, e.g, the effect of processing conditions of proteins (which may influence the degree of denaturation of proteins), batch to batch variability of proteins or reagents, inconsistencies in the method of dissolving proteins in solvent, storage conditions, etc., which need to be discussed in detail by any researcher in order to enable comparison of specific results with those reported in other studies. Due to the lack of reported studies applying Raman spectroscopy to investigate the interaction of proteins and polysaccharides, and/or the effect of pH, the present study can be considered an initiation of a basic science in this area. The information obtained in this study may help to understand changes in the secondary structure of whey proteins, as well as amino acid side chain vibrations, and protein-water interactions due to the effect of pH, heating and presence of K C G . These data can be useful in cases where a scientist or a food product developer is interested to know what structural changes may arise by adding an ingredient such as K C G to the protein, or by changing the pH of the environment as well as heating. Such structural changes may or may not be in favor of the expected functional characteristic of that protein. 157 CHAPTER VI Conclusions and Recommendations 1 Conclusions In this study, P R O D A N was used as a neutral fluorescent probe for the determination of surface hydrophobicity of whey proteins under heated or unheated conditions, over a wide range of pH, and in presence or absence of K-carrageenan. This study showed that the type of fluorescent probe affects the measured value of surface hydrophobicity of whey proteins. The neutral nature of the PRODAN probe is advantageous over the anionic probes, due to the elimination of contribution of charge effect on the value of S 0. Charge interactions may have interfered with the measurement of protein surface hydrophobicity using anionic probes, particularly at acidic and alkaline pH. Using P R O D A N fluorescent probe, significant effect of pH on the value of S 0 was observed, with the lowest SQ values being found at pH 3.0. Heating caused either increases or decreases in the value of S 0, depending upon type of proteins and the extent of denaturation due to heating. Surface hydrophobicity of proteins was found to be generally highest at pH 9.0, compared to other pH and was-more sensitive to the effects of heating and K C G at pH 9.0 than at pH 7.0. The hydrophobicity of mixtures of K -carrageenan:protein depended also on the ratio of these constituents. Generally, for the unheated KCG:protein mixtures, the higher the ratio, the higher the S 0 value. Heating in the presence of medium ratio of KCGrprotein at pH 7.0 (for K C G : B L G and KCG:BSA) and pH 9.0 (for KCG:BSA) resulted in lower S 0 values than the corresponding protein heated alone. 158 Raman spectroscopy allowed the examination of the effect of several variables including pH, heating, and presence of K-carrageenan on the secondary structure of proteins based on amide I and skeletal stretching modes of the polypeptide backbone, as well as the environment around the amino acid side chains. However, this technique could not be applied for detailed analysis of the coagulated samples at pH 3.0 due to low signal/noise ratio. This low ratio is probably due to background fluorescence of some samples, when excited by the argon laser at 488 nm. Some general observations were made in this study based on the Raman spectroscopy of the whey proteins at various pH, in the absence or presence of heating or K C G . The intensity of the C H and O H stretching bands in the Raman spectra of protein samples were decreased after heating at pH 9.0, which suggest structural changes involving hydrophobic interactions and protein-solvent interactions, respectively. Heating in the presence of K C G , however, had less noticeable effect on the intensity of these bands. Heating (especially at pH 9.0) or presence of K C G (especially at pH 5.0) caused decreases in SS (-508 cm"1) and Trp (-761 cm"1) band intensities, lowered helical content, and increased P-sheet content. Presence of K C G at pH 7.0 and 9.0 increased the helical content of BSA. Decreased helical content and increased P-sheet content were observed after heating B L G at pH 7.0 and 9.0, while these changes were not observed after heating K C G : B L G mixtures at these pH. On the contrary, heating KCG:WPI mixtures at pH 5.0 and 9.0 resulted in large increases in SS and Trp band intensities and helical content, and decreases in the ratio of the tyrosine doublet, which indicate a more buried or hydrophobic environment around the aromatic residues. 159 To summarize, in this thesis, the methodology of Haskard and Li-Chan (1998) for the measurement of protein surface hydrophobicity (S0) using the neutral fluorescent probe P R O D A N was modified to use a methanolic stock solution of the probe. The use of P R O D A N for measurement of S 0 could avoid the problem of influence of the permanent charge effect associated with commonly used anionic fluorescent probes, such as ANS and CPA. Since the measurement of surface hydrophobicity is a common test in food protein related studies, more accurate information can be obtained by establishment of such methodology. The PRODAN probe method could also detect changes in the S 0 in presence of K-carrageenan (KCG) at various KCG:protein ratios. Raman spectroscopy could not only provide information regarding the protein structure and conformation under relevant processing conditions such as pH and heating, but also could detect changes that may occur due to interaction with other molecules, e.g., polysaccharides, which was identified by changes in the intensity as well as shift in the location of the bands due to complexation. The concentration of the proteins used in Raman spectroscopy is also more relevant to what may be found in food systems than the concentrations that are used in fluorescence methods. 2 Recommendations By considering the information obtained in the present study, use of the fluorescent probe P R O D A N for the measurement of the surface hydrophobicity of proteins (S0) is recommended. The measurement of S 0 to explore possible effect of presence of K C G , with or without heating, at various pH is a good example of the 160 applicability of this methodology. The use of this probe for studying interactions of proteins with other molecules as well is also recommended. There are numerous reasons to use Raman spectroscopy for studying food proteins and their interactions with other materials. To mention a few, Raman spectroscopy is non-destructive, requires little or no sample preparation, and can be used for the analysis of solid or liquid samples and can give information regarding molecular structure and conformation. However, Raman spectroscopy has one drawback, fluorescence, which limits its application in materials that fluoresce. This can be overcome by using near infrared Fourier-transform (NIR-FT) Raman technique and by exciting at 1064 nm which allows faster collection of the data, good signal to noise ratio (especially for protein samples studied at pH 3.0), and elimination of the fluorescence problem (Li-Chan et al., 1994). On the other hand, since FT-Raman is less sensitive than visible laser Raman, again poor signal/noise may be obtained. Some new advances in instrumentation include the use of dispersive Raman using NIR diode laser (e.g., 782 nm). This instrument is more sensitive than FT-Raman and may eliminate the fluorescence problem of visible laser. Raman/FT-Raman spectroscopic techniques are a valuable means for studying proteins at high concentrations used often in food processing and can provide information regarding the secondary structure conformation of the polypeptide backbone, microenvironment, polarity and the state of ionization of amino acid side chains. 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Reading, M A . pp. 72. 185 Appendix I Tables 13-20 show the detailed results of the effect of pH, heating, and K -carrageenan on the secondary structure and various bands and bands of the Raman spectrum of the protein samples. 186 Coi Random a Toi O O IT) O -3" C N C N O o i n O C N c o c o o > n N O O C N O i n O o O >—1 C N C O C O i n O O i n •— 1 c o c o o o o o i n c o c o •sheet C O . Total o O m i n n - c o N O o o o i n > o O H O * O i n O O O —I C O C - —< i n o o i n N O c o i n "vl" < n O m o c o N O c o m o o o i n i n i n xj- c o •helix a Toi m o O o o \ C N m — i o i n o N O < n ( N m O C M r ~ m O O O O i n C N < n C N C N o m i n o N O C N C O C N o o i n i n o c o c o Heated Samplesb HWPI3* HWPI5 HWPI7* HWPI9 HKCGWPI3* HKCGWPI5 HKCGWPI7 HKCGWPI9 HBLG3* HBLG5* HBLG7* HBLG9 HKCGBLG3* HKCGBLG5* HKCGBLG7 HKCGBLG9 HBSA3* HBSA5* HBSA7 HBSA9 HKCGBSA3* HKCGBSA5* HKCGBSA7 HKCGBSA9 Coil Random "3 Toi o o o o •sf C O C I ( M O m O O -3" c o c o c o o m i n o C N C O C O i n o o o C O C O C O i n o i n o '— 1 C O C N C O o o i n o C O C O —i C N •sheet C O . "3 : Toi i n m O m c N C N -*t m i n > n m o C N i n < n m o o o o C O < N -*t C O o o o m i n N O i n - t f -m m o N O i n >-i C N o o > n C O C O O i t-helix "3 Toi m i n o i n r o n N m o m o C O * - * • - 1 C N O m m O c o i n C N m O o m C O C N C N O i n O O C N N O N O < n O O i n > n T t o o N O 1 Unheated Samples" 1 WPI3* WPI5 WPI7 WPT9 KCGWPI3* KCGWPI5 KCGWPI7 KCGWPI9 BLG3* BLG5 BLG7 RLG9 KCGBLG3* KCGBLG5 KCGBLG7 KCGBLG9 BSA3* BSA5 BSA7 RSA9 KCGBSA3 KCGBSA5 KCGBSA7 KCGBSA9 e Q. T3 C i n co X c C3 C 1) OO a fc a s> u o W -a u oo c o o -a CO .S < -a « 2 c o o . 1) •a 3 is on >< % T J c o H 3 O 3 _D O 0 0 o o • C O . o •J o <3 • n V o c is 03 C 0 0 3 Q 187 Table 14: Effects of pH, heating, and K-carrageenan on the normalized intensity and Raman shift of bands typical of cc-helical structure of the Raman spectrum of protein samples. Sample3 Wavenumber -938-945 cm 1 (a-helix) Unheated Heated WPI3 1.0 (939)* 0.7 (941)* WPI5 0.5 (941) 0.5 (947) WPI7 0.3 (935) 0.7 (942)* WPI9 0.6 (940) 0.4 (941) KCGWPI3 0.7 (941)* 0.6 (948)* KCGWPI5 0.4 (940) 0.7 (947) KCGWPI7 0.3 (942) 0.4 (944) KCGWPI9 0.4 (937) 1.0 (947) BLG3 0.5 (945)* 0.7 (943)* BLG5 0.9 (948) 0.5 (945)* BLG7 0.5 (939) 1.1 (947)* BLG9 0.5 (940) 0.5 (939) KCGBLG3 0.5 (935)* 0.9 (945)* KCGBLG5 0.5 (945) 0.6 (945)* KCGBLG7 0.2 (948) 0.4 (945) KCGBLG9 0.3 (938) 0.5 (940) BSA3 0.7 (943)* 1.1 (939)* BSA5 0.9 (940) 0.6 (935)* BSA7 0.6 (943) 0.1 (935) BSA9 0.5 (939) 0.2 (938) KCGBSA3 0.9 (942)* 0.5 (946)* KCGBSA5 0.6 (938) 0.6 (938) KCGBSA7 0.4 (943) 0.3 (934) KCGBSA9 0.3 (938) 0.2 (942) Note: Intensity values normalized to the phenylalanine band at 1004+2 cm (Raman shift in parenthesis). a WPI: whey protein isolate, BLG: P-lactoglobulin, BSA: bovine serum albumin, KCG: K-carrageenan, H: heated, 3,5,7, and 9 are pH of the samples. * Due to low signal/noise ratio (<5), the results should be considered with caution. 188 Table 15: Effects of heating, pH, and K-carrageenan on the normalized intensity and Raman shift of bands typical of SS stretching band of the Raman spectrum of the protein samples. Sample" Wavenumber -508 cm'1 Unheated Heated WPI3 0.6(515)* 1.0 (509)* WPI5 0.6 (507) 0.7 (508) WPI7 0.6 (514) 0.6 (505)* WPI9 0.4 (508) 0.3 (509) KCGWPI3 0.6 (511)* 1.1 (509)* KCGWPI5 0.3 (511) 0.8 (505) KCGWPI7 0.6(513) 0.7 (507) KCGWPI9 0.3 (512) 1.0 (508) BLG3 0.8(512)* 0.6 (505)* BLG5 1.1 (512) 0.7 (508)* BLG7 0.4 (512) 0.6 (512)* BLG9 0.3 (512) 0.2 (506) KCGBLG3 0.2 (513)* 0.8(510)* KCGBLG5 0.7 (509) 0.8 (506)* KCGBLG7 0.3 (513) 0.2 (515) KCGBLG9 0.3 (510) 0.3 (507) BSA3 0.5 (510)* 0.7 (508)* BSA5 0.9 (507) 0.7 (509)* BSA7 0.4 (510) 0.3 (511) BSA9 0.4 (512) 0.2 (515) KCGBSA3 0.5 (508) 0.4 (508)* KCGBSA5 0.4 (513) 0.8(513)* KCGBSA7 0.3 (507) 0.3 (509) KCGBSA9 0.3 (511) I 0.3 (509) Note: Intensity values normalized to the phenylalanine band at 1004+2 cm (Raman shift in parenthesis). a WPI: whey protein isolate, BLG: P-lactoglobulin, BSA: bovine serum albumin, KCG: K-carrageenan, H: heated, 3,5, 7, and 9 are pH of the samples. * Due to low signal/noise ratio (<5), the results should be considered with caution. 189 O "a <J 'cL >-> •*-* CO C es 03 E T3 C OS S co "S3 o X ! C O "os £ « o c «M o X E 3 B £ O V c/3 u I o v o r o o c o * * * * * C O O N V O o o o \ o o C N v o i n m i n i n m m C O C O C O C O C O r o r o ' — ' — ^—' ' — • *—' — • •— ' v o C O O s C O i n o o o > C N o o o o d d d * - - * * * . - * * * -i n o s o T t C N O m o o o o C O V O T f C N o o \ T t V O r o m o o i n T t C O T f T T m V O v o C O C O i n T i - T t T i - m T T v o T i - i n Tt- T i - T i - T i - T i - T l -c o C O C O C O C O C O C O 3^ C O C O r o c o r o C O C O r o r o C O r o r o r o r o r o c o •• * v ' v ' V ' *.—• V—J 1 — ' , — ' — ' —^ — ' — ' ^—' —•• v —^ •—^ *—' • — ' ^—' ^— * — ' — • o s o o I /O K O o o • T t o o C O T t o s C N r o wo V O C N C N V O p r o T j -o o o o d © o O o O o o O O O d d d d d — 1 d d T i - T t o T t ( N T t K O C O O S -t C O C M o s o o 0 0 T t o o V O TI- T i - f~ o o r o C O C O c o T t T t C O C O C N C O C O C O r o C O C N T l - r o T i - r o C N C O r o r o C O r o C O C O C O C O C O C O C O C O C O C O C O C O C O C O r o r o r o r o r o r o C O r o C O . — i 1—4 —-< ,—i .—1 1—( 1—1 — H 1—1 ' — i i—i . — i — i <—1 , •• V ' ^ -s ' *-—' •>—- N ' ^—• *— 1 — ' s — ' v — ' ' 1 — ' ^—' '~ ' ^ — ' — ' v — • ^—' ~— '*—y — *— V O r - v o T t OO o o T t o o o o r ~ T t O S m C O C O OS o o C N m a s r o C O o o o o d o d o d o o o o o o d d d d d d d d s CI > a a a -c s w> S 2 S • * J O "2 «« C 3 o "53D C U ^ <u •S E « 2 ( U c o ^ tJ I C M C w c i n * ^ Q* 2 2 E u 1—1 IS J-. «> '1 3 C > a. E c o o i n O o o v o v o i n v o i n c o r o r o r o r o v o c o i n u o i n d d d S di r-- ' c o ^ C-T i n " c o * o ? r o " r o T l - T l - T l - T l - T l - T t - V O T l -c o c o r o r o r o r o r o r o O S r ~ d d T ? ^ r o c o c o r o vo oo d d m v o m m i n ci d> c5 di d> ' r o * i n C N ~ c o r o r o r o c o r o r o r o c o c o c o T t v o T J - m T ) -d> d d> ci ci c N ^ i n — i ^  - i m ooinvovoooososoq d d d d d d d d • H C S - i V O C N C N — i ^ O v O ^ l O ^ O v O l O ^ O s o o v o m r ^ v o v o i n d d d d d d d d v> t> os PH CM PH P I ^ & £ £ P H C H P H C H U C J O U V O C O O O S u o v o s o m r o c o r o r o v o O T f i n d ^ d d . . . * O v o CO VO v o v o r o r o r o i n c o T J -d d d o o c o i n i n t - - T t o o T f Tt" T f C O T t T f T f c o c o r o r o r o r o r o r o r - ~ o s T t m v o s o r o T t d d d d d d d d O c o i n i n o o c o — < C N r o c o c o r o c N r o c o c o c o c o c o c o c o c o r o r o i n O - r t i n v o i n T t T t d d d d d d d d * * * r o - ^ i n - ^ i n c o ^ - t c o V O V O V O V O V O V O V O V O r - r ~ c o > n o o o s T t i n d d d d d d d d v O V O V O V O v o v o v O v o r ~ - r ~ - r - r - - r - r ^ t - - r - -o s — i r - o o v q r - ^ v q v o d d d d d d d d O O O O J J H-l J w v i t - o s M M M M T t m ro v o d o o c N > n v o r o r o C N T t d d O N T t w o C N o o T t T t T t T t T t UO T t m ^ r o c o c o c o c o c o r o t v o T t c o v o C N c o < d d d d d d C N —i T t T t c o r - r ~ — ' c o r o r o T t T t r o C N S t r o c o c o r o c o c o c o i ' i—i ^ ^ . — i i n v o r - c o i n r o r O T t d d d d d d d d o s i n i n o o r o o v i n - - ^ i n v o v o i n v o i n v o v o r - v o — i c N m o o c N r o d d d d d d d d r - c N c N ^ H - ^ m ' - < r N i n o s c o c o i n c o c N v q d d d d d d d d r o m t - o s < < < < vi in c« c» pa CQ pa oa papapapal^beiwbei o -a c oo •±3 u o 55 190 Table 17: Effects of heating, pH, and K-carrageenan on the normalized intensity and Raman shift of bands typical of tyrosine doublet ratio (860/825 cm'1) of the Raman spectrum of protein samples. Sample3 Wavenumber -860/825 cm 1 Unheated Heated WPI3 0.8 (858/825)* 0.7 (862/834)* WPI5 1.1 (863/826) 1.3 (856/834) WPI7 1.5 (857/834) 1.0 (858/836)* WPI9 1.1 (854/831) 0.9 (856/836) KCGWPI3 0.9 (860/820)* 1.3 (858/834)* KCGWPI5 1.1 (859/840) 0.5 (856/838) KCGWPI7 1.3 (857/837) 1.2(857/834) KCGWPI9 1.5 (861/837) 0.5 (861/828) BLG3 1.1 (854/833)* 0.8 (862/828)* BLG5 0.8 (864/833) 1.1 (860/822)* BLG7 1.0 (859/832) 1.1 (854/832)* BLG9 0.7 (860/834) 1.4 (855/831) KCGBLG3 0.6 (860/835)* 1.5 (862/835)* KCGBLG5 0.7 (862/832) 0.7 (857/828)* KCGBLG7 1.3 (859/833) 0.4(860/831) KCGBLG9 1.7 (859/833 ) 0.9 (858/832) BSA3 1.0 (854/833)* 1.0 (848/824)* BSA5 1.1 (853/826) 0.9 (860/826)* BSA7 0.9 (856/830) 1.0 (856/828) BSA9 1.5 (852/830) 1.0 (860/831) KCGBSA3 1.1 (853/830) 0.7 (860/832)* KCGBSA5 0.8 (857/825) 1.0 (855/832)* KCGBSA7 1.5 (853/829) 0.8 (853/830) KCGBSA9 1:7 (858/831) 1.4 (853/831) Note: Intensity values normalized to the phenylalanine band at 1004+2 cm (Raman shift in parenthesis). a WPI: whey protein isolate, BLG: P-lactoglobulin, BSA: bovine serum albumin, KCG: K-carrageenan, H: heated, 3, 5,7, and 9 are pH of the samples. * Due to low signal/noise ratio (<5), the results should be considered with caution. 191 Table 18: Effects of pH, heating, and K-carrageenan on the normalized intensity and Raman shift of the C H 2 bending band of the Raman spectrum of protein samples. Sample3 Wavenumber ~ 1453 cm 1 Unheated Heated WPI3 1.0(1453)* 0.8 (1454)* WPI5 1.1 (1459) 1.0(1452) WPI7 0.8(1453) 0.5 (1453)* WPI9 0.7(1451) 0.8 (1450) KCGWPI3 0.9 (1455)* 0.9 (1453)* KCGWPI5 0.7 (1451) 1.0(1455) KCGWPI7 0.7 (1455) 1.1 (1450) KCGWPI9 0.7 (1457) 0.9 (1455) BLG3 0.6 (1453)* 0.7 (1451)* BLG5 0.8 (1453) 0.7 (1450)* BLG7 0.6 (1452) 1.7 (1454)* BLG9 0.7 (1455) 0.5 (1453) KCGBLG3 0.7 (1458)* 0.9 (1455)* KCGBLG5 0.8 (1454) 0.5 (1453)* KCGBLG7 0.6(1450) 0.5 (1453) KCGBLG9 0.7 (1452) 0.7 (1456) BSA3 0.4 (1452)* • 1.0(1454)* BSA5 0.7 (1452) 0.7 (1455)* BSA7 0.6(1453) 0.3 (1453) BSA9 0.5 (1450) 0.3 (1454) KCGBSA3 0.8 (1459) 0.6(1453)* KCGBSA5 0.4 (1455) 0.8 (1458)* KCGBSA7 0.4(1450) 0.3 (1450) KCGBSA9 0.7 (1452) 0.4 (1457) Note: Intensity values normalized to the phenylalanine band at 1004+2 cm ~ (Raman shift in parenthesis). a WPI: whey protein isolate, BLG: P-lactoglobulin, BSA: bovine serum albumin, KCG: K-carrageenan, H: heated, 3, 5, 7, and 9 are pH of the samples. * Due to low signal/noise ratio (<5), the results should be considered with caution. 192 o T J £ u o so o CO 3 C o > E o o c o O N C N 1 U CU •1 3 £ S3 E u o 0 0 0 0 C N i S~ O) '1 3 C > E co * * o o i n o a \ o i o o i o s o s o s o s o s o N O s o o o o o o o o c o c o c o t o c o c o c o s t r - n - t v o i n n i i o o o o ' o o o c UO SO o o co cn C N u o —i o o o o — i r ~ s o s o i n s o r - -o o o o o o cn cn cn cn cn cn i n o o C N i n C N cn C N o o o o o o r— i n i n s o c o c o c o c o c o c o c o c o c o OS OS OS OS OS OS OS C N C N C N C N C N C N C N i o — < - ^ c N s q — < - - < J D CN —< oi —< CN CN c O S C N S O C O O C O O S O S C O C O C O X ) - T 1 - - ^ ) - C O C O O s O s O s O S O S O s O s O s C N C N C N C N C N C N C N C N O C O G I s o C O O C O C N C N C N C N C N C N CO t i n "^}" O s O o o r - ~ o o r- r - ~ o o o o o o o o o o o o o o C N C N C N C N C N C N * — i t~~ —I O S O —I . O 6 - 6 ^ H c T J - — i i n o o c o s o - * o o o o o o o o o o o o o o o o C N C N C N C N C N C N C N C N O s o - ^ c N - — i c o — i O co m os Pi PH PH >• > >• P L , P H P - C P - I ( J ! J C J C J * * * * — ' O C O O O SO C N C N s o s o r - r - ^ i y o s o s o o o o o o o o o C O C O C O C O C O C O C O C O C N C N O N — < O O r - c s o S O ^ S O S O S O S O S O S O o o o o o o o o C O C O C O C O C O C O C O C O o o o o o o o o * " * CO — ' C N — i w o c N C N O O O O O O O O U O S O T f c O r f M - s O O s • ^ l - s o s o s o i o i o s o u o O O O O O O O O C O C O C O C O C O C O C O C O U O C O C N C O C O C O - - < C N O O O O O O O O * * — • i n O O O O C O C O T f N O C O C O C O C O T f C O C O C O OS OS OS OS OS OS OS OS C N C N C N C N C N C N C N C N — i v o s o —i —* o o ^ , _ ; ^ O - - < C N — < — < c N M - - * — • o r ^ c o T t C O C O C O T J - C O C O C O C O O s O s O s O S O S O s O s O s C N C N C N C N C N C N C N C N —< U-) O O < N O O C N i r - J c N c N ^ C N C N C N c N - * c o m — i c n h T t o o r ^ r ~ r ^ o o r ^ r ~ ~ o o o o o o o o o o o o o o o o C N C N C N C N C N C N C N C N ^ O O M ^ H O O O O o o ' o o — ! o i n N O —( O S s o i n i n O N r - r - r - r - r - r - r - c -o o o o o o o o o o o o o o o o C N C N C N C N C N C N C N C N C O C N C N C N — I O O C N o o o o C O V ) O S oooo J H J pJ pa pa pa PQ oooo u o o u p a p a p a m W W W W s o - d - r - m i n C — — i / i s o s o s o s o i o s o s o s o o o o o o o o o CO CO CO CO CO CO CO CO C O C N C O C O - 3 - C O C N C O o o o o o o o o o o - a - " * C N C N o o r -- s t c o c o c o c o c o c o c o OS OS OS OS OS OS OS OS C N C N C N C N C N C N C N C N ^ - i G S C N C N - t f ^ l - C N l O —; o —; —; o —; —< ^  s o o o o s r~~ o o o '— 1 C O C O C N C N C O C O C O C O O s O s O s O S O S O s O s O s C N C N C N C N C N C N C N C N o o o o o s r ^ o o i n o o o o * * * O T f o o m - ^ O ^ f O r - s o t - ^ t ~ - - t - ~ o o r ^ - o o o o o o o o o o o o o o o o o o C N C N C N C N C N C N C N C N r ^ - - < 3 - i / - > < o c o r - - s o o o o o o o o o o o o o i n c o r ^ - c o o c o c o t ^ r ^ r ^ c - ~ r - ~ o o r ^ t ^ o o o o o o o o o o o o o o o o C N C N C N C N C N C N C N C N O s O s O o o o s o q o o o s o o — < o o o o o C O V I t~ O S < < < < C O V I O O S < < < •< co co co co pa pa PQ pa o e> e> o p a p a p a c a W W W W co .£; o cx CO ca O S T J c ca n o T J CO co c ca c • eo K ca CO -g c co fc; ca o ca . . .S U <~ w 12 c -a c E ca 3 W VVJ 6 a ° & s | 3 ^ S CO CO O CO C N .S -+i > s 8 8 - < £ " 2 . 3 1 ^ ^ O O G O •S o 5 g 0 0 g 3^ o «o >> ^2 S c T -S " ffl o o - *c • J O B "S3 s N O 2 e C C co ca o s c C g 0 0 CO co ca > s > « o ^ * o S .t! fr1 co -° g ^ P 55 o 193 Table 20: Effects of pH, heating, and K-carrageenan on the normalized intensity and Raman shift of the O H band of the Raman spectrum of protein samples. Sample3 Wavenumber ~ 3220 cm'1 Unheated Heated WPI3 1.6 (3225)* 1.0 (3211)* WPI5 2.8 (3233) 1.6 (3230) WPI7 2.0 (3215) 0.9 (3207)* WPI9 2.0 (3222) 2.1 (3211) KCGWPI3 1.4 (3226)* 1.2 (3209)* KCGWPI5 2.0 (3222) 1.5 (3209) KCGWPI7 1.8 (3213) 1.6 (3214) KCGWPI9 2.0 (3217) 1.9 (3215) BLG3 1.7(3224)* 0.9 (3222)* BLG5 1.5 (3203) 0.9 (3225)* BLG7 1.9 (3218) 0.8 (3212)* BLG9 1.9 (3219) 0.4 (3220) KCGBLG3 1.3 (3231)* 1.2 (3225)* KCGBLG5 2.0 (3227) 1.4 (3214)* KCGBLG7 1.9 (3222) 0.9 (3221) KCGBLG9 2.0 (3220) 1.7 (3220) BSA3 1.2 (3218)* 0.6 (3209)* BSA5 1.4 (3210) 0.6 (3224)* BSA7 1.4 (3210) 0.8 (3219) BSA9 1.2 (3210) 0.9 (3215) KCGBSA3 1.1 (3236) 0.8 (3241)* KCGBSA5 1.2 (3234) 1.0 (3218)* KCGBSA7 1.3 (3216) 1.0 (3233) KCGBSA9 1.5 (3226) 1.2 (3224) Note: Intensity values normalized to the phenylalanine band at 1004+2 cm (Raman shift in parenthesis). a WPI: whey protein isolate, BLG: P-lactoglobulin, BSA: bovine serum albumin, KCG: K-carrageenan, H: heated, 3, 5, 7, and 9 are pH of the samples. * Due to low signal/noise ratio (<5), the results should be considered with caution. 194 Appendix II. Colorimetric determination of carrageenans Soedjak (1994) developed a simple, highly sensitive, fast and reproducible technique for quantifying carrageenans and other anionic hydrocolloids. This method uses methylene blue (a cationic dye) and is based on the formation of a soluble complex when the absorption maxima shifts from 610 and 664 nm to 559 nm. The hydrocolloid concentration is determined by the absorbance of the complex at 559 nm. Materials and Methods A solution (0.41 mM or 0.18 mg/mL in H2O) of methylene blue (dye content of 86%, Difco) was prepared. K-Carrageenan stock solution was diluted to 0.02% with H2O. 0-10 mL of 0.02% K-carrageenan was transferred to 100 mL volumetric flasks. Then 75 mL water and 10 mL methylene blue solution was added. Enough water was added to bring up the content of the flask to 100 mL. The solutions were mixed and the absorbance was measured at 559 nm. A standard curve can be obtained by plotting the absorbance at 559 nm versus the corresponding hydrocolloid concentration (w/v). Methylene blue solution turns to purple (from blue) immediately after addition of carrageenan and consequently increases in the absorption maxima at 559 nm. A typical slope and standard error for K-carrageenan is 0.27 ± 0.01, respectively (Soedjak, 1994)); Values of 0.32 + 0.02 was achieved in the present study. 195 A p p e n d i x III. S D S - P A G E p r o f i l e o f w h e y p r o t e i n s a m p l e s M W (DALTONS) B S A B L G A-LACTALBUMIN 4 5 2 0 5 , 5 0 0 1 1 6 , 0 0 0 9 7 , 4 0 0 8 4 , 0 0 0 6 6 , 0 0 0 5 5 , 0 0 0 4 5 , 0 0 0 3 6 , 0 0 0 2 9 , 0 0 0 r~ . 2 4 , 0 0 0 2 0 , 1 0 0 14 ,200 6 ,500 8 Figure 1: SDS-PAGE profile of whey protein samples Lanes 1,8: Standard molecular weight marker Lane 2: B L G (A and B) Lane 3: B S A Lanes 4,5: WPI Lanes 6,7: Whey powder 196 

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