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Studies on structural stability of chicken’s egg yolk immunoglobulin (IgY): Chansarkar, Namrata Labhe 1998

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Studies on Structural Stability of Chicken's Egg Yolk Immunoglobulin (IgY) by  Namrata Labhe Chansarkar B.Sc, Nagpur University (India), 1989 M.Sc, Nagpur University (India), 1991 B.Ed., Nagpur University (India), 1992 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES ( Department of Food Science) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA July 1998 © Namrata Chansarkar, 1998  in presenting this degree at the  thesis in partial fulfilment of  University of  the  requirements  for  an advanced  British Columbia, I agree that the Library shall make it  freely available for reference and study. 1 further agree that permission for extensive copying of  this  department  or  publication  thesis for by  his  or  scholarly purposes may be granted by the head of her  representatives.  Department of  FOO O  SeTE-KJCE  The University of British Columbia Vancouver, Canada  DE-6 -(2/88)  Oce  is  understood  that  copying  or  of this thesis for financial gain shall not be allowed without my written  permission.  Date  It  my  I  ABSTRACT Immunoglobulins play a critical role in therapy, diagnostic assays and purification of important compounds. Hen egg yolk immunoglobulins, or IgY, have been studied intensively by researchers. Many techniques for isolation of IgY have been established. IgY has many advantages over the mammalian antibodies. It does not bind with mammalian complements or rheumatoid factor. Low cost and convenient production make it an attractive antibody for human use. To obtain specific antibodies, elution protocols often expose IgY to harsh conditions such as low pH, which may lead to denaturation. This research aims to study the effects of acidic pH, temperature, the denaturant guanidine-hydrochloride on IgY and the reversibility of those effects. Structural properties were studied by ultraviolet spectroscopy, intrinsic fluorescence, extrinsic fluorescence with hydrophobic probes, and enzyme linked immunosorbent assay. IgY was found to be sensitive to acidic pH (2.8) with irreversible changes when treated at 37°C. IgY denatured with > 3 M guanidine hydrochloride concentration showed a significant drop in antigen binding activity determined by enzyme linked immunosorbent assay suggesting that the changes resulting in inactivation  are only partly reversible. These results should be considered in  establishing processes for the isolation or utilization of IgY antibodies. For practical use of IgY in food or therapeutic formulations it is important that it is stable to processing conditions. Stability of IgY to freezing and freeze drying was investigated by monitoring its solubility, intrinsic fluorescence, extrinsic fluorescence and enzyme linked immunosorbent assay activity. IgY was found to be stable to freezing  ii  at -8°C but showed a significant drop in solubility and enzyme linked immunosorbent assay activity after freeze drying. Immunoaffinity chromatography has been used in the past to isolate IgY and other antigens. Structural conformation of eluted IgY was investigated in the present study. Surface hydrophobicity of IgY antibodies eluted at pH 2.8 was significantly higher than the crude IgY isolate before affinity purification. Trehalose in the eluting medium had a protective effect on IgY as it decreased surface hydrophobicity of pH eluted IgY.  in  TABLE OF CONTENTS ABSTRACT  ii  TABLE OF CONTENTS  iv  List of Tables  vii  List of Figures  viii  List of Abbreviations  x  Acknowledgement  xii  Chapter I Chapter II  INTRODUCTION LITERATURE REVIEW  1 4  2.1 2.2 2.3  4 4 7 9 15 15 15 16 16 18 18 19 21 22  2.4  2.5  Chapter III  General overview of immunoglobulin Hen's egg yolk antibodies (IgY) Comparison of IgY to IgG 2.3.1 General structure & composition 2.3.2 Carbohydrate composition 2.3.3 Isoelectric point 2.3.4 Disulfide bonds and reductive dissociation 2.3.5 Aggregation 2.3.6 Precipitin reaction Stability of immunoglobulin 2.4.1 Stability to acidic pH 2.4.2 Stability to GuHCl denaturation 2.4.3 Freezing, freeze drying and IgY 2.4.4 Structural conformation of eluted IgY Techniques used to characterize structural and functional properties of proteins 2.5.1 UV absorption spectroscopy 2.5.2 Intrinsic fluorescence 2.5.3 Fluorescence probes 2.5.4 Raman spectroscopy 2.5.5 Solubility • 2.5.6 Enzyme-linked immunosorbent assay (ELISA)....  23 23 24 25 27 27 28  MATERIAL AND METHODS  29  3.1 3.2 3.3 3.4  29 29 30 31  Materials Buffers Isolation of IgY Protein concentration determination  iv  3.5  3.6  Chapter IV  Treatments 31 3.5.1 pH and temperature 31 3.5.2 GuHCl concentration 32 3.5.3 Freezing and freeze drying at low and high salt concentration 32 3.5.4 Structural conformation of IgY eluted from an immunoaffinity column 33 3.5.5 Effect of dialysis on control IgY 34 Analysis 34 3.6.1 UV-fourth derivative spectroscopy 34 3.6.2 Intrinsic fluorescence 35 3.6.3 Light scattering 35 3.6.4 Extrinsic fluorescence probes for surface hydrophobicity 36 3.6.4.1 ANS (1,8-anilinonaphthalene sulfonic acid ) probe hydrophobicity 36 3.6.4.2 CPA (cis-parinaric acid ) probe Hydrophobicity 37 3.6.5 Enzyme linked immunosorbent assay (ELISA) 37 3.6.6 Raman spectroscopy 38 3.6.7 SDS polyacrylamide gel electrophoresis (SDS PAGE) 39 3.6.8 Solubility 39 3.6.9 Statistical analysis 39  RESULTS AND DISCUSSION  41  4.1  41 41 41 45 49 53 56 56 56 59 64 68 70 72 72  4.2  4.3  Effect of pH and temperature 4.1.1 Intrinsic fluorescence (a) Relative fluorescence intensity (b) Shift in emission maximum 4.1.2 Extrinsic fluorescence 4.1.3 ELISA for lactoferrin binding activity of IgY Effect of guanidine-HCl concentration 4.2.1 Intrinsic fluorescence (a) Relative fluorescence intensity (b) Shift in emission maximum 4.2.2 Extrinsic fluorescence 4.2.3 UV fourth derivative spectroscopy 4.2.4 ELISA for lactoferrin-binding activity of IgY Effect of freezing and freeze drying 4.3.1 Solubility 4.3.2 CPA hydrophobicity, intrinsic fluorescence light scattering 4.3.3 ELISA for lactoferrin binding activity of IgY 4.3.4 Raman spectroscopy  V  74 75 77  4.4  Chapter V  Structural conformation of specific (eluted) antibodies 4.4.1 Extrinsic fluorescence of eluted IgY 4.4.2 ELISA for lactoferrin binding activity of eluted IgY  GENERAL DISCUSSION AND CONCLUSIONS  BIBLIOGRAPHY  78 79 82 84 88  APPENDIX I  A typical SDS PAGE profile of crude IgY isolate  97  APPENDIX II  ANOVA analysis for data in Figure 5-23 and Table 5..  98  vi  List of Tables Table 1: Biological functions of Ig classes  6  Table 2: Amino acid composition of IgY  14  Table 3 : Effect of pH on X  55  max  of IgY with ANS probe  Table 4: Effect of GuHCl concentration on X  with ANS probe  67  Table 5: Effect of GuHCl concentration on UV-fourth derivative parameters of IgY  68  Table 6: Secondary structure of frozen-thawed and 5°C IgY  77  max  vii  List of Figures Figure 1: The five functional classes of mammalian immunoglobulin  5  Figure 2: Comparison of the structure of IgG to IgY  12  Figure 3: Structure of IgY (a) and IgG (b)  13  Figure 4: Structure of ANS (a) and CPA (b) fluorescent probes  26  Figure 5a: Effect of pH (7, 4, 2.8) and temperature (x=5, y~25, z=37°C) on relative fluorescence intensity of IgY excited at 280 nm  43  Figure 5b: Effect of pH (7, 4, 2.8) and temperature (x=5, y~25, z=37°C) on relative fluorescence intensity of IgY excited at 297 nm  44  Figure 6a: Effect of pH (7, 4, 2.8) and temperature (x=5, y~25, z=37°C) on wavelength of maximum emission (X ) of IgY excited at280nm  47  Figure 6b: Effect of pH (7, 4, 2.8) and temperature (x=5, y~25, z=37°C) on wavelength of maximum emission (k ) of IgY excited at297nm  48  Figure 7: Effect of pH (7, 4, 2.8) and temperature (x=5, y~25, z=37°C) on ANS hydrophobicity of IgY  51  Figure 8: Effect of pH (7, 4, 2.8) and temperature (x=5, y~25, z=37°C) on CPA hydrophobicity of IgY  52  Figure 9: Effect of pH (7, 4, 2.8) and temperature (x=5, y~25, z=37°C) on ELISA of IgY  55  Figure 10a: Effect of GuHCl concentration on relative fluorescence intensity of IgY excited at 280 nm  57  Figure 10b: Effect of GuHCl concentration on relative fluorescence intensity of IgY excited at 297 nm  58  Figure 1 la: Effect of GuHCl concentration on wavelength of maximum emission (k ) of IgY excited at 280 nm  61  Figure 1 lb: Effect of GuHCl concentration on wavelength of maximum emission (X ) of IgY excited at 297 nm  62  max  max  max  max  viii  Figure 12: Emission spectra of control and 6M GuHCl treated IgY  63  Figure 13: Effect of GuHCl concentration on ANS hydrophobicity of IgY  65  Figure 14: Effect of GuHCl concentration on CPA hydrophobicity of IgY  66  Figure 15: UV fourth derivative spectra of control ( treated (-—) IgY  69  ) and 6M GuHCl  Figure 16: Effect of GuHCl concentration on ELISA absorbance values of IgY.... 71 Figure 17: Solubility of frozen-thawed and freeze dried IgY  73  Figure 18: CPA hydrophobicity of frozen-thawed and freeze dried IgY  74  Figure 19: Intrinsic fluorescence and light scattering of frozen-thawed and freeze dried IgY  74  Figure 20: ELISA absorbance values of frozen and freeze dried IgY  76  Figure 21: Typical elution profile of anti-lactoferrin IgY with glycine HC1 pH 2.8 buffer  78  Figure 22: ANS and CPA hydrophobicity of eluted IgY  81  Figure 23: ELISA absorbance values of eluted IgY  83  IX  List of Abbreviations ANS C CD °C Cy Ce cm CPA CPB CPBS Cv ELISA F Fab Fc FD g 8  GuHCl H hr Ig IgG IgY kD L 1 M mg min mL mM mm mT mV MW |iL nm PB PBS RF  l-anilinonaphthalene-8-sulfonate constant region of immunoglobulin chain circular dichroism degree C e l s i u s constant region of IgG constant region of IgE centimeter cis parinaric acid citrate phosphate buffer citrate phosphate buffer with saline constant region of IgY enzyme linked immunosorbent assay frozen antigen binding fragment of immunoglobulins crystallizable fragment of immunoglobulins freeze dried gram gravity guanidine hydrochloride heavy chains of immunoglobulins hour immunoglobulin mammalian immunoglobulin chicken egg yolk immunoglobulin kilodalton light chains of immunoglobulins liter molar (moles/liter) milligram minute milliliter millimolar millimeter millitorr millivolt molecular weight microliter nanometer phosphate buffer phosphate buffer saline relative fluorescence  X  RFI Rg SDS sec SH SS UV V ^-max  relativefluorescenceintensity radius of gyration sodium dodecyl sulphate second sulfhydryl disulfide ultraviolet variable region of immunoglobulin chain wavelength of maximum emission  XI  Acknowledgement This research was conducted within the department of Food Science, at The University of British Columbia. I am indebted to Dr. J. Vanderstoep for allowing me to use the facilities in the department. The words are indeed inadequate to express my heartfelt gratitude towards my supervisor Dr. Eunice Li-Chan, I sincerely thank her for excellent supervision and financial support. I also thank Dr. T . Durance, Dr. C . Seaman, Dr. B . Skura, and Dr. A . Yousif for being the members of my research and examining committee. Their helpful advice and comments on my research program and thesis are gratefully acknowledged. I wish to thank Dr. R. Chaudhary for encouragement and cooperation. I am grateful to Angela for all her help. The assistance of Eddy, Ling, Nooshin, Yasmina and Jack is gratefully acknowledged. I feel greatly indebted to Emmanuel for invaluable guidance throughout my study. I wish to thank my parents in-law for their encouragement. I also wish to thank my parents and my brother Vikram for constant support throughout my study. M y expression of gratitude would be incomplete without a word for my husband Dinesh and my  daughter Mitali. I sincerely thank them for their patience, understanding, and  immense support.  xii  Chapter I INTRODUCTION The importance of egg yolk as a convenient and inexpensive source of antibodies is well recognized. There has been extensive research on isolation of IgY from hen's egg yolk; however less is known about its structural conformation. For practical use of IgY antibodies as food supplements or as immunochemical reagents, stability of IgY under food processing and preservation conditions must be determined. Proteins generally present stability problems; degradation and loss of activity may occur during' storage. Hence many protein products are freeze dried to provide adequate shelf life stability (Pikal et al., 1991). Freeze drying of proteins (for example somatotropin, insulin, tetanus toxoid, monoclonal IgM) often leads to aggregation, which may alter the functionality of the protein thereby decreasing the bioactivity and solubility (Costantino et al., 1995; Draber et al., 1995). It was reported that freezing and freeze drying did not affect activity of IgY unless freeze-thaw cycles were repeated several times (Shimizu et al., 1988) but there is no evidence to support these statements as no data was shown. To obtain specific antibodies, elution with acidic pH is most commonly used for dissociation of antigen-antibody complexes in immunoaffinity chromatography (Kummer and Li-Chan,  1998). However, such harsh conditions may result in  denaturation and inactivation of antibody activity. Otani et al. (1991) isolated IgY by immunoaffinity chromatography eluting IgY with 0.5 M glycine HC1 at pH 2.3, followed by 4 M GuHCl at pH 7 in a cold room. They found that 4 M GuHCl was 4 times more efficient than glycine HC1 buffer but these conclusions were drawn on the basis of protein concentration and activity of the recovered IgY was not tested. IgY was reported to be  1  stable in a pH range of 4 to 9 at 37 or 40°C. It was also found that IgY was heat sensitive at temperatures above 65°C (Hatta et al, 1993; Shimizu et al, 1992, 1988; Otani et al, 1991). Stability of IgY at ambient temperature or 5°C is important because most of the processing would be carried out at these temperatures. Effect of pH on IgY at room temperature or 5°C has not been investigated. Although the stability of IgY and IgG has been studied by Shimizu et al. (1992), these previous studies on stability of IgY were done at 37°C and knowledge of this aspect of immunoglobulins under other conditions is still limited. The present research will evaluate the stability of IgY to acidic conditions at 5°C, 37°C, ambient temperature and to denaturants like GuHCl as well as the reversibility of these changes back to the original state. Information on structural conformation of IgY exposed to acidic pH or GuHCl would be helpful in further formulation of elution protocols. For practical use of IgY as a food supplement, it is important that it is stable under processing conditions. It is known that IgY is not stable at temperatures above 65°C, thus it cannot be dried and preserved by drying methods which require high temperature. Hence it is important to investigate stability of IgY to freezing and freeze drying. Spectroscopic methods including UV fourth derivative spectroscopy, intrinsic fluorescence and extrinsic fluorescence (ANS and CPA probes) were used to study structural conformation, while antigen binding activity was tested by ELISA. The specific objectives of this research are as follows: Objective 1: To study the effect of (la) acidic pH and temperature, and (lb) GuHCl -  treatment on denaturation and renaturation of IgY.  2  Objective 2: To investigate the effect of freezing and freeze drying at low and high salt concentrations on the structure of IgY. Objective 3: To study structural conformation of specific antibodies obtained by immunoaffinity chromatography.  3  Chapter II LITERATURE REVIEW 2.1 General overview of immunoglobulin Antibodies are now recognized as probably the most complex and polyfunctional group of proteins. They are synthesized by the body on exposure to foreign agents (antigens) such as bacteria, viruses, etc. All antibodies are constructed in the same way from four polypeptide chains, and for all such proteins a generic term immunoglobulin (Ig) is used. The antibody molecule itself has two separable functions: one is to bind to antigens, which is carried out by the Fab (fragment antigen binding) portion of the molecule, and the other is to engage effector mechanisms which is carried out by the Fc (fragment crystallizable) portion. Although all immunoglobulins are constructed the same way with four polypeptide chains, five classes of antibodies can be distinguished biochemically as well as functionally. The five functional classes of Igs are IgG, IgM, IgD, IgA, IgE as shown in Figure 1 (Janeway and Travers, 1994). The heavy chains of Ig classes are denoted by the corresponding lower case Greek letter as y, |i, 8, a and e respectively. The biological functions for Ig classes are given in Table 1 (Burton and Gregory, 1986).  4  Table 1: Biological functions of Ig classes (adapted from Burton and Gregory, 1986). Class  Site of action  Functions  IgG  Intra and extravascular, transplacental  Complement activation, opsonization, immunity in neonate.  IgM  Intravascular  Complement activation, agglutination  IgD  B-cell surface  not known  IgA  Luminal secretions, breast milk  IgE  Subcutaneous, submucosal  Neutralization at body surfaces, intestinal immunity Mast cell sensitization, eosinophil activation  6  2.2 Hen's egg yolk antibodies (IgY) Hen's egg yolk is an efficient source of specific and high affinity antibodies. Chicken serum consists of 5-6 mg/mL IgG, 1.7-2.6 mg/mL of IgM, and 0.3-5 mg/mL of IgA (Stevens, 1996). In 1969, Gerrie Leslie and Bill Clem proposed the term IgY for the designation of 75 Ig of the chicken serum, previously called as IgG (Leslie and Clem, 1969). Ohta et al. (1991) has used the abbreviation IgY for egg yolk antibody and IgG for serum antibody of chicken and they suggest that there may be differences between the two. However, it has been shown that IgY (yolk) is indistinguishable from IgG (serum) in antigen binding properties and molecular weight (Faith and Clem, 1973). The chicken IgG differs markedly from any known Ig, and hence it is called IgY. In 1988, Parvari et al. suggested that the H-chain should be called V and constant region Cv, as V is the Greek transliteration of the letter Y. IgY is transported to the egg in a manner similar to placental transfer of IgG in mammals. The protection against pathogens that the relatively immunocompetent newly hatched chick has is through transmission of antibodies from the mother via the egg. In the egg, chicken IgY is found mainly in the egg yolk, whereas the concentration in egg white is very low. The IgY concentration in the yolk (8-15 mg/mL) is higher than in serum (5-6 mg/mL) due to the active transport from hen to the egg yolk (Larsson et al., 1993; Stevens, 1996). A laying hen produces approximately five to six eggs/week with a yolk volume of approximately 15 mL per egg, thus in one week a hen produces antibodies equivalent to 90 to 100 mL of serum or 180 to 200 mL of whole blood antibodies (Larsson et al, 1993). The eggs laid by an immunized hen in a year would  7  yield 40 g of IgY while whole serum of an immunized rabbit would give only 1.4 g of IgG (Hatta et al, 1993).  -  •  Antibodies presently available for research in laboratories belong to one of three main categories: mammalian monoclonal antibodies, mammalian polyclonal antibodies and avian polyclonal antibodies. In the last decade there has been increased interest in the use of chicken antibodies. There are several advantages of using chicken antibodies instead of mammalian ones. Rheumatoid factor is a major source of interference in many immunoassays, where it reacts with IgG from different mammalian species, including mouse monoclonal, causing erroneous results. Because of the immunological differences between mammalian IgG and chicken IgY there is no cross reactivity (Larsson and Sjoquist, 1990). Also the low cost and convenient production (Poison et al, 1980) and compatibility with modern animal protection regulation makes it an interesting and attractive antibody for widespread use. IgY antibodies have been used as immuno reagents in the following ways : 1. As a food ingredient for the local passive immunization of the gastrointestinal tract against pathogens like E. coli (Shimizu et al., 1988; Yokoyama et ah, 1992) and rotavirus (Hatta etal, 1993; Ebinaef a/., 1990). 2. To detect different components in food, like lactoferrin (Miesel, 1990), soybean glycinin (Miesel, 1993), ochratoxin A fungal metabolite (Clarke et al, 1993) and IgG in bovine milk (Li-Chan and Kummer, 1997). 3. To detect different components in biological fluids like insulin (Lee et al., 1991), bovine growth hormone and prolactin (Schmidt et al, 1993). 4. To prevent diseases like Edwardsiellosis in Japanese fish (Gutierrez et al, 1993).  8  5. To prevent dental caries in rats (Otake et al., 1991). 'Meiji', a commercial manufacturer in Japan, has already developed a commercial product (candies) containing IgY to prevent dental caries. Hatta et al. (1997) reported that IgY was effective in controlling the colonization of Streptococcus mutans in the oral cavity of humans. 6. In immunoaffinity chromatography to purify bovine IgG (Akita & Li-Chan, 1998; Kummer and Li-Chan, 1998) and to isolate lactoferrin from milk or cheese whey (LiChan et al. 1998).  2.3 Comparison of IgY to IgG 2.3.1 General structure & composition Chicken IgY was originally compared to mammalian IgG, because of the functional similarity, but chicken IgY has been found to be structurally different from mammalian IgG. The molecule of IgY is larger than that of mammalian IgG, 170,000 kD versus 150,000 kD respectively (Kobayashi and Hirari, 1980), with larger M W of 70,000 kD for heavy (H) chain compared to 50,000 kD for mammalian IgG. The M W of light chains from both species is found to be the same, at 22,000 kD. IgY has two light chains (L) and two heavy chains. One pair of each H and L chain is held together by a disulfide bond (Dressman and Benedict, 1965; Gold et al., 1966). Cser et al. (1982) studied the structure of IgY by x-ray small angle scattering. It was found that the general shape of IgY and relative position of Fab and Fc parts is similar to that of IgG. The value of radius of gyration (R ) for IgY was equal to 6.13 nm. Even though g  the MW of IgY is higher than IgG by 20,000 to 22,000 kD, the observed value of radius  9  of gyration was smaller than that of mammalian IgG: 6.68 nm for rabbit and 6.78 nm for pig (Cser et al, 1982). It was supposed that the extra mass was distributed near the centre of mass of the molecule, situated at the Fc fragment area. The supposed additional fifth domain of the H-chain should be located at the Fc region. From light scattering data, three most probable and typical models of the IgY molecule were generated. Model (a) was similar to a fragment of the IgM molecule; model (b) was the T-shaped form and model (c) was a modified variation of a shirt shape. Figures 2 and 3 show the structure of mammalian IgG as compared to IgY. According to Gregory et al. (1995), the H chains of IgY typically possess one variable (V) and four constant (C) region domains. The Cy2 and Cy3 domains of IgG are most closely related to the Cv3 and Cv4 domains of IgY respectively. The equivalent of the Cv2 domain is absent in y chains of IgG. The Cv2 domain was probably condensed to form the IgG hinge region. The hinge is a singularly mammalian feature of Ig structure. IgY and avian IgA both possess H chains with four C-region domains and no genetic hinge. In IgY, there are regions near the boundaries of the Cvl-Cv2 and Cv2-Cv3 domains that contain proline and glycine residues. These regions have the potential to confer limited flexibility on the molecule in a manner analogous to 'switch' regions described for some mammalian Igs. The constant region of the H chain consists of 426 amino acids. The size of the Cvl, Cv2, Cv3 and Cv4 domains are 105, 94, 109, and 118 amino acids respectively (Parvari et al., 1988). The Cv region of H chain resembles mammalian Ce on the basis of length, presence of four domains and the distribution of cysteines in Cvl and Cv2 (Parvari et al., 1988). Shimizu et al. (1992) estimated the  10  secondary structure of IgG and IgY, and reported that IgY had lower content of ordered P-structure. The amino acid composition of chicken IgG (IgY) is given in Table 2 (Higgins, 1975). The amino acid composition shown in Table 2 does not indicate tryptophan residues, which are usually destroyed during the amino acid analysis (Creighton, 1993). Shimizu et al. (1992) reported that IgY molecule is less stable compared to mammalian IgG. They suggested that the differences in stability may be due to the following structural differences: 1. Intramolecular forces supporting immunoglobulin structure are weaker in IgY than in IgG. 2. IgY has a lower content of P-structure than IgG. 3. IgG has an intrachain disulfide link between V L and C L domains as shown in Figure 3b that is absent in IgY. 4. IgY differs in glycosylation sites from IgG but it is more similar to IgE. 5. IgY has less chain flexibility between CHI (Cvl) and CH2 (Cv2) domains compared to the hinge region of IgG.  11  Figure 2 : Comparison of the structure of IgG to IgY (adapted from Greogary et al, 1995, with permission). The H-chain contains four C-region domains Cvl Cv4 and lacks the hinge of IgG.  12  00 VL  CL i  c CHO  1  1  1  VH  1  1 CHI  ' CH2  CHO  1  1  I i CH3  i CH4  (b)  VL  CL  Figure 3: Structure of IgY (a) and IgG (b) (adapted from Shimizu et al, 1992 with permission). V represents the boundary region between CHI and CH2 which includes the'hinge region for IgG, CHO represents the carbohydrate chains. The C stands for Constant region and V stands for variable region. The H and L represents heavy and light chains respectively  13  Table 2: Amino acid composition of IgY (adapted from Higgins, 1975). Amino acids  Amino acid, g/100 gm  Nonpolar/Hydrophobic side chains Alanine  4.5  Isoleucine  2.5  Leucine  7.5  Methionine  0.8  Phenylalanine  3.2  Proline  5.8  Valine  7.0  Polar/Hydrophilic side chains Serine Threonine  7.1  Tyrosine  4.2  Cysteine  2.4  Glycine  4.4  Basic side chains Lysine  3.2  Arginine  6.2  Histidine  1.6  Acidic side chains Aspartic  6.1  Glutamic  9.0  14  2.3.2 Carbohydrate composition All immunoglobulins are glycoproteins and IgG has a carbohydrate content of 2-3 %. The carbohydrate moieties are usually associated with H-chains via covalent attachment to aspargine residues. Three functions have been ascribed to the carbohydrate residues: (a) to facilitate secretion from the antibody synthesizing plasma cell; (b) to enhance the solubility of the immunoglobulin and (c) to protect the molecule from degradation. There are differences reported in carbohydrate content of IgY, varying from 2.2% (Leslie & Clem, 1969) to 5.5-6.0 % (Acton et al, 1972). The carbohydrate content of IgY was found to be twice that of mammalian IgG (Acton et al, 1972). Ohta et al. (1991) reported three types of oligosaccharides unique to IgY.  2.3.3 Isoelectric point The isoelectric point (pi) of IgY was reported to be 5.2 (Tenenhouse & Deutsch 1966), and 6.6 (Gallagher and Voss, 1970), which is lower than that reported for mammalian IgG of 7.5-7.9 (Gallagher and Voss, 1970).  2.3.4 Disulfide bonds and reductive dissociation IgY is composed of H and L polypeptide chains held together by one disulfide bond (for each Fab arm) similar to IgG as shown in Figure 2 (Gregory et al, 1995). IgY has four cysteine residues in the Cv2 domain, similar to mammalian IgE, of which two form H-H interchain disulfide bonds and two participate in intradomain loop formation. In IgG, there is one H-H interchain disulfide bond (Parvari et al, 1988).  15  The disulfide bonds of IgY can be easily reduced by reducing agents like mercaptoethanol or dithioerythritol (Benedict et al., 1963a; Rosenquist and Campbell, 1966; Schram et al., 1971). Similar to mammalian IgG, the interchain L-H disulfide bond in chicken IgY is more susceptible to reduction than the H-H bonds (Benedict and Yamaga, 1976).  2.3.5 Aggregation A unique property reported for IgY is salt dependent aggregation. Kubo and Benedict (1969) studied salt induced aggregation of avian and mammalian IgG. Mammalian IgG did not form aggregates in high salt but chicken IgY formed aggregates. The Fc fragments were precipitated by high salt whereas Fab fragments were not aggregated by high salt. With high salt concentration of 1.5 M NaCl, IgY with MW of 550 kD to 590 kD, and a sedimentation coefficient of 145 was obtained (Hersh & Benedict, 1966). Based on molecular weight determinations, the polymer consists of at least 3 units of 7S IgY. Gallagher & Voss (1970) reported dimers of molecular weight 347 kD and 390 kD at NaCl concentrations of 2 M and 3 M respectively. The salt induced aggregation of chicken IgY was found to be reversible (Kubo and Benedict, 1969).  2.3.6 Precipitin reaction The most important property of an antibody molecule is the neutralization of antigen, which involves the interaction of the antigen binding site (comprised of hypervariable loops of both heavy and light chains) with the antigen. The intermolecular  16  forces that are involved in stabilization of antibody-antigen complex are similar to those involved in the stabilization of proteins and other macromolecules, such as hydrogen bonding, hydrophobic interactions, Van der Waal's forces, ionic interactions, and steric repulsive forces (Steward, 1984). IgY has an expected valency of two. The requirement of IgY for high salt in precipitation of antigen was first reported by Hekteon in 1918 (Gallagher and Voss, 1969), and was later confirmed by several researchers (Goodman et al, 1951; Benedict et al., 1963b). To explain this salt dependency two models were proposed. Gallagher and Voss in 1969 suggested that high salt induced a conformational change in the IgY molecule resulting in more polar orientation of the combining sites leading to lattice formation and precipitation. It was thought that this is due to the secondary phase of precipitation and not related to the primary antigen-antibody binding. Kubo et al. (1973) suggested that IgY formed trimers at high salt concentrations by aggregation of the Fc portion, thus orienting the binding sites for cross linking with the antigen. In 1970, Gallagher and Voss reported the precipitation of IgY at physiological salt concentration but at low pH. Poison et al, in 1980, reported precipitation of IgY with a high molecular weight viral protein, without any high salt or low pH requirement. It has been suggested that antigens with low molecular weight (less than 30 kD) did not evoke precipitation of antibodies in chicken. However several researchers have reported that chicken could generate antibodies against low molecular weight antigens such as asi-casein (23.5 kD), (MacLaren et al, 199A) and human insulin (6 kD) (Lee et al, 1991).  17  2.4 Stability of immunoglobulins 2.4.1 Stability to acidic pH Purification of specific antibodies from antigen-antibody complexes often involves dissociation by acidic pH, but the stability of antibodies at such harsh conditions is still unknown. Day (1990) studied mammalian IgG using circular dichroism (CD) and ultraviolet (UV) spectroscopy and found that the dimeric light chains were stable in structure within a pH range of 4-10. Above pH 10.2, a randomly coiled structure resulted due to denaturation; at acidic pH below 4, denaturation with irreversible conformational changes was observed. Conformational changes of whole IgG, Fab, and Fc fragments at acidic pH were also studied by infrared spectroscopy (Day, 1990). Fc fragments lost their conformation and native properties below pH 5.1 whereas Fab fragments resisted significant changes at pH as low as 2. At acidic pH whole IgG underwent conformational changes that could be attributed to loss of Fc integrity. Perhaps the most important function of antibody molecule is to combine with corresponding antigen to form an antibody-antigen complex (Steward, 1984). Antibodies (immunoglobulins) are proteins nature and are subject to conformational changes with acidic pH which can affect their interactions with antigens. The pH of the solution is one of the most important factors determining the state of a protein. Lysozyme, (3-lactoglobulin, and ribonuclease were stable at as low as pH 2 and retained their conformations, whereas glyceraldehyde 3-phosphate dehydrogenase was dissociated below pH 4 (Habeeb, 1976; Tanford, 1968).  18  Researchers have studied conformational changes due to heat and pH and found that IgY is more susceptible to changes than mammalian IgG (Hatta et al., 1993; Otani et al, 1991; Shimizu et al, 1988). Shimizu et al. (1992) studied conformational changes of the IgY molecule by fluorescence measurement  and CD spectra. Conformational  changes of the IgY molecule resulted in rapid loss of activity at acidic pH. It was shown that destruction of the secondary structure proceeded readily in the IgY molecule, where as IgG was more rigid and stable. Reversibility of the denatured state to native state is an important issue concerning the functionality of the protein molecule. "Denaturation in the broad sense is the disorganization of the individual protein molecules. If denaturation is disorganization and not decomposition, it should be possible for denatured protein to revert to the natural native form" (Hsien, 1995). In the last decade, denaturation of IgY due to pH changes was studied, but reversibility of these changes to the native state of IgY had not been studied.  2.4.2 Stability to GuHCl denaturation Urea and GuHCl are two commonly used protein denaturants. Organic compounds such as urea and GuHCl when in concentrated aqueous solutions (4-8 M), contribute to the disruption of hydrogen bonds as well the decrease in hydrophobic interactions by increasing the solubility of hydrophobic amino acid residues in the aqueous phase (Creighton, 1993; Fennema, 1985). In 1968, Tanford wrote that, 'Proteins which contain disulfide bonds between polypeptide chains will of course retain them after  19  denaturation by GuHCl if reagents to break disulfide bonds are not added' (Tanford, 1968). GuHCl is a strong electrolyte and affects the ionic strength of a solution. Randomly coiled proteins in urea are subject to effects of pH and ionic strength which are not observed in a GuHCl solution (Tanford, 1970). The aqueous solubilities of all the constituent parts of a protein increases with increasing urea or GuHCl concentration (Tanford, 1970). Solutes affect proteins by altering the physical properties of water and by interacting directly with proteins. The two effects can be distinguished by dividing the free energy of a molecule in aqueous solution between the energy cost of making a cavity in the solution in order to accommodate the molecule, and establishing the interaction between the molecule and the water upon filling the cavity. Change in the first component should be general to all molecules, and should be reflected in the surface tension of water. Increasing the surface tension should make it more costly to make a cavity and will tend to decrease the solubilities of other molecules. Since the unfolded protein has a higher nonpolar area exposed than a native protein, urea and GuHCl stabilize unfolded conformations (Creighton, 1991). Several proteins including lysozyme, insulin, ribonuclease, chymotrypsinogen, Fab fragments of Ig, bovine serum albumin, and p-lactoglobulin were studied in 6 M GuHCl, and a loss of conformation was observed (Habeeb, 1976). Shimizu et al. (1992) studied stability of IgY and IgG to various GuHCl concentrations, and found that IgG is more stable than IgY. Most proteins with an ordered native structure undergo a marked transition upon the addition of GuHCl. There has been extensive research on the effect of GuHCl on  20  proteins. It has been demonstrated that GuHCl is a strong denaturing agent. GuHCl denatured proteins show a loss of conformation. In the last decade, focus has been on the study of the unique structure of the 'Molten Globule State' (Hirose, 1993; Kuwajima, 1989; Ptitsyn, 1987). Reversibility to native state after renaturation is an important issue. Hattori et al. (1993b) investigated unfolding and refolding behavior of pMactoglobulin with GuHCl; they reported that complete refolding was never attained.  2.4.3 Freezing, freeze drying and IgY A product is generally freeze dried if it has stability problems in aqueous solutions and is not stable when undergoing other drying methods such as spray drying. Although freeze drying is considered expensive because of plant cost and energy consumption, it is often a method of choice for protein products when a high quality product is the goal. From previous studies it is known that IgY is not stable to temperatures above 65°C, thus conventional drying methods may not be used. In freeze drying, the solvent is first frozen and then the frozen solvent is removed by sublimation in a vacuum environment. During freezing, water crystallizes to ice, thus concentrating all solutes between the ice crystals; solutes and buffer components may crystallize, producing massive shifts in pH. Once freezing begins, the protein environment changes, and it may have adverse effects on proteins. Hannson (1968) reported that human IgG formed aggregates with freezing and thawing. Similar results were also reported by Tencer et al. (1994), who found that there was significant decrease in IgG activity of urine samples after frozen storage at -20°C. Stability of IgY to freezing and freeze drying has not been investigated.  21  2.4.4 Structural conformation of eluted IgY Immunoaffinity chromatography is a powerful and popular technique for antibody/antigen purification (Kummer and Li-Chan, 1998). The mechanisms of antigenantibody binding are complex and varied, involving similar forces as those required in stabilizing protein conformations (Steward, 1984). Immunoaffinity chromatography has been used in the past to isolate IgY and other antigens. For dissociation of antigen-IgY complexes, researchers have often used acidic pH in the eluting media (Kummer and Li-Chan, 1998; Li-Chan et al. 1998; Li-Chan and Kummer, 1997) but the effect of an acidic elution protocol on conformation of IgY has hot been investigated. Shimizu et al. (1994) reported that IgY was stable at pH 3 in the presence of high (>30 %) sugar concentrations. A sucrose (>30 %) solution is quite viscous and may not be practical for elution purpose. Trehalose is highly soluble, nonreducing, nonhygroscopic and belongs to the most chemically unreactive sugars. Trehalose is commonly used for stabilizing proteins (Skrabanja et al, 1994). The detailed molecular mechanism of the protective action of trehalose is not completely understood; however there are several theories to explain the effect such as hydrogen bonding and glass formation (Roser, 1991). It has been reported that trehalose is effective in stabilizing proteins at a lower concentration than sucrose (Draber et al, 1995; Roser, 1991).  22  2.5 Techniques used to characterize structural and functional properties of proteins 2.5.1 U V absorption spectroscopy Visible and near UV spectra originate from relatively low energy electronic transitions. In this region of the electromagnetic spectrum three main groups of chemical species are involved: compounds containing metals, such as prosthetic groups in metalloproteins, large aromatic ring structures and conjugated double bond systems. For wavelengths above 230 nm, the absorbing components in proteins are essentially the aromatic amino acids tyrosine, tryptophan and phenylalanine. The UV absorption spectrum of a protein is changed by additives such as organic solvents, salts and by variation in pH and temperature. Since these changes are very small, derivative spectroscopy is used as a tool for analysis of conformational changes (Padros et al, 1984). Fourth derivative spectroscopy provides better resolution than first or second derivative spectroscopy and hence it has been widely used to study characteristics of aromatic amino acids in proteins (Padros et al, 1984; Mach et al, 1989). Changes in the environment of the aromatic residues produce changes in the fourth derivative spectra of proteins. Second and fourth derivative spectroscopy has been used in biochemistry, to determine protein concentration, aromatic amino acid residues, and in the study of protein structures. It had also been used in food analysis to determine whey protein nitrogen content and to study thermal denaturation of whey proteins (Meisel, 1995). Yesilada et al. (1992) used derivative UV-spectroscopy to study differences between bovine and porcine insulin.  •23  2.5.2 Intrinsic fluorescence Thefluorescencespectra of proteins are mainly due to aromatic amino acids. In UV spectroscopy, the absorption spectra are measured, whereas in intrinsic fluorescence, the emission spectra are measured. The aromatic side chains of tryptophan and tyrosine residues in proteins are capable offluorescence.When they are excited by UV radiation, the excited state decays to electronic ground state in at least two steps: 1. vibrational energy gained in the excitation is dissipated as thermal motion within the protein molecule and 2. thefluorophoredecays exponentially back to the electronic ground state with the re-emission of radiation (Gurd and Rothgeb, 1979). The principal reason for studying the intrinsicfluorescenceof proteins is to obtain information about conformation (Freifelder, 1976). When tyrosine is excited at 275-280 nm an emission spectrum is observed between 298-305 nm and when tryptophan is excited at 280 nm an emission spectrum is observed between 310-350 nm. In practice, tryptophanfluorescenceis the most commonly studied aspect of the spectrum, because phenylalanine has a low quantum yield offluorescencewhilefluorescencefrom tyrosine is quenched by ionization or interaction with amino, carboxyl, or tryptophan residues. Three spectral classes of tryptophan residues in proteins have been reported. These are: 1) the residues completely buried in nonpolar regions of the molecule, which show an emission maximum (k ) near 330-332 nm, max  2) the residues which are completely exposed to the surrounding water and show an A,  max  near 350-353 nm,  3) those that have limited contact with water and are probably immobilized at the protein surface which have ? i  m a x  near 340-342 nm (Li-Chan, 1991).  24  2.5.3 Fluorescence probes Fluorescence probes can be defined as small molecules which undergo changes in one or more of their fluorescence properties as a result of non covalent interaction with a protein or other molecule (Brand and Gohlke, 1972). Fluorescent compounds such as 1anilino-8-naphthalene sulfonate (ANS) and c/s-parinaric acid (CPA) have extremely low quantum yields in water (Nakai and Li-Chan, 1988). In the presence of many proteins ANS and CPA show a dramatic increase in quantum yield with a concomitant shift in fluorescence  emission maximum, suggesting that the interaction of probe with  hydrophobic regions of the proteins is responsible for changes in fluorescence properties (Kronman and Robbins, 1970). CPA is a nonaromatic fluorophore which has been used as a probe for protein and biological membranes (Sklar et al, 1975). ANS & CPA are both anionic probes, the structure of both is shown in Figure 4. The quantum yield of ANS has been reported to be unaffected by acidic pH values, but CPA has poor solubility below pH 5, and hence CPA cannot be used at acidic pH values (Nakai et al, 1996). Hayakawa and Nakai (1985) suggested that the binding sites on protein molecules, may differ for ANS and CPA probes. They classified CPA hydrophobicity as aliphatic hydrophobicity due to aliphatic amino acid residues and ANS hydrophobicity as aromatic hydrophobicity due to aromatic amino acid residues. Both ANS and CPA probes have been extensively used to study hydrophobicity of proteins and their relation to functionality. Hayakawa and Nakai (1985) studied the relationship of hydrophobicity and net charge to the solubility of milk and soy proteins. They found a correlation between ANS hydrophobicity and protein insolubility,  25  suggesting that aromatic hydrophobicity may play an important role in protein solubility. Lipophilization of pMactoglobulin was studied by Akita and Nakai (1990). They reported that high solubility and ANS hydrophobicity were both needed for the best emulsifying properties.  (a)  (b) H HH  H  III  I  HjC • CH, • C=C-C=C-C=C-C=C • (CH ) • COOH  I  H  III  2  7  H H H  Figure 4: Structure of ANS (a) and CPA (b) fluorescent probes (adapted from Nakai and LiChan, 1988, with permission).  26  2.5.4  Raman Spectroscopy Raman spectroscopy is a branch of vibrational spectroscopy which give useful  information on the vibrational motion of molecules. The intensity and frequency of the molecular vibrations are sensitive to chemical changes and to the environment around the atoms, hence Raman spectrum can be used as a monitor of molecular chemistry (Li-Chan et. al, 1994). The observed Raman spectrum of a protein consists of contributions from various amino acid side chain vibrations as well as from the polypeptide backbone. In addition to information on vibrational motions of side chain residues, the amide I and amide III band regions in protein Raman spectra (near 1650 cm' and 1250 cm" 1  1  respectively) have been used for quantitative estimation of the peptide backbone (LiChan et. al, 1994). Compared to other spectroscopic methods such as UV absorption spectroscopy, Raman spectroscopy has the advantage of being applicable to insoluble as well as soluble samples.  2.5.5 Solubility Proteins in solution acquire a specific configuration, which is stabilized by weak and strong covalent and noncovalent forces. Disulfide linkages, hydrogen bonds, electrostatic interactions, and hydrophobic bonds are important to stabilize protein structure in solutions (Thakker and Grady, 1984). Denaturation of proteins frequently impairs their functional properties, and is expressed as a loss of solubility (Nakai and Li-Chan, 1988). Solubility of proteins is an important factor, as it can affect functional properties.  27  2.5.6 Enzyme-linked immunosorbent assay (ELISA) The enzyme linked immunosorbent assay uses an enzyme bound to antibody or antigen. In practice, there are many variations of an ELISA Assay (Sell, 1987), such as direct, indirect, sandwich, competitive, etc.  Depending on the format/type either  antibody or antigen is adsorbed on a solid phase. The format for indirect ELISA is given here since it was used in the present research. The antigen is allowed to adhere to the microplate well and unbound antigen is washed off.  Then solution containing  unknown amount of the specific antibody is added. After binding of antibody, a second enzyme-labeled antibody (also called conjugated antibody) directed against the first antibody is added. The amount of conjugated antibody binding to antibody (unknown) depends on the amount of antibody (unknown) present. A substrate is then added that changes color when acted upon by the immobilized enzyme, and the color development is measured by colorimetric analysis. Two of the enzymes commonly used are horseradish peroxidase, which turns a colorless solution of 5-aminosalicylic acid to reddish brown and alkaline phosphatase, which turns a colorless solution of p-nitrophenol phosphate to yellow (Dixon, 1994; Sell, 1987).  28  Chapter III MATERIAL AND METHODS 3.1 Materials Actigel ALD Superflow was purchased from Sterogene Bioseparation, Inc. (Arcadia, CA, USA). Butylated hydroxyanisole (BHA) and lactoferrin were purchased from ICN Pharmaceuticals Inc. (Montreal, PQ, Canada). Bicinchoninic acid (BCA) protein assay kit was purchased from Pierce (Rockford, IL, USA). Immulon II 96 well microtiter plates and Spectra Por® 1 (MW cut off 6000 to 8000 daltons) dialysis membrane and Whatman # 4 filter paper were purchased from VWR Canlab (Mississauga,  ON,  Canada).  Rabbit  anti-chicken  conjugate  from  Jackson  Immunoresearch Lab. Inc. was acquired through Bio/Can Scientific Inc. (Missisauga, ON, Cananda). Chicken gamma globulin, GuHCl , p-nitrophenyl phosphate substrate, trehalose and Tween 20 were purchased from Sigma-Aldrich Ltd. (Oakville, ON, Canada). ANS (1,8-anilinonaphthalene  sulfonic acid) was prepared according to  Hayakawa and Nakai (1985) and CPA (c/s-parinaric acid) was purchased from Molecular Probes Inc. (Eugene, OR, USA). Citrate and glycine were purchased from Fisher Scientific Ltd. (Nepean, ON, Canada). Disodium hydrogen orthophosphate and sodium dihydrogen orthophosphate were purchased from BDH chemicals (Toronto, ON, Canada).  3.2 Buffers Glycine-HCl (0.05M) pH 2.8 buffer, citrate phosphate pH 2.8, pH 4 and pH 7 buffers (CPB) and phosphate buffer pH 7 (PB 0.01 M) were prepared according to  29  Dawson and Elliot (1986). CPB with 0.14 M NaCl (CPBS) and PB with 0.14 M NaCl. (PBS) were also prepared. Buffers for ELISA (carbonate coating buffer, blotto, conjugate buffer, substrate buffer) were prepared according to Kummer et al. (1992).  3.3 Isolation of IgY IgY was isolated by the method of Akita and Nakai (1992) with modifications. Egg yolk was diluted ten times with distilled water and pH was adjusted to 5.2 using 0.1 M HC1. This diluted yolk preparation was incubated overnight in the cold room at 5°C. The diluted yolk solution was centrifuged at 10,000g at 4°C for 25 min. The supernatant was filtered through Whatman # 4 and glass wool, 19 % ammonium sulfate was added and the supernatant solution was incubated in the cold room overnight at 5°C. Next day the solution was centrifuged at 10,000g at 4°C for 25 min, and the pellet containing IgY was re-dispersed in PBS. These isolates were stored at 5°C in PBS containing 0.02% sodium azide until analyzed. The IgY isolate obtained from this procedure is referred to as 'crude IgY' isolate. Non-specific crude IgY was isolated from eggs purchased at Safeway (a local supermarket), while crude IgY containing anti-lactoferrin IgY was isolated from eggs of hens immunized against bovine lactoferrin at the Avian Research Centre, UBC (Li-Chan et al, 1998). All IgY isolates were checked for purity with SDS-PAGE and all analyses were completed within 6 months after isolation. A typical SDS-PAGE profile of IgY sample is shown in Appendix I. The electrophoretic purity of crude IgY isolate was found to be >90 %. A separate batch of crude IgY isolate (non-specific) was prepared for  30  the treatments described in sections 3.5.1 and 3.5.2. One batch of anti-lactoferrin IgY was used for the treatments described in 3.5.3 and 3.5.4 and ELISA for all treatments.  3.4 Protein concentration determination  Absorbance at 280 nm was used for the estimation of IgY concentration except for samples used in ELISA and solubility assays. For ELISA and solubility, bicinchoninic acid protein assay was used according to Pierce's BCA protein assay instruction manual, with chicken y globulin as the standard. For all the calculations of protein concentrations extinction coefficient of 14  cm" was used (Sigma technical data sheet). 1  3.5 Treatments 3.5.1 p H a n d temperature  IgY from stock solution (30 mg/mL) was added to CPBS buffer (pH 7, 4, 2.8) to yield 1 mg/mL final concentration. IgY (2-6 mL) in 1 mg/mL concentration was held in glass test tubes at pH 2.8, 4 or 7 at ambient temperature (20 to 25°C), 5°C or 37°C for 2 hours in CPBS. It took 15 min for IgY sample to reach 5°C (walk in cold room on second floor of food science building) and 10 mins to reach 37°C (Thelco GCA incubator, Precision Scientific, Chicago, II, USA) from ambient temperature. To study renaturation, the pH treated IgY was dialyzed (Spectra Por 1 dialysis membrane) at 5°C for 4 days against 2 liters of pH 7 CPBS, with the buffer changed 6 times. The structural conformation of pH and temperature treated and renatured IgY was studied by UVspectroscopy,  intrinsic fluorescence,  extrinsic  31  fluorescence  probes  for  surface  hydrophobicity and ELISA. For ELISA, IgY isolate specific to lactoferrin was used. For all other techniques, non-specific IgY isolate was used.  3.5.2 GuHCl concentration A sample of IgY from crude IgY isolate solution (-30 mg/mL) was added to 0 (control), 1, 2, 3, 4, 5 or 6 M GuHCl in PBS to obtain a final IgY concentration of 1 mg/mL. IgY in GuHCl solution was held for 1 hr at ambient temperature (20 to 25°C) and compared to IgY held in PBS as the control. To study renaturation, GuHCl treated IgY was dialyzed (Spectra por® 1 dialysis membrane) @ 5°C against 2 liters of distilled water for 1 day, and then against PBS at pH 7 for 3 days; the control sample was dialyzed (Spectra por® 1 dialysis membrane) for 4 days against PBS pH 7 (Hattori et al, 1993b). Structural conformation of GuHCl treated, control and renatured IgY was studied with UV-spectroscopy, intrinsic fluorescence, extrinsic fluorescence probes for surface hydrophobicity and ELISA. For ELISA IgY isolate specific to lactoferrin was used. For all other techniques non-specific IgY isolate was used.  3.5.3 Freezing and freeze drying at low and high salt concentration Anti-lactoferrin IgY isolate was dialyzed against pH 7 citrate phosphate buffer containing low salt (LS) (0.14 M NaCl) or high salt (HS) (1.5 M NaCl). For each salt concentration, two IgY concentrations of 1 mg/mL (dilute) and 30 mg/mL (concentrated) were prepared. From each of the four conditions 0.5 ml of IgY  (in 1.5 mL  microcentrifuge tubes [eppendorf] placed in a cardboard stand) were frozen (F) at -80°C (Biofreezer, Forma Scientific, Inc. Ohio, USA) and -8°C (Viking) in replicates for 7  32  days. After freezing the IgY sample for 2 days one set of each of the above conditions was freeze dried (FD) in a VIRUS freeze drier (Model 1451, Gardiner, N.Y. USA); the chamber temperature was at room temperature, and condenser temperature was set to 55°C at an operating vacuum of 150 mT. The frozen samples were thawed at room temperature, and freeze dried samples were reconstituted in distilled water to their original volume prior to analysis. These samples were studied with the following techniques: intrinsic fluorescence  and extrinsic fluorescence  probes for surface  hydrophobicity, light scattering and ELISA.  3 . 5 . 4 Structural conformation of IgY eluted from an immunoaffinity column An Actigel ALD Superflow column (10 mL) with 2.26 mg/mL of immobilized lactoferrin (Li-Chan et al., 1998) was used to isolate specific IgY. The column was washed with pH 2.8 glycine-HCl (0.05 M) buffer for 20 min and then with PBS pH 7 at a flow rate of 0.75 mL/min, until pH was 7. Crude IgY isolate (25 mL at concentration of 10 mg/mL) from eggs of lactoferrin immunized hens was applied onto the column at a flow rate of 0.25 mL/min. PBS pH 7 buffer was applied to remove unbound IgY until the A 8o was 0.0. Fractions were collected at every 4 mL volume. Glycine-HCl pH 2.8 buffer 2  was applied at a flow rate of 0.75 mL/min for 20 minutes to elute bound IgY representing lactoferrin-specific antibodies. Eluted IgY fractions (1.8 mL) were immediately neutralized in 0.2 mL pH 8 phosphate buffer (0.5 M) the pH of the neutralized fraction was 7.6 A 8o of all the collected fractions was measured. IgY isolated by immunoaffinity 2  chromatography is further referred to as "eluted IgY".  33  Specific antibodies against lactoferrin were also eluted from immunoaffinity chromatography with glycine-HCl buffer at pH 2.8 containing 15% trehalose. The properties of eluted antibodies were investigated by extrinsic fluorescence probes and ELISA. 3.5.5  Effect of dialysis on control IgY From the results of control IgY samples (pooled from all the treatments) it was  observed that ANS hydrophobicity of samples after dialysis appeared to be lower than the control undialysed samples. One way ANOVA was applied to ANS and CPA hydrophobicity of dialysed versus undialysed IgY control samples to compare the effect of dialysis There was no significant difference between dialysed and undialysed IgY (P < 0.05) as shown in appendex 2. Since ammonium sulfate in crude IgY isolate was removed by dialysis, the possible effect of ammonium sulfate on IgY was studied. To 1.2 mg/mL of dialysed IgY solution 1, 3, 6, 20, 100 mg/mL of ammonium sulfate was added. ANS and CPA hydrophobicities were measured. There was no significant difference between any sample (P < 0.05).  3.6 Analysis 3.6.1 UV- fourth derivative spectroscopy A UV scan was measured from 250-330 nm range with a spectrophotometer (Unicam UV/Vis spectrometer UV2 ATI Unicam, Cambridge, UK) using a speed setting of 30 nm/min, data interval of 1 nm and band width of 2 nm. IgY concentration (determined based on an extinction coefficient of 14) was adjusted to 0.5 mg/mL or 1 mg/mL in the appropriate buffer (pH 2.8, 4 or 7) or GuHCl concentration (0-6 M). All  34  sample spectra were measured in duplicate against a reference cell containing the appropriate buffer or GuHCl solution. The UV scans were saved on a disk and the fourth derivative spectra were calculated by using Grams 386 (Galactic Industries, Salem, NH, USA). From the fourth derivative spectra of IgY seven parameters were calculated according to Padros et al. (1982, 1984) and Dunach et al. (1983). The parameters are related to the environment of tryptophan, tyrosine and phenylalanine residues, these include phenylalanine, tyrosine, tryptophan peak positions (Phe^ , Tyrx. i and Tyr>. , Trp^ i 2  and Trpx 2) and tyrosine and tryptophan peak height ratios (R = h / hi, R = h / hi, tyr  2  trp  2  where hi and h are the peak to valley heights at A,i and X respectively). 2  2  3.6.2 Intrinsic fluorescence Emission spectra were measured from 310 to 360 nm at two excitation wavelengths (280 & 297 nm) with a spectrofluorometer (Shimadzu spectrofluorometer RF-540, Shimadzu Corporation, Kyoto, Japan). The IgY concentration was 0.06 mg/mL in the appropriate buffer (pH 2.8, 4 or 7) or GuHCl concentration (0-6 M). The parameter settings were as follows: scan speed was slow, excitation and emission slits were at 5 nm, ordinate scale was 2 and sensitivity was high (Arteaga, 1994). All spectra were corrected for buffer blanks. All spectra were run in duplicate.  3.6.3 Light scattering Light scattering methods provide a sensitive measure of aggregate formation in protein solutions. Light scattering was measured using the Shimadzu RF-540 spectrofluorometer by setting the excitation wavelength at 350 nm and the emission  ' 35  wavelength at 355 nm (Copeland, 1994). The other parameter settings were as per intrinsic fluorescence.  3.6.4 Extrinsic fluorescence probes for surface hydrophobicity IgY treated with acidic pH, GuHCl, freezing or freeze drying treatments was diluted in PBS pH 7 to determine hydrophobicity by two hydrophobic fluorescence probes, according to Nakai et al. (1996) with some modifications as described below. 3.6.4.1 ANS (1,8-anilinonaphthalene sulfonic acid ) probe hydrophobicity 8 mM ANS stock solution was prepared by dissolving the magnesium salt of ANS prepared according to Hayakawa and Nakai (1985) in PBS pH 7. The ANS stock solution was kept in glass bottle wrapped with aluminum foil to exclude light and was stored at room temperature. To a series of 5 tubes each containing 4 mL of PBS, 20 ul of 8 mM ANS was added and mixed well by vortexing. To these mixtures, IgY solution (1, 15, 30 mg/mL) was added to yield a series of 5 tubes with final IgY concentration in the range of 0.04 to 0.20 mg/mL. After 15 min the relative fluorescence intensity (RFI) of each dilution was measured with a spectrofluorometer (Shimadzu spectrofluorometer RF-540 Shimadzu Corporation, Kyoto, Japan), starting from the lowest to the highest IgY concentration. An excitation wavelength of 390 nm and emission wavelength of 470 nm was used. Both excitation and emission slits were set at 5 nm and the ordinate scale was set at 6. The RFI of protein blanks (protein diluted in probe free buffer) and a buffer blank (buffer with probe but no protein) were also measured. The RFI of each protein dilution blank was subtracted from that of the corresponding protein solution with ANS to obtain net RFI.  36  All the samples were measured in duplicates. Standardization of the RFI scale was done by measuring ANS in methanol (10 mL methanol + 10pJ of ANS stock ) and setting this value as 50 at ordinate scale of 3. The hydrophobicity was calculated by linear regression analysis on Minitab Inc. (State College, PA, USA) as the slope of net RFI versus protein (%). The emission spectra of ANS probe with the protein samples at highest concentration were measured in the wavelength region of 450 nm to 520 nm at excitation wavelength of 390 nm. 3.6.4.2 CPA (cis-parinaric acid ) probe hydrophobicity The CPA hydrophobicity was measured according to the method of Kato and Nakai (1980) with some modifications. CPA probe was prepared by dissolving 10 mg of CPA per 10 mL of ethanol containing 100 micrograms of butylated hydroxyanisole (BHA). This solution was stored at -10°C. The procedure was essentially the same as described for ANS except that RFI of samples were measured at excitation wavelength of 325 nm and emission wavelength of 420 nm. Standardization of the RFI values was done by measuring CPA in decane (lOul of CPA to 4 mL of decane) and setting this value as 50 at ordinate scale of 2. All the samples were analyzed in duplicates. The emission spectra in the 400 nm to 450 nm wavelength region of CPA probe with the protein sample at highest concentration were measured at excitation wavelength of 325 nm.  3.6.5 Enzyme linked immunosorbent assay (ELISA) Enzyme linked immunosorbent assay was performed as described by Kummer et al. (1992) with modifications. Immulon II microtiter plates were used as the solid support. Wells were coated with 100 ul of lactoferrin in carbonate coating buffer (10  37  (j.1/10 mL) and incubated at 37°C for 1 hr. Plates were washed thrice with PBS Tween, followed by a blocking step using 250 (ll of blotto (0.5% milk powder in PB) incubated at 37°C for 30 min, then the buffer was discarded. 100 |il of IgY sample (at a concentration of 1 |J.g/ml in PBS unless otherwise specified) was then added in each well and incubated at 37°C for 1 hr. The plates were then washed thrice and 100 ui/well rabbit anti-chicken conjugate was added and incubated at 37°C for 1 hr. The plates were washed with PBSTween, followed by addition of 100 uJ of p-nitrophenyl phosphate substrate (0.5mg/mL) per well and incubated at room temperature for 10 min, then read at 405 nm on a ELISA micro plate reader (Labsystems iEMS Reader MF; Helisinki, Finland). All the ELISA measurements were done in three replicates (same sample three wells).  3.6.6 Raman spectroscopy Raman spectra was recorded on a JASCO model NR-1100 laser Raman spectrophotometer (Jasco Inc., Easton, Maryland, USA) with excitation from the 488 nm line of a Spectra-Physics model 168B argon ion laser (California, USA). The Raman scattering of IgY samples in a transverse/transverse arrangement (capillary held horizontally and incident laser beam perpendicular to the capillary axis) was measured at ambient temperature under the following conditions: laser power, 200 mW; slit height, 2 mm; spectral resolution, 5.0 cm' at 19000 cm" ; sampling speed, 120 cm" /min with data 1  1  1  taken every cm" ; 6 scans per sample. Secondary structure of IgY was determined by 1  Raman spectral analysis package (RSAP; version 2.1) (Li-Chan and Nakai 1991).  38  3.6.7 SDS polyacrylamide gel electrophoresis (SDS PAGE) The IgY sample was prepared in 2-3 mg/mL concentration in PB pH 7. SDS PAGE was carried out in non-reducing conditions on a 10 to 15 % gradient PhastGel, followed by Coomassie brilliant blue staining were conducted according to the specifications of the Phast System manual (Pharmacia-LKB, Uppsala, Sweden).  3.6.8 Solubility Solubility was determined using the collaborative method reported by Morr et al. (1985) with modifications. A sample was taken from frozen and reconstituted freeze dried samples to adjust IgY concentration to 1 mg/mL (0.1%), and was centrifuged at 14000g for 30 minutes at ambient temperature. For objective 2 set C IgY was centrifuged at 30 mg/mL concentration, and then diluted for protein assay. The protein content before centrifugation (total protein) and of the supernatant after centrifugation (soluble protein) was determined in triplicate by the bicinchoninic acid (BCA) protein assay according to the manufacturer's recommendations (Pierce, Rockford, IL, USA). Percent soluble protein was expressed as the percentage ratio of soluble to total protein.  3.6.9 Statistical analysis Balanced ANOVA was applied to test significance of two factors and their interactions. One way analysis of variance and Tukey's pairwise comparison test was applied to test for significant difference between the means, using Minitab Statistical software version lOxtra (Minitab Inc. State College, PA, USA) and Systat for Windows, version 5 (Systat Inc. Evanston, IL, USA). For both ANOVA and pairwise comparison  39  tests, the confidence limit was set to the 5% significance level (P < 0.05). The ANOVA results are shown in Appendix II.  40  Chapter IV RESULTS AND DISCUSSION 4.1 Effect of p H and temperature 4.1.1 Intrinsic fluorescence (a) Relative Fluorescence Intensity The effect of pH and temperature treatment on RFI (relative fluorescence intensity) of IgY at ^  max  (330-338 nm) is shown in Figures 5a and 5b. The RFI of IgY at  both excitation wavelengths of 280 and 297 nm increased for samples treated at pH 2.8 at ambient temperatures or 37°C, but RFI for pH 7 and 4  treated samples were not  significantly different from each other. Temperature by itself did not have any significant effect on the RFI, but the interaction of temperature with pH was found to be significant; RFI was higher for pH 2.8 samples incubated at ambient temperature and 37°C. After renaturation the RFI of samples treated at pH 2.8 (~25°C and 37°C) were significantly higher than pH 7 and 4 treated samples, suggesting that the changes were only partly reversible at pH 2.8. Steven and Somero (1982) reported an increase in fluorescence intensity with decrease in pH for phosphofructokinase. Similarly, Kronman (1989) reported that low pH denaturation of a-lactalbumin, gives an increase in fluorescence quantum yield.  Excitation of IgY at 280 nm was found to give relative fluorescence intensity values twice those of excitation at 297 nm. This may be due to the fact that excitation at 280 nm may contribute to emission of both tyrosine and tryptophan, whereas, on excitation at 297 nm the emission may be due to tryptophan residues only. In protein • 41  sample excited at >295 nm, emission is due to tryptophan only, but intensity could be low because fluorescence intensity is dependent on the absorption extinction coefficient at the excitation wavelength (Copeland, 1994).  42  ca >  "•S  -a -a w "id  CD L-  CS  "3  CD CO  CD  erf  -G  V  £  u  s o  o  CD  CD  /-> *a U  CO  £  .g  G  rt  G  o  ro  /rt IT)  o o  CD  rt -a  •  M  II  £  N  u  G  rt  _  3  1  cCD  T3  i n oo II co  G rt  GO GO  Si  t>  «  3  T3  CD  -r  CD  <u © rt X w co s ro — J * rt s-  s u  E  G G coj CD  00  &  I—H  rt  S s ©  £  00  C  CN  rt „  a  >  •a cu T3  <rt-  >>  O  rt  rt c £ c1-.o ' r t  a  _ rt S 4C> .o 53  u  c cu CD CO  M  rt c  °c« - a CD o H -4—»  •  U  r— ca  u  w  CN  « in  CD  So ffi PQ CD  ox  ex  -a •  a  G C/i CJ  c  o  rt rt  rt  rt  CD  co  CD  2? ra rt CD rt rt  ^ c2 00  1—1  _G •0  CD  00  -a  1 1 - 1  CO  1—t  CD S  rt rt  w rt ±3 13  2 -X X £ 00 00 CD So §  S 55  43  u  s i s C d u  0 >«  ^  ~ ,  cd  oo _^  ^  13 «  H  S 8| 1  |  v  _e  fc"  »o §  ^  1  S g  u fl c ,u fc< j ?  U g  - s  cs  J 8 Q  CD  D  cd  's  & • " u 5 ^  CO Cd  «  M  oo  -a  "  u  „  cu  £  ^  j - .  H  ' 5  M  « g. -s ?  E i!  V"  PQ  cd  $^>< "2 ^ OX  5 00  3  g in  •<-">» fc" O -i* -g r - <u  3 Sis ~ = 53 S  W IT)  -  CU  CU  A  fc< cu r OJO  ^ w cd -o c g cn co  W  c^-gcd  B .  w  cd  fe C « -a  -aCJ "cd  44  (b) Shift in emission maximum Figures 6a and 6b show the effect of pH and temperature on wavelength of maximum emission of intrinsic fluorescence of IgY. Shimizu et al. (1992) reported a red shift in IgY and IgG with acidic pH of < 3.5 after incubation for 3 hrs at 37°C. In the present study similar results were observed in the wavelength of maximum emission (^maxX with a red shift at pH 2.8. The pH 2.8 treatment (at ambient temperature and 37°C) was found to show significant increase in A,  max  on excitation at both wavelengths.  On excitation at 280 nm, IgY treated at pH 4 (at 37°C) showed a significant increase in ^max as compared to 5°C or at ambient temperature. IgY treated at pH 2.8 (at 37°C and at ambient temperature) showed significant increase in X  max  as compared to 5°C. The X  max  of IgY at pH 7 was 332 nm, implying buried aromatic residues, while for pH 2.8 treated IgY A,  max  residues  was 333-338 nm. The red shift to 338 nm suggests that the hydrophobic are neither completely buried nor completely exposed, but are probably  immobilized at the protein surface. The structure of protein denatured by temperature, salt or by acidic pH has often been found to be partly unfolded, as proteins usually retain some features of the structure. The intermediate state is called as 'molten globule state' (MGS) (Hirose, 1993; Ptitsyn, 1987). "'Globule' refers to native like compactness, and 'Molten' refers to the increased enthalpy and entropy on transitions from native structure to the new state, as in the melting of solid" (Hirose, 1993). Many proteins have been shown to take on the molten globule state under mild denaturing conditions. The renaturation process did result in a decreased emission wavelength at both excitation wavelengths (280 & 297 nm). The increases in ?i  45  max  brought about by pH  treatment were completely reversible by renaturation after pH 4 treatment but not after pH 2.8 treatment (Figure 6a, excited at 280 nm) suggesting only partial reversibility after pH 2.8 treatment.  46  N CO  00  rt ti  CM  s >< CO  cu  GO  es  CU  CD  £ ts  OX)  a 2 3  o ^  X CO  cu —  3  es fc. cu  a,  CD  co  U o P rt \/ r- u m -o o y •= II a co e N  in  2 5 o  «« Q II  cu  *° =  .3 x  c« cu  rt .tH  es  •4-*  C O  CU  -in  -s  rt  00  53 +«H £  a, e« rt rt £  TJ  cu  cu  « « c4  ^  .  ^ S cu a s °  0 0  o  •s ^  —  CU  wi  CD  CD  j-  CD  I£ e S  53  3 «s ^CD cs2  6X1 H H  G  ^  N  C (H  CJO  £ u  o  ^  co  G rt CO ~ cu  wj  -C  rt  oo rt  CS  g  g  O  -  w  ^3  •a *° S CD  W  £ S "S .2 rt 3  '*  s  s  feu es (uiu)  aoissiuia xunuiiXBUi jo  47  qjSuapABAV  —  •  •  Cd CD  co cu  > u  rt,  co  C  cd CD  rt ^>  cd CD  O  1-  CD ' — * CO  cd  O  c  CD  g U o c  X  CO  CD 2 £f- -5 II 2  3  CD O  CD  E  11  X  CO  CU  5  cd  CO  C5  .  CU  2 §  S .s? #  cu <u T3 CJ X C5  cs  "cD  ^ e  O N  c -a  N  cd  tO  lH  a  cn  rt C CD  cd  9 in Q TJ C ^ CS C  V PH  O cd CD  CO 1cd  a.  CL '  " O  o o  CU  °=  s CD  -a c  rt ffi - o ^ « ,CD  a, w -a a  03  CD  ODrt  w  CD  rt'  *  52 cd  I  is  ffl ^  cd  00 PH  U  O  R  O  ^ CO CD  ca  rt r trt &  CO  5g  x C=3 § ^  £ (uia) noissima uinmixem jo njSuaiaABAV  C  00 -a cd  CD  ^ cd  CD cd CD  &o  ,o  X  1  3 X -§ 0X1 CS O  5  48  3 s  cd  >-  S .S  w  x) CD c w xj cd  4.1.2 Extrinsic fluorescence The changes in ANS and CPA hydrophobicity are depicted in Figures 7 & 8. The fluorescence emission intensity of ANS is known to increase when the dye (ANS) binds to hydrophobic regions of protein (Stryer, 1965). For both treated and renatured IgY it was found that pH 2.8 treatment caused significant increases in both ANS & CPA hydrophobicity compared with pH 4 & 7 treatments. The ANS hydrophobicity of IgY treated at pH 2.8 and 37°C was twenty fold higher than the IgY samples held at pH 7 at 5°C. Goto and Fink (1989) studied conformational states of p-lactamase at acidic pH, and they reported an increase in fluorescence intensity suggesting strong ANS binding. A blue shift in A,  max  with the ANS probe was observed when IgY was treated with  acidic pH. The changes in X  max  in X  max  are given in Table 3. A blue shift from 540 nm to 470 nm  with the ANS probe was also reported for p-lactamase at acidic pH. It was  indicated that the blue shift suggested the burial of the ANS molecule in a hydrophobic environment (Goto and Fink, 1989). The literature indicates that many proteins such as Plactamase, apomyoglobin, cytochrome C, interleukin-2, ovalbumin and P-lactoglobulin have been shown to take on a typical molten globule state at acidic pH. It has also been observed that an increase in ANS fluorescence is typical of a molten globule state (Hirose, 1993). The CPA hydrophobicity after pH 2.8 treatment at 37°C was -12 fold higher than the control IgY at pH 7, whereas after renaturation it was 5 times higher than the control. There was no significant difference in emission maximum for CPA hydrophobicity. In the presence of anions some proteins do come back to the native state but some  49  proteins undergo irreversible changes leading to aggregation and permanent loss of conformation (Hirose, 1993; Habeeb, 1976). After renaturation, the hydrophobicity of IgY at pH 2.8 was still 7 fold higher than the control IgY and the A.  max  for the ANS probe  was 500 nm for pH 7 & 4 treated IgY and 480-485 nm for pH 2.8 treated IgY (Table 3) suggesting partial reversibility after pH 2.8 treatment and complete reversibility after pH 4 treatment.  50  (,-%)  AjpiqoqdojpAH  51  u rt m  HI  C CM  W  CD  rt  rt II cu  •~ s rt «  CN re  vT b ;LTS 11  3  m  T 3  & re  K rt a  cu icu  2  ^*  i  o, g  -  >H  rt "O BO re  k.  rt — s  cu  E  rt —  CU I S * -  T3 ^  _ :  CS ..  on  L«  w  &0 > H  D<  C3  i  rt CD  ES CU 3  00 cu  rt  TS  Soo  «  cu  rt  ^  co rt  CU  s  TS  BO c  6  0  rt  ^  TS  «  CL)  cu  CD O  g  rt  rt  rt  x,  ^co ES o  rt - O  a"  s  M  S  D  tS 53  CO  o co  rt  o  2  rt CD CD <rt  .5 '-3  5 rt-  *1 CO  CD  o  «»  rt  in  S 2  (,.%)  o o  IT)  Apiqoqdojp^H  52  s  CO  CS CD CD t_ TS  00  rt  6 -a  i. P  CD  rt  ~  •s s  DB ^  o o  C  — .SP  TS  ^  £2 fc- rt re o o o  CD  ° rt  3  o o o o CN  0  TS  trt C £ '  °  ©  rt rt V  CD CD QJ <u  rt  rt rt  3  rt  ^ s m 2  Table 3 : Effect of pH treatment of IgY on A,  max  with ANS probe.  (nm) pH  Treated IgY  Dialyzed IgY  7  490  500  4  485  500  2.8  475  480-485  C V <3%, n=2.  4.1.3 ELISA for lactoferrin binding activity of IgY In 1968, in a chapter on protein denaturation Tanford described immunoglobulins as a special case. He stated, 'Immunoglobulins were the first multichain proteins to be restored to their native state after denaturation to a completely disordered state, and rupture of interchain and intrachain disulfide bonds. However, the renatured molecules were found to have recovered only a fraction of their original biological activity', (Tanford, 1968). The results for ELISA assay for lactoferrin binding activity of pH treated IgY are shown in Figure 9. With a decrease in pH there was a significant decrease in ELISA absorbance readings representing lactoferrin binding activity of IgY. ELISA values for IgY after treatments at pH 7, 4 and 2.8 were found to be significantly different from each other. Shimizu et al. (1992) reported similar results; they found that IgY lost activity at pH 3 while IgG lost activity at pH 2. The rapid decrease in antibody activity with a decrease in pH suggests damage to the antigen binding site when subjected to acidic pH.  53  The decrease in ELISA values could also be due to decreased recognition of the acid treated IgY by the secondary antibody. After renaturation lactoferrin binding activity of IgY treated at pH 2.8 and 4 were significantly lower than IgY at pH 7. IgY treated at pH 2.8 at 37°C did not refold, since the  A 5 4 0  readings were significantly lower, suggesting  that increase in temperature at acidic pH had an adverse effect.  54  ffl  m  ft  u a r-.  JD  w=  a. cs 'rt "O c II «N oo rt rt -a -a  ^1  IT;  CD CD CO I J  >> rt rt _fc-  O  '•3 „ 00  fcCD CO  CD  rt ^  CD  « fc"  00  I—I  5  GO  £ rt tD  £  "O  CD  I is y,'  = CS  CO  CD  rt O o G CD -a w  r(D  • Tito c3  N  ' w  Q,  CD  T3  "3" ^ rt  Q  co  rt  co o rt —^ CD CD  a  CUD  »—<  ©  T3  •w  CD  •  -  S  —  cs  CD  .Q _  S  -  fc. '  CS  IB  aoueqjosqv  55  3  .£?  CA  Os C  7  >.  -° . > 3  P*"  * J  sot  T 3  1  0 3  o £  .2  fc- -fc'>  I—(  u  aiu  o.  CD  1  rt -a »oo fc—°  JC  CH  E |  2  CD  3  ^  rt  C  4.2 Effect of guanidine-HCl concentration 4.2.1 Intrinsic fluorescence (a) Relative fluorescence intensity Figures 10a and 10b show changes in relative fluorescence intensity of GuHCl treated IgY samples at excitation A, of 280 nm & 297 nm respectively. RFI of GuHCl treated samples measured at both excitationX of 280 nm & 297 nm increased after 2-6M -  GuHCl treatment.  There was no significant difference between control and 1M GuHCl  treated IgY. After renaturation, however there was no significant difference between any level of GuHCl concentration. These results are similar to results reported by Hattori et al. (1993b), who found that fluorescence intensity of P-lactoglobulin increased with GuHCl concentration and the renatured P-lactoglobulin was similar to the native protein.  56  AJISliajUI 3 3 U 9 0 S 3 J 0 n i J 3 A I J B P H  57  cu  C rt  'cu  ^  CO s~ ,C0  X C CU o rt T3  ft'3 OX  cd /rt 0 0  I—l  ju  c CO  o rt o O  "5 " £ 5 0u (3 CO C+H  <*  |  c-~ j o  m p  5 cd  rt a CO  "rt  CS  u  co cj s o CJ  u  > > rt T 3 rto T23 j s co co 13 co 2  00  2 ^  .3  o  CO CJ  00 T3 CD  cd co  "  cd  _ rt 00 i—i  co u i*  «2  W  cd CO u  rt rtcd >H  CO  cd  cd  PH  rt,  C CD  2  .CD  C  -s ^ coco °  "  58  cd  CD co  -4—;  A4ISU3JUI 3 0 U 3 3 S 3 J 0 1 H J 3 A p B J 3 ^ [  cd CO rt  U 2 K 2 T3CO O T3 5 CO  c o ^ o ^ r t N O o o ^ o . r r t N O  CO  • i t  s  JO  Cd  Q  o S ^ a £ r-  a °^ ox^  •—  rt  b  cd  o c  CD CO Cd • °  cd >  o O V  '—  12 -£ -ii  £ co  cd CD  O ti  O  rt  o  2 00  (b) Shift in Emission Maximum Figures 11a and lib show the effect of GuHCl concentration on X  max  of IgY  excited at 280 and 297 nm respectively, while Figure 12 shows the fluorescence spectrum of control (0 M GuHCl) and denatured (6 M GuHCl) IgY. When excited at 280 nm, the ^max for all the treatments of GuHCl treated IgY was significantly different from the control, suggesting the IgY was denatured even at 1M concentration (Figure 11a). When excited at 297 nm, control and 1M GuHCl treatment were found to be the same, but treatments containing > 2 M GuHCl were significantly different from the control (Figure lib).  A red shift in the emission wavelength was observed at both excitation  wavelengths, suggesting that with GuHCl treatment the protein was unfolded exposing the aromatic amino acids, whereas for control and 1 M GuHCl treated IgY the aromatic amino acid residues were buried. The emission maxima were 330, 335, 350-353 nm for control, 1 M and 2-6 M GuHCl treated IgY respectively. These results are similar to those reported in a previous study by Shimizu et al. (1992) on IgY and IgG. They had reported that conformational changes as monitored by a red shift in X  max  started at 2.5 -3  M GuHCl for IgY and at 3-3.5 M GuHCl for IgG. However the present results suggest greater sensitivity of IgY since changes were observed even at 1 M GuHCl concentration With renaturation, there was no significant difference between 0, 1, 2 and 3 M GuHCl treated IgY samples but for the 4 to 6 M GuHCl treated IgY there was a significant difference in A.  max  observed at 297 nm excitation. The emission maxima for  renatured IgY from 4-6 M GuHCl treatment was 334-335 nm versus 350-353 nm for denatured IgY, suggesting that partial refolding occurred to approach the 1M denatured state. After renaturation, changes were not reversible for IgY treated at >3 M GuHCl  59  concentration, when excited at 280 nm. Hattori et al. (1993b) reported that A,  max  of (3-  lactoglobulin increased with GuHCl concentration from 336 nm for native to 357 nm for the 6 M GuHCl treated sample and the values were regained after renaturation.  60  (uiu)  U0ISSIUI9  uinuipcBtu jo qjSusjaAB^  61  62  B 66. 9  6 0. 0  € 8 . GL  excited at 28.0 nm 40.  a.  4 0.  0  4  46 .  0.8  CJ CJ  3 u  CO  W  o  excited at 280 nm  CJ  "S "3  20.  I  2 y.  y  20.  _  28.0  «-—.—excited at 297 nm  Pi  -excited at 297 nm 0 .-0_  0. 0  : 10. 0  350. e  e. a. 32  0 . 0  G . 0  37 8.  0  Emission wavelength (nm) Figure 12: Emission spectra of control (A) and 6 M GuHCl (B) treated IgY.  63  4.2.2 Extrinsic fluorescence The effect of GuHCl concentration on extrinsic fluorescence of IgY with the hydrophobicity probes ANS and CPA is shown in Figures 13 & 14. With both ANS & CPA hydrophobicity it was found that 1 & 2 M GuHCl treatment had no effect, compared to the control; but from 3 M to 6 M there was a significant increase in hydrophobicity. ANS and CPA hydrophobicity of renatured IgY showed that there was no significant difference between control and 1-2 M GuHCl treated IgY, but the refolding was not complete for IgY treated with > 3 M GuHCl. Table 4 shows the effect of GuHCl treatment of IgY on X  max  with ANS probe. A  blue shift was observed in the emission maximum of ANS probe with IgY treated with GuHCl. After renaturation, the emission maximum was 500 nm for control, 1 and 2 M GuHCl treated IgY suggesting complete reversibility, whereas the emission maxima of 485 nm at 3 M GuHCl concentration and 480 nm at 4-6 M GuHCl concentration suggest incomplete renaturation after >3 M GuHCl treatment.  64  CJ T3 O s_ C 3  CJ / - i . CO  m  CS  O  rt P  •° c CS »  O +-> rt C  CO CJ  3 ,CJ  2  CJ CJ  Q -o DX)  CS J  10  fc;  fcCJ  •d c  *j  3  cs  M  o  a , C .T3 rt o O T3  „ 5  cs fc. 3  -a rt  cu  CJ  S  13  CJ  Z O  o  52 £ rt 3  C  3  CQ rt . c  U xn fc-  s g a3  £ 1 3  Oi  in  u  co  tS  •— co -O 3 , 3 'rt 1  o u  U « 3  o  *  2 g w  00 i d *S rt 3  *  T3  CJ CS CD  rt  CJ T3 CJ CO rt CJ  CO  CJ  DO 53 t 00 —i—i  w i—<  '  co  . T3 CJ ^ u S W> rt «  cj , i- -a is rt 3 DX) cj *~ CJ  v  ( o ) yt»piqoqdojpXH t  /o  65  M  "  DO o  CD T 3 rt CD  o  3 CD •O  co . i— rt  1-1  rt  CD  /—-  CO  IO  rt  ©  3  CD  ' £ rt K o  co C  rt Q «£3 s -=3  CD  O rt >,rt  '2 _O § s o "rt  "5  T3  A  <  rt '3  rt  CD CD  CD  3?  rt  rt  O  kH  OJ  CO  T3  JS  s. 3  s  3  ft  2  CO  rt •O  CD  £ 3 . 3  CD  |  c3  c  o  SS  h3  ti o rt*  3  "S >->  3  1)  T3  •tS  rt  rt  CD  cj  S3 O  CJ  u K  S3  o  3  CM C S  rt  r>  •£ o v 0  rt  rt  3  r-«  r!  ,  ,  g ffi "  O  T3  Si l-l  'rt  rt  3 TS  CD  a £  co  •Sort °  i-  ^ rt CJ CJ  k. >H  rt  rt  66  "*  33 T3 v  £>ts T3  w  c  -a CD CD  H  rt 1 - 1  >H CD  1 — 1  «  0  rt  rt  s*C CJ  ( o/ ) A^piqoqdojpXH  co CD  CD  O ti ,  a is  Table 4: Effect of GuHCl treatment of IgY on A GuHCl concentration  ^rnax  (M)  (nm)  of ANS probe  m a x  ^rnax  (nm)  Treated I g Y  Dialyzed I g Y  0  500  500  1  490  500  2  490  500  3  475  485  4  475  480  5  475  480  6  475  480  CV<3%,n=2.  67  4.2.3 UV Fourth derivative spectroscopy The  parameters related to the environment of tryptophan, tyrosine and  phenylalanine are given in Table 5. The fourth derivative spectra of control and denatured (6 M GuHCl) IgY are shown in Figure 15. At > 3 M GuHCl concentration a blue shift was observed in the UV fourth derivative spectrum near 280 nm (tyrosine residues peak position, Tyr^i). There was no significant difference in the other parameters of tyrosine peak height ratio, tryptophan peak height ratio, tryptophan peak position, or phenylalanine peak position. The blue shift in Tyr^i, suggests unfolding of IgY and thus exposing the aromatic amino acid residues. After renaturation there was no significant difference between treatments in any of the UV fourth derivative parameters. Table 5: Effect of GuHCl concentration on UV-fourth derivative parameters of IgY. The parameters were measured as defined by Padros et al. (1982). Only Tyr^i (shown in bold italics) showed significant (P<0.05) differences as a function of GuHCl concentration. Tyr^i bearing the same superscript letter are not significantly different. GuHCl  Phex  Cone  260 nm  0  259.8  1M  Tyrxi  Tyrxi  Trpu  Trp  280 nm  285 nm  297 nm  290 nm  2.3  279.8"  285.0  2.0  297.6  291.9  259.4  2.5  280.0"  284.9  1.9  297.8  292.8  2M  259.4  2.8  279.8"  285.1  1.8  297.8  290.4  3M  259.5  3.1  278.4  284.9  1.8  297.6  290.1  4M  259.8  2.7  278.¥  283.8  1.9  297.5  290.8  5M  259.6  2.8  278.3  b  284.0  1.9  297.9  290.4  6M  259.4  2.8  278.1  b  283.5  1.7  297.3  290.4  Rtyr  h  68  Rtrp  X2  10.0001  •=  5 . 0000|  11,  Phex  Tyr J | [ xl  X  w  0.00001  A  ^  ;ii  ' U f*  1  I  1  - 5 . 0000-  -10.0800 Wavelength (nm) Figure 15: UV fourth derivative spectra of control ( ) and 6 M GuHCl treated ( ) IgY. The parameters as described by Padros et. al, (1982), are identified for the control IgY spectrum.  69  4.2.4 ELISA for lactoferrin binding activity of IgY The effect of GuHCl concentration on lactoferrin binding activity of IgY measured by ELISA is shown in Figure 16. GuHCl treatment decreased the ELISA readings of IgY after treatment with GuHCl at > 2 M concentration. Control (0 M) and 1 M GuHCl treated IgY were found to be the same. ELISA readings for 2 M GuHCl treated IgY were found to be significantly lower than those of the control and 1 M GuHCl treated IgY, while 3-6 M GuHCl treated IgY were all significantly lower than control or 1 or 2 M GuHCl treated IgY. After renaturation, 1 and 2 M GuHCl treated IgY were found to be the same, while 3-6 M GuHCl treated IgY were significantly lower, suggesting that from 3 M GuHCl concentration the changes were only partially reversible. Hattori et al. (1993b) reported complete refolding of renatured P-lactoglobulin with physico-chemical techniques, but indicated differences in antigen recognition with GuHCl treatment.  70  co  CO  CD  T3  CD  g  r„  CO  l-i  J3  U3  o b ^ g C tc!  cd J3  "O  H  CD T3  ^ cd  CD  £ «  Q  O  T3 i3 CD . O  CUD  co — I i CD  rt  i_,  rt f« 33 o  rt O  co  ^N  cn - 3 CD  U  rt  "3  i-i  5 rt j °^  O  >  CD  -3  £  •2 * CD CO O 3 S 2 < 3© ec: 3 rt ca Xi V 5 rt- - 3 P H o CJ CJ  ^  H  UO  —  s  rt  co 3 o a CD 0 0 « R ra rt n cS CD c  o  • •a C3  s  <a P 3 -  ivyW P- ^ -o 1  s CO  3 °  c w Srto  CD O O  U  *o t3  CD  .2 £  ^  .CD  CO  • 33  rt  3  O  oo  'S op  .CD  CD v-  h—I  3 JH" ' c o  &> cj C  >  CJ  "S  .2 *  *^  SH  M  T3  o  c 3  rt  —'  rt '-a rt .3 00 rt r"  -3 co  , CD  u ^rt ^ o "3 00 CD T D •~  co  CD  Cd  3  DH  CD  w  ftoo 8 k.  H  1—1  u a  CO  1H  i Tt  "inr::  ' II  11  <— i t 1  rt CD  N  CD  rt  3  3 i—i T3 -« ~"g ^ 'rt  CO  d  co d  ran got? * aoncqjosqv B  71  d  CN  d  £ ^ 3 o OX 2 rt 3  T3  rlH  p  CO  3  rt  4.3 Effect of freezing and freeze drying 4.3.1 Solubility Stability of IgY to freezing and freeze drying was studied at low (0.14 M) and high (1.5 M) salt concentrations at pH 7 and at two protein concentrations of 1 mg/mL and 30 mg/mL. Samples were frozen either at -8 or at -80°C. Solubility of frozen-thawed and the reconstituted freeze dried IgY is shown in Figure 17. Solubility was determined as follows: Set A. All samples were frozen (F) and freeze dried (FD) at 30 mg/ml and solubility was measured at 1 mg/mL. Set B. All samples were frozen (F) and freeze dried (FD) at 1 mg/ml and solubility was measured at 1 mg/mL. Set C. All samples were frozen (F) and freeze dried (FD) at 30 mg/ml and solubility was measured at 30 mg/mL. In the case of set A, only FD sample frozen at -80°C at high salt concentrations (HSFD-80) was found to have significantly lower solubility than the other treatments. In set B, FD samples frozen at both temperatures showed a drop in solubility. In freeze dried samples, low salt samples showed significantly lower solubility than high salt samples. The solubility of IgY in set C with low salt was between 80-94 % but with high salt it was between 13-28 %. These results suggest that there was aggregation of IgY at high salt with high IgY concentration (30 mg/mL) but it was completely reversible for all samples (except IgY samples frozen at -80°C in high salt and FD) as found in set A.  72  i i  <  0) CO  CD 0) CO  o 00  • • •  s -a -a -s « ^  (%) A*!l!qn|os  73  K  4.3.2 CPA Hydrophobicity, Intrinsic Fluorescence, Light Scattering There was no significant difference (P<0.05) in the CPA hydrophobicity (Figure 18), intrinsic fluorescence, and light scattering of frozen-thawed and freeze dried samples of IgY from set B (Figure 19).  2500 j 2000 1500 o JS cu o  1000 --  >>  0 •  500 -  w  LS-8  HS-8  LS-80  HS-80  LSFD-8  HSFD-8 LSFD-80 HSFD-80  Freezing and Freeze drying treatment Figure 18: CPA hydrophobicity of frozen-thawed and freeze dried IgY. There was no significant difference (P<0.05) between treatments. LS refers to low salt (0.14 M NaCl), HS refers to high salt concentration (1.5 M NaCl), -8 and -80°C are freezing temperatures, and FD stands for freeze drying.  Vi  S 30 25 a u 20  O excited @ 280 • excited @ 297 S3 light scaterring  S3 o> 15  « 10  °3 < 5 S  LS-8  HS-8  LS-80  HS-80  LSFD-8  HSFD-8  LSFD-80 HSFD-80  Freezing and freeze drying treatments Figure 19: Intrinsic fluorescence and Light scattering of frozen-thawed and freeze dried IgY. There was no significant difference (PO.05) between treatments. LS refers to low salt (0.14 M NaCl), HS refers to high salt concentration (1.5 M NaCl), -8 and -80°C are freezing temperatures, and FD stands for freeze drying.  74  4.3.3 ELISA for lactoferrin binding activity of IgY ELISA results for frozen-thawed and freeze dried IgY are shown in Figure 20. The IgY samples frozen at 30 mg/mL (Set A Figure 20 clear bars) showed significantly lower ELISA values for freezing temperature of -80°C and all freeze dried samples as compared to the unfrozen sample or sample frozen at -8°C. The IgY samples frozen at 1 mg/mL (Set B Figure 20 dark bars) showed significantly lower ELISA values when frozen at -80°C at both salt concentrations as compared to the unfrozen sample which was stored at 5°C or the frozen sample at -8°C. The IgY samples frozen at lmg/mL showed significantly lower ELISA values on freeze drying (40 % of  A405  of unfrozen  sample) under all conditions studied. .These low ELISA readings may be due to aggregation or conformational changes in the antigen binding site of IgY. Skrabanja et al. (1994) reported that rapid freezing induced more aggregation of monoclonal antibodies than slow freezing. Similar results were found in the present study. Freeze-thawing of IgY at -80°C (rapid freezing) resulted in decrease in activity compared to freeze-thawing at -8°C (slow freezing). Fast freezing (quench freezing) leads to formation of relatively large ice-water interface as compared to slow cooling, which may be contributing to protein denaturation (Chang et al, 1996).  75  LS5C  HS5C  LS-8  LS-80  HS-8 HS-80  LSFD-8 HSFD-8 LSFD80  HSFD80  Freezing and Freeze drying treatments Figure 20: E L I S A absorbance values of frozen-thawed and freeze dried IgY. Set A , samples frozen and freeze dried at 30mg/mL, Set B samples frozen and freeze dried at 1 mg/mL. Solubility for both sets was measured at 1 mg/mL. Bars with different letters within each data set (capital letters for Set A , small letters for Set B) are significantly different (PO.05). LS refers to low salt (0.14 M NaCl), HS refers to high salt concentration (1.5 M NaCl), -8 and -80°C are freezing temperatures (5C stands for samples stored at 5°C), and FD stands for freeze drying.  76  4.3.4 Raman spectroscopy Raman spectroscopy was used to study secondary structure changes associated with aggregation of IgY. Freeze dried IgY samples (low and high salt) and frozen-thawed low salt samples (IgY concentration of 30mg/mL) had too high fluorescence so they could not be studied. However the insoluble pellets from frozen-thawed high salt samples (IgY concentration of 30mg/mL) were run on Raman spectroscopy and the results are compared to unfrozen IgY control (Table 6). The results indicate increase in p-sheet structure after frozen storage. Due to technical problems (The Raman spectroscopy instrument was not working) this experiment was completed only once, but the trend seems interesting, and agrees with other studies on protein aggregation (Li-Chan and Qin, 1998).  Table 6: Secondary structure of frozen -thawed and 5°C IgY. The pellet of IgY sample (stored for 2 weeks) at 5°C in 30 mg/mL concentration in high salt (1.4 M NaCl) and frozen-thawed IgY samples (stored for 1 weeks at 5°C) were analyzed by Raman spectroscopy. Secondary Structure Fractions IgY samples  a-helix  p-sheet  undefined  total  5°C  0.30  0.48  0.22  1.0  -8°C  0.04  0.75  0.21  1.0  -80°C  0.10  0.70  0.20  1.0  77  4.4 Structural conformation of specific (eluted) antibodies  The elution profile of IgY lactoferrin specific antibodies obtained by the passage of the IgY isolate through an immobilized lactoferrin column is shown in Figure 21. Elution with pH 2.8 Glycine-HCl buffer resulted in ~ 20 mg specific (eluted) IgY per -120 mg of crude IgY isolate.  Fractions  Figure 21: Typical elution profile of anti-lactoferrin IgY with glycine HC1 p H 2.8. buffer. Crude IgY isolate (25 mL at concentration of 10 mg/mL) was applied to Actigel A L D Superflow column (10 mL, 22.6 mg immobilized lactoferrin), washed with PBS pH .7 (4 mL fractions collected), and eluted with pH 2.8 glycine HC1 buffer (1.8 mL fractions collected).  78  4.4.1 Extrinsicfluorescenceof eluted IgY ANS and CPA hydrophobicity of the eluted IgY antibodies is shown in Figure 22, compared with crude IgY isolate in PB in low (0.14 M) and high (1.5 M) salt concentration. Eluted IgY antibodies were compared with low and high salt crude IgY controls because IgY eluted with pH 2.8 buffer were neutralized with 0.5 M PB pH 8 which leads to high ionic strength of eluted IgY. Surface hydrophobicity of elution 1 (anti-lactoferrin IgY eluted with pH 2.8 glycine HC1 buffer) was measured after 1 week of storage at 5°C, while elution 2 (anti-lactoferrin IgY eluted with pH 2.8 glycine HC1 buffer) and elution 3 (anti-lactoferrin IgY eluted with pH 2.8 glycine HC1 buffer with 15 % trehalose) were measured on the day following elution. ANS and CPA hydrophobicity of crude IgY isolate were the same under low and high salt conditions. Eluted IgY obtained from elutions 1 and 2 showed ANS hydrophobicity 4 and 5.4 fold higher than that of crude IgY respectively. For IgY from elution 3 with 15 % trehalose, ANS hydrophobicity was twice that of crude IgY. The emission maximum with ANS showed a blue shift with all the eluted samples compared to crude IgY. The emission maximum for crude IgY was 500 nm, compared to 480, 475, 490 nm for IgY from elution 1, elution 2 and elution 3 respectively. CPA hydrophobicity from elution 1, 2 and 3 of IgY was 1.7, 2.2 and 1.38 fold higher than crude IgY respectively. There was no significant difference in the emission maximum for the CPA probe. Surface hydrophobicity measured by both ANS and CPA probes was found to be significantly higher for eluted IgY. Also, there was a significant decrease in surface hydrophobicity of eluted IgY measured after 1 week storage as compared to the  79  hydrophobicity measured on the day after elution. This possibly could be due to partial renaturation (after neutralization) during storage. Elution in the presence of 15 % trehalose significantly decreased both ANS and CPA hydrophobicity as compared to elution with pH 2.8 glycine HC1 buffer alone and also showed a shift in A, suggesting a protective effect of trehalose to acidic denaturation.  80  max  (490 nm)  CD  cu  T3  co  o x> _  ^  OX)  5  ^ -°  oo "CD M  >.  cu rt * 3  ©  CN c  O  CD  ^  3 "CD  ^ U  X! ^bO OU  o  •3  u  oo  ^ r t coco  ^  £  3  «  <  "rt  -3  O  g  CD  to to >-> 3 CD  . „  CO  0  CD  "to r  3  5  CO  1  to* O >-j  O  CD bO ca  ?">>•» ca  rt u,  CO CO 2 <  <  rt  CL  O  S  re  U  « cO  "to  rt  p  -a CD o  55  "rt 2 rt . 3  a  3, co  2 rt  " o  S J2  TS  U "co  rt >, rt  CD -"ti bO CD  ca  """ ra  rt  U rt rt  <rt  -2  _^  C ^ «* bO T 3  1^  w  vi  >H  >,  n,  ^  CO 4J s  O  ^rt  ( J 0  " ° s  CN  ft  ca  to  CD <  co 2 C/3  o 1  CD  w> T3  5 «K  it >cn  CN  2  i-  T3  a  00  o  •a  o  CD  a .a ta . 3 -° s ° -° _ .3  O  W  c to  3  <D  3  3  ^  O  V  PH  .3.  rt P -a S-c CD  O  CD  *  < s rt-G„ • - rt  • •  CO  CD  CO  ^L; H  ..  o o  CD  O  o  o o  CN  (,.%)  O  o o  o o  CD  o o  P  O  co  ^  P '3 ^£  3  g  -to  3 CD CD  rt  _£  «  *S u .-a  81  rt  CD  ta  fCD  is *-<  itjpiqoqdojpXH  u  cO  £ to  a 5i)^  CD  's 00  4.4.2 ELISA for lactoferrin binding activity of eluted IgY ELISA of eluted IgY antibodies is shown in Figure 23. For all three elutions the ELISA readings were not significantly different from each other, at all three protein concentrations of 1, 0.5 and 0.1 (ig/mL. It has been reported that crude IgY has ~ 10 % anti-lactoferrin specific IgY (Li-Chan et al, 1998). ELISA readings of crude IgY isolate assayed at protein concentration of 1 ug/mL were about the same as the ELISA values of eluted IgY antibodies assayed at 0.1 p:g/mL, indicating that about 10 % of crude IgY isolate consisted of lactoferrin-specific antibodies. The % of antilactoferrin IgY (eluted) was found to be -17 % based on the protein assay to monitor the yield of specific IgY eluted (20 mg) after passage of 120 mg/mL crude IgY isolate through the immobolized lactoferrin. From ELISA assay only -10 % of crude IgY was antilactoferrin IgY. These differences may be due to couple of reasons; either there was some non-specific IgY with specific IgY on eluting or there was some loss of activity because of the elution protocol.  82  g  cs  •a ^ •a  = 2 « rt  3  -1  rt  "So 3  O  —I  s g  o  H  00  . rt£  "2  3  J H  •J  3  O  J3  cs  PQ  _1  00  3  in O  • •  ©  E3  rt  •£>  r^;  00 3.  L> ^  o ^  CO  o CD  <  C3  3  Cw>  1—  CU  3  O  u  3 cu — .tu  O  1— CN  o _2  cu  "3 CS  I*  \3 _3  .  g CO CO  ^ CO CD  •S  3-  CU  X>  O  cl)  CU  _3 "cD  "cs  00  o 3 o o 3  •rt*  re -a  o cn Si  £CD  3  co rt  fc  2  ©  00  u  rt CD  rt  CO -O 3  O  CN  co 3rt  3  U  CUD  • Jo  CD  3 l-J . 3  3  CD  CD J—  •—  00  rt-  '3 p 2 a, °  u 3 es  cu  83  CO rt CD  "2)  es  m a cot? * c a a u c q j o s q y  3  >->  CU  c o _2  CD  CD  _3  m 'o ^  3  •»  -3  rt -a  CD  .3  'a  M W  rt O  is to 'CD 3 irt  1 §coi->  X)  G  rt  CD  Chapter V GENERAL DISCUSSION AND CONCLUSIONS Ptitsyn (1987) suggested that protein denaturation due to temperature, pH and GuHCl concentration induces thermodynamically similar states which could be transferred into one another without any noticeable cooperative thermodynamic effects. "Protein denaturation is any modification in conformation not accompanied by rupture of peptide bonds involved in primary structure. Denaturation may be reversible or irreversible. When disulfide cross links contribute to the conformation of the protein, and if these are broken denaturation is often irreversible" (Fennema, 1985). In the present study, the degree of denaturation of IgY by acidic pH treatment was found to be different from that of GuHCl treatment. The findings of the present study are as follows: ANS and CPA surface hydrophobicities were higher for IgY treated at pH 2.8 at 37°C than GuHCl (1-6 M concentration) treated IgY. Lower hydrophobicity was observed for IgY treated at pH 2.8 at 5°C or at ambient temperatures than at 37°C for both ANS and CPA probes, suggesting that increase in temperature caused greater lability of IgY to acidic pH denaturation and thus increase in surface hydrophobicities. The intrinsic fluorescence showed a shift in A,  max  , from 332 nm at pH 7 to 333-  338 nm at pH 2.8 (4-37°C) and 350-353 nm in GuHCl concentration (2-6 M). This suggests that the degree of unfolding of IgY at GuHCl concentration of 2-6 M was more extensive than at pH 2.8. After renaturation they had come to a similar state since the ^•max  was 333.5-335 for GuHCl and 333-336 for pH 2.8 treated samples after renaturation.  84  The UV fourth derivative spectroscopy did not show any difference with change in pH and temperature. With GuHCl concentration there were significant differences from 3 M concentration primarily in the Tyrxi parameter related to tyrosine residues. ELISA activity of IgY was sensitive to changes in pH and temperature. Loss of ELISA activity of IgY samples treated at pH 2.8 at ambient temperature and at 5°C was partially reversible, restored after renaturation to pH 4 state but an increase in the temperature of pH 2.8 treatment to 37°C had a significant effect on ELISA activity which was not regained after renaturation treatment. ELISA activity of IgY treated with up to 2 M GuHCl concentration was lower than the control (0 M GuHCl) IgY after renaturation, and greater loss was observed after >3 M GuHCl treatment, suggesting that the changes resulting in inactivation were only partly reversible. ANS, CPA hydrophobicities and ELISA were measured after pH or GuHCl treated IgY samples were diluted in pH 7 PBS, whereas intrinsic fluorescence and UV fourth derivative spectroscopy were measured at that pH or GuHCl concentration. The differences in the degree of denaturation of IgY observed with different techniques may be due to the differences in the way they were measured. ANS, CPA hydrophobicities and ELISA measurements suggest pH 2.8 at 37°C was harsher than GuHCl treatment (the GuHCl samples may have partly renatured during the time required to prepare for the assay, but the pH treated IgY may not have renatured). On the contrary, the reverse is shown for intrinsic fluorescence measurements, GuHCl treatment induced more severe changes than acidic pH. This supports the results from renaturation studies, suggesting that the GuHCl treatment while causing more unfolding was partly reversible, whereas much of the changes after pH 2.8 treatment at 37°C were not reversible.  85  It had been observed in the past that exposure of proteins to heat at acidic pH results in reversible changes (Habeeb, 1976; Tanford, 1968). However Dorrington and Bennich (1973) reported irreversible thermal denaturation of IgE at 56°C for 30 min; they suggested that irreversible denaturation of IgE was due to the Fc part, since thermal denaturation of the Fab fragment was reversible. Similar results were reported for mammalian IgG at acidic pH. Freezing of IgY at -8°C did not affect the solubility and ELISA significantly. Freezing at -80°C and freeze drying resulted in a significant drop in solubility and ELISA values. Draber et al. (1995) studied stability of monoclonal IgM to freeze drying. They reported a 70 % drop in ELISA values with freeze drying and demonstrated that excipients like trehalose are effective in stabilizing antibodies to processes like freeze drying. To prevent loss of activity of IgY with freeze drying, the use of excipients like trehalose is recommended. Katakam and Banga (1995) studied aggregation of insulin by incubating with moisture at 37°C, or in solution by lyophilization, and shaking or multiple passage through a needle; and reported that aggregation could be reduced by carbohydrate excipients. ANS and CPA hydrophobicities of eluted IgY antibodies were significantly higher than the crude IgY isolate suggesting changes in surface hydrophobicities, but these changes may be reversible as ELISA activity of IgY was not affected. Trehalose in eluting medium had a protective effect on IgY as it decreased surface hydrophobicity of pH 2.8 eluted IgY.  86  Scope for future work  •  IgY is found to be sensitive to acidic pH at 37°C, and this may affect its activity in physiological conditions, hence its activity under physiological conditions could be tested with different carrier systems.  •  Denaturation of IgY resulted in decrease in ELISA value. It is not known if this is due to changes in the Fab fragment or in the Fc fragment. It would be interesting to study this aspect.  •  IgY is sensitive to temperature changes, and shows an interesting trend where decreases in oc-helix and increases in (3-sheet content occur with freezing. This aspect could be further investigated.  •  IgY is found to be sensitive to freeze drying. In order to improve its stability, several excipients, and different freeze drying cycles could be tested.  •  It is suggested in the literature that IgY is stable at 4°C but the stability of IgY at 4°C on long term storage has not been investigated. As well, stability of IgY during frozen storage has not been investigated.  •  IgY can be eluted from an immunoaffinity columns by acidic media, but IgY may not be stable to acidic conditions as shown by the increase in surface hydrophobicity after elution. It would be interesting to study protective effects of different excipients in acidic elution media on IgY. Also, GuHCl <1 M concentration could be tested in eluting medium at neutral pH.  87  BIBLIOGRAPHY  Acton, R. T., Niedermeier, W., Weinheimer, P. F., Clem, L. W., and Bennett, J. C. 1972. The carbohydrate composition of immunoglobulins from diverse species of vertebrates. J. Immunol. 109: 371. Akita, E. and Li-Chan, E. C. Y. 1998. 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Analysis of Variance (Balanced Factor pH Temp Analysis  TyP Levels Values fixed 3 2 fixed 3 4 e  of Variance  Source PH Temp pH*Temp Error Total  MTB  Designs)  DF 2 2 4 9 17  > ANOVA  4 25  7 37  f o r I@Exi280  298 85 82 39 505  SS 493 935 538 438 404  MS 247 967 634 382  149 42 20 4  F 34 06 9 81 4 71  P 0 000 0 005 0 025  'RE@Ex230' - pH Temp pH" Temp  Analysis of Variance (Balanced Designs) Factor pH Temp Analysis Source PH Temp pH^Temp Error Total  Type L e v e l s V a l u e s fixed 3 2 fixed 3 4 of V a r i a n c e DF 2 2 4 9 17  152 17 11 1 184  4 25  7 37  f o r RE@Ex280 SS 632 973 996 919 520  76 3 2 0  98  MS 316 986 999 213  F 357 91 42 14 14 07  P 0 000 0 000 0 001  ANOVA analysis for Figure 5b data. MTB > ANOVA  'ieexias?'  = pH Temp pH* Temp.  Analysis of Variance (Balanced Designs) Factor pH Temp Analysis Source PH Temp pH^Temp Error Total  Type L e v e l s V a l u e s fixed 3 2 f ixed 3 4 of V a r i a n c e DF 2 2 4 9 17  4 25  7 37  f o r I@exi297  SS 62.247 20.992 18.581 2 . 276 104.096  F MS 31.123 123 07 10.496 41 50 4.645 18 37 0 . 253  P 0 000 0 000 0 000  Analysis of Variance (Balanced Designs) Factor pH Temp Analysis Source pH Temp pH*Temp Error Total  Type L e v e l s V a l u e s fixed 3 2 fixed 3 4 o f V a r i a ce DF 2 2 4 9 17  4 25  7 37  f o r Emi=297R  SS 7.4444 3.1111 2.2222 3.0000 15.7778  99  MS 3.72 22 1.55 56 0.55 56 0.33  F .11 17 4 67 1 67  p 0 004 0 041 0 241  ANOVA analysis for Figure 6a data. MTB > ANOVA  •Emis-280' = pH Temp pH~ Temp  Analysis of Variance (Balanced Designs) Factor PH Temp  Type L e v e l s V a l u e s fixed 3 2 fixed 3 4  A n a l y s i s o f V a r i a n ce Source PH Temp pH*Temp Error Total  DF 2 2 4 9 17  MTB > ANOVA  for  7 37  Emis'-2 8 0  SS 52 .000 14 .333 13 . 667 2 . 500 82 . 500  'Emi-2 8 0R'  4 25  MS 26.000 7.167 3.417 0.278  93 25 12  F P , . 60 0 .. 000 , . 80 0 .000 . . 30 0 .001  = pH Temp pH* Temp  Analysis of Variance (Balanced Designs) Factor pH Temp  Type L e v e l s V a l u e s fixed 3 2 fixed 3 4  A n a l y s i s of Variance Source pH  Temp r,u*T. pH*T E r r o :r Total  ff!S  ~mp  DF DF 2 2 2 ~ 4 i9 9 17  4 25  7 37  f o r Emi-280R  SS SS 36 . 1111 5 . 7778 ~> . i i i o 6 .,5556 6.5556 P. nnrm 6 .0000 . 54 .44 4 4 3 6 . 1 1 1 1  100  MS MS - i f t. r . 0556 R K < r 18 2 ^.8889 ,. B B 8 9 1 ,6389 1.6389 r. . 0 6667  F P F - ^ ~ ~ 27 . ~08 0.000 4 4,33 ,, 33 0048 2.46 0 121 2 .46 w.x^j.  A N O V A analysis for Figure 6b data.  MTB  > ANOVA  ,  E m i s - 2 9 7 ' = pH Temp pH* Temp.  Analysis of Variance (Balanced Designs) Factor PH Temp Analysis  Type L e v e l s V a l u e s fixed 3 2 fixed 3 4 of  Source PH Temp pH*Temp Error Total  7 37  V a r i a n c e f o r Emis--297 DF 2 2 4 9 17  MTB > ANOVA  4 25  SS 9 3333 2 3333 1 3333 1 0000 14 .0000  MS 4.6667 1.1667 0.3333 0.1111  F 42 00 10 50 3 00  P 0 000 0 004 0 079  'Emi=2 9 7R' = pH Temp pH" Temp.  Analysis of Variance (Balanced Designs) Factor PH Temp Analysis Source pH Temp pH*Temp Error Total  Type L e v e l s V a l u e s f ixed 3 2 f ixed 3 4 of  Variance for DF 2 2 4 9 17  7 3 2 3 15  SS 4444 1111 2222 0000 7778  101  4 25  7 37  Emi = 2 9 7R MS 3 . 7222 1.5556 0.5556 0.3333  F 11 17 4 67 1 67  P 0 004 0 041 0 241  Ci Ci  Cd  04  o o o  o o o  o o o  o  o  o  w rt CM HI  O O O  O O O  O O O  o  o  o  o  o  rt  Q o o  H  M  0 1 o r r vo t-i  Cu  rH  rr m  CN 00  i—i  VD ID LO  m  VD  m  I  fa  cn  0  rt  r-  H  cn cn  vo rr  rm  rt  CO 00 00 o 0 0  u  + + +  W  cn  tr> Ol a i  I O  vo CN  o CO M  W  VO r-l  w u  rt  a  1  M  1-1  r~ rr  cn  CO  CO  CM  w  VD cn 03 r r r-l VD CO 0  rH  CN CN  •  a  cp  CO CO 0 0  + + +w Cd rw - r- n  rt T3 cu s  CTl  m  Ol cn  1-1  r r VD  CN CN  C>< H  .o  r~  rt 0  r-  1-1  -  m  • VD  0 0 0  •  m r—  m  rH  VD  cn 0  m  CN CTl 0 CN CN  rt  CN CN r r  cn  co CO  rt rt  cn  0  LD  o  CN CN r-  r~ m in rr  M  IT)  4^ CN r r  m  o  r r VD  m -t- r r in  O  O  T  Cd • • m H rr  m  -  cn vo  1-1  CN  r r o co r r r- rn o co o  rr rr CN  rH  CTi rr  cd cn  co  o o o m rm  CN  m co  co  in  rt e « > O  3 >  Q  CM  W Q  Z, <  102  Q  r— Cn  rcn cn  Ci  w hH  W  O  O  o o  o o  o  o  o  o o  cu M EH HH  1 rt>  01  CM  1—I fH  O O H  LO LD  Cn  I  n  cn cn  W  cn  1  CN CN <T  i  •<s*  I  cn  O  O O O  O O O  O O O  o  o  o  CO H <3- H ID o  r~  CO  ro  co in roi n H  o  Cn  cn co co o o o  CC  w  4-  +  +  W W W T T l£> «3"  CM l-H  ID  r- o I D r- I D cn T cn co N n N  EH rH  CN CN CN  w  CN  CM M  o  co I D cn o in ro  W O  +  W ID  n ro rcn  > w o  N  cn CO CO  rH  •  r-  <r in ro o rH o • r-  EH  ID CN CM  O  N  • CO  ri ro ID  r-  cn ro o o o CN-  ro T  cn  cn  ea  w oi  •>— e3  TJ co ea  <« a to. I  s  Cu O  DX) u  .o  cn o + W cn  co cn o o + + W W ea I D  CN CO  ID  in o co m CM in cn cn rH  <rt r-  rH  CO CO CO  o o o  CN r H "C  cn in ro o o  H  CO  o o o  o o o + 4- 4W W W  o  r- r~ ID o o cn "sr n H  CN 10  rro o  m o cn ro  CM  xn  Vi  >.  13 c es  < > o  z, <  cu w  a  w u cc t> o CO  W  § a§ w Cu £H Cu  O  C4  Q  o  co  W  103  EC W K Cu E H Cu  w  ANOVA analysis for Figure 9 data. MTB  > ANOVA  'A 4 0 5' = pH Temp pH* Temp.  Analysis of Variance (Balanced Designs) Factor pH Temp  Type L e v e l s V a l u e s fixed 3 2 fixed 3 4  A n a l y s i s of V a r i a n c e DF 2 2 4 18 26  Source pH Temp pH*Temp Error Total MTB  > ANOVA  4 25  7 37  f o r A 405  SS 3 . 08846 0.11938 0.08721 0.51454 3 . 80959  'A-4 0 5Re'  1 0 0 0  MS 54423 05969 02180 02859  F 54 02 2 09 0 76  0 0 0  = pH Temp pH* Temp,  Analysis of Variance (Balanced Designs) Factor pH Temp  Typ© L e v e l s V a l u e s fixed 3 2 fixed 3 4  A n a l y s i s of Variance Source pH Temp pH*Temp Error Total  DF 2 2 4 18 26  0 0 0 0 1  4 25  7 37  f o r A—405Re SS 63303 23880 18596 09456 15235  0 0 0 0  104  MS 31652 11940 04649 00525  F 60 25 22 73 8 85  p 0 000 0 000 0 000  vo  (U o o o  H  in  o co r f  vo  04  W 2  CO CT\ cn 2 CM in  c C O  a  cs  « o 3 OJO  .o 1/5  >,  13 a CJ  > O  w CD O 3  >  c ra ra m  rt  8c  CD  CD TJ  a, >xID  ra  4H  5  •s >>  ra  u  U  •P  CO n rr r>-  10  C«- rH 00  C\ rt b H H f l VO VO  CD  > CD  ro CD  MH  rt  -P  -P  •p ro u CD ro u \u - P  CO CM  c  rt 4H  ro rt U ro  tL| VD  > P  o o rH  4H  rt  ro c <  CD In  -  CJ IH  3  -P  ro o ro CD  O lH  CO  I H - H -  -P  IH +> IH 0 W H  105  Q  ra  ra m  3 " c ra •s en  H  rt  HH ,  CD  3  H  ©  wO .  CD  rt ro  in  >  IH  W ^  rt  CO  rf  CO  CO r f CM  0  >  r f CO  <T\  CO O (Ti CM H H CM  CD U  CD  C  CD  ro  •H CD  a  < •c > 5 o  CD  s  -  o  CPJ  (0 0  '  U  -H CO  u  II  e  H CM  vo t".  CO CM  •  H  ca CD  rt ro  o  o  •H  or  rt  O CO CM  t».  XJ CD  (H  ro  >  CLI  P  vo r>- ro H  MH  0  cn  rt  In  CO  0  -P  U  CD  >»  •p ro  ra  ro  k  u -P  > rt  CD  U  v U PH  3  -P  k ro o ro CD U ro c 0 k U 0 < co -p w h  10  VO  in  Pn in in o  fc o o o ro  fc  fc c\  CN  ro  CO  CN  T  r> •p ra  CD M  •p  cs  .a 1—I  cu  SD  £ u o cs c cs  < > O  rt 6)  Q  X3 O U  -s o  « c ra  CN  o  -rH  u m  > 0  c  ra  ra m  3 c ra  w  CD  >»  ra  5  VO  CM  rH  H  CO LO  (71  O  •p ra  ©  O  3 rH  •H  rH  -p  X  |_, 0  >  (H  U CD  CO o *<r CO VO LO rH l O  CD  CD > CD  rH f-  •4  CD f 0. C D  10  >  <N  ©  fc vo r- ro Q  0  -P  -P  (0  U CD  ro u fc - P  w >.  rH  CD  CD  C  >  IH  a co  IH r H  ^ ra o ra  <  U U 0 to - P W h  3 0  CD  106  IH  -P  H rH  ro  "V rH  ra  >  fc vo r» ro Q  H  0  Cfl •H  to  IH -P  H  W in o co in ro co CN in  ra  o z  U  10 <^ CN  u  < > CD  0  4H  CO  H  rH  ra c  IH  CD IH  CO r>  H o  ©  rH  (H  CD  0) rH  s  OV  0  •rH  I-  OV  •H  tH  3  rH '  >  CN C A OV  W CD O  ra  u c ra  •s CO  <T\  o ro X vo o  10  CO co L O S oo vo r H ro - •  0 P  P  (0  U OD [Q  fc  IH P  >  rH  ra c  <  CD U -P U in  3  -H  ra o ra ®  (H  P  UU 0 to - P W H 0  VO  in  CM CM o o  On o o o  FA  lu O ID CM  CM  CO £  •p  ca CD  u  rt «  2  Q o  CU  c ra  CO CM  1 C  e> re w re -<H ex £ m  i3  CD  C es  < >  o  > o  8  c •re c re > «  CO >»  re  CM  CO in n S  H  c-  rH  o  c\ o  CO CM  rH  • •P  10 CD  ©  cn  tn CD O  3  rH  (0 >  tn r--  rt  IH  •H  E CD U 0  -P  CO r H O r H CO r> o f-  4H  -  -  •  RF  in  ON  CD  CD  U  >  o  o  rH  rH  rH  -O CO CM <2>  CD  -H  CD TJ  a CD >x  u  (0  >  Cn  VD  r-  Q  CO H  co w  •H  u -P  >1 r-H  •P  (TJ  (CJ  «  IH  U CD -P  c•  <  -  <  M-l  > o 2  0  CM  II  c  CD  0  H  CD U  -P  IH  -H  U 10 0 10  3 CD IH - P 0 U U 0 CO - P W H  cc  107  c s a 1 c en  TO ra  m  8  c rra ra > £ en >» re  O H 00 CM  CO CM  CM  O  <B  co  CD 3  o  O  rt  rt"  CD  «  CO  (H  >  CO t> rt CD > CD iJ CD TJ a CD  £3  0  4H  CD U  CO CO  H  O  H  C- o F in o ir> CO in rr; VO H H  CO RT  c  to  rt  u ca [II VO > n  C~-  r  HH  0 CO  rt CO  IH  0  -P  •P  <0  U  CD  CO  u  CM  -P  > rt (0 c <  CD CJ - P U U tO 0 (C 3 CD t-i • P 0 ^ IH C CO - P w t-  vo  If)  ao o o  CM O O  o  ro P-i  CA oo  fc r-  ro 1-1  vo  ro  CN  CO CA ^ 2 t> \o  •p CS  CJ cn  a  13  3  6X1  to o  CA CM ©  w  -H  e CD  >  o  10  0  c  ro re  co  c  I  re  ra  •rH  U  ra t-  -H CD > CD  0  O V UT>  4-1  rH CM rH  CD  U  c 10  ,4 CD TJ a CD ><*  rH <M H  u 10  >  CM  P  >»  ro  5  0  -P  •P  (0  U  CD  10 IH [H . p  10  c <  •  >  >> CD  to ©h  CD U -P  IH  ©  ro  o  a  c  CO CO  0  CD U  vo c-- ro H  Cs. rH • •  sc ro  CM  CD  U-l  CO  u  s  00  « in  0  CO  8 c  • I -  l+H  •H  co ro ro  CA CM  CD  W H O H CO [s. if) CM  ST  >  CD O 3  >  < 3 es  ©  t r eat  CD IH  * c  On  to  s  em  +3  CO  rH O O <N  Cs<T\ <N  en  CA <N  o cn o CM  T  O N*  ro CM  CM . CN  10 •H  u (0 > fc vo I- ro s  P  H  rH  U 10 0 10 3 CD IH - P 0 IH IH 0 CO - P W H  108  < co  -P  wh  vo  vo  in  CH O  PH O O O  o o o  fc  PH co CO vo ro  CO lO  CO  ro  CO  S  CN  (N to in vo Cs  <N if)  in  cs. r H rs.  vo ts. o CO rH •«r CN  CN  CO i-H  in  cn  rt  es T 3  ro  3  eo 0>  Q  •o o c  ro ro  OD  na  to v.5  0) "  tn  ^  C  to  CO rH  3  ra  CO  CD  •<  >  IH  CO rv.  IH  CO CO  0 •+H CD  0  > CD  T oo oi CN co CN  o\  C  0  r-l  CN ro o CT\ N* CO vo  O <N ro  TJ  a co >x  CN o-  HH  < H  O  < >  HH  ro  to  0  -P -p irj O  CO  (0 u  fc -P  ><  r—( ia  c  <  CD  IH  a  •o 8 c ro  ro m  0 2 O IH  3 0 CO  -P trj  IH  CD IH IH  -P  IH  -P  >» ro  0  w h  109  P 2  r-H  ra  W  2  CO  >  IH  0  •<r ro ro co CN o rN C\ rH o  in  HH CD  CD CJ  CD  C  >  a  >x  HH  IH  ra  >  in  N*  H  H  ra -rH CD  o  o a\ to Ol OO  r-H  5 o  -H  0 (0  H  3  CD T J  ro  jO CO  CD  IH  CO CD  rH  2 fcvots.ro  CO •rH  rH  -  C)  CO  0  co < c > >o»  II  00  IH  >  •P  rH  •rH CD  CD  c p  2  -4  es 3 es  o  T  rH VO rH  £  fc VO Cs P  ro H  HH  0 CO -•H CO  IH  0  -P •p ra U  CD  ra u fc P  >  r-H  rej C  <  CO  U  4-1  IH  r-H  ra o -P ra 3 CD O uU 0 W •P w h IH  IH  vo  in CM  fn o o o  O O O  CM CO  ro  co  ro  CO  CO CM CM  m co ro vo rr  CO CM  X  CM  CT\  rH  0  rt  (0  > CO  rt  r>-  cn  u 0  00  •  co  -p  CO C M VD C M CO  HH  CD  CD  U C 10  > CD i-J CD  < c^  cr, rr o ro a\ in CM o o CM  ro co rr co o o CM  •H  T)  a CD MH  ro  >  CM  vo r- ro  rH  Q  u CD 0 M  CM - P  rt ro c •<  -CD U  CD  in ro 3 CD 0 IH  u -  A  o ro u -P IH  0  C O - P W H  no  s  o TJ  O  O E « ca  8  c ta r ca >  U  CO (J  3  < Pi u  >  u  H  rt ro CO  rt  r-  >  M  Z  MH  1/1 ^  ro in co 00 CO ro ro  c  CD  (0  TJ  a CD >x MM  rt u ro  >  s  o in vo ch co rvo ro o H  •4 CD  CO  CD U  CD  I*  CO CO VO r p  0  fa  VO  P  r- ro H  MH  0 CO  rtCO  !M  0  -P  ro CD ro u fa  JJ  CQ  00 ro vo  CD  c  < m  •<  •H  to  II  2  CO  0 rt +> id  ro CD u •P  < > o  MH  0  u  H  VO O l  CM  rr  3  CM  rH  CM  CO CO  in  Z  U  -P  > rt ro c  CD U  -P  U rt  in ro o ro 3 CD P . - P 0 P. k O  < co  -P  W E-  ANOVA analysis for Figure 16 data. One-Way Analysis of Variance Analysis Source Cl Error T n t a l  o f V a r i a n c e on GuHCl DF SS MS 6 5.54623 0.92437 14 0.08294 0,00592 20 5.62918  F 156.03  p 0.000  MTB > Oneway G u H C l r e C l ; SUBC> T u k e y 5. 1  1  One-Way Analysis of Variance A n a l y s i s o f V a r i a n c e on GuHCl r e Source DF SS MS Cl 6 0.84562 0.14094 Error 21 0.03509 0.00167 Total 27 0.38071  111  F 84.34  p 0.000  ANOVA analysis for Figure 17 data. MTB  > ANOVA  'concsolu'  = temp s a l t  temp*  salt  Analysis of Variance (Balanced Designs) Factor temp salt  Type L e v e l s V a l u e s fixed 4 1 fixed 2 1  A n a l y s i s of Variance Source temp salt temp*salt Error Total MTB  DF 3 1 3 16 23  > ANOVA  2 2  f o r concsolu  SS 17.2 24071.1 796.0 341.2 25225.5  'Dil%solu'  MS F 5.7 0.27 24071.1 1128.78 265.3 12.44 21.3  = temp s a l t  temp"  P 0.846 0.000 0.000  salt.  Analysis of Variance (Balanced Designs) Factor temp salt  Type L e v e l s V a l u e s fixed 4 1 fixed 2 1  Analysis  of Variance  Source temp salt temp*salt Error Total  DF 3 1 3 16 23  for Dil%solu  SS 2893 . 24 211 .59 1185 . 74 482 . 66 4773 . 23  con C % S O l '  2 2  964 211 395 30  = temp s a l t  MS . 41 . 59 . 25 . 17  F 31 . 97 7 . 01 13 . 10  p 0 000 0 018 0 000  temp* s a l t .  Analysis of Variance (Balanced Designs) Factor temp salt  Type L e v e l s V a l u e s fixed 4 1 fixed 2 1  A n a l y s i s of Source temp salt temp*salt Error Total  V a r i a ce ' DF 3 1 3 16 23  for conc%sol MS 474. 11 317. 34 324. 67 19. 82  SS 1422.34 317.34 974.01 317.18 3030.86  112  F 23.92 16.01 16.38  P 0.000 0.001 0.000  ANOVA analysis for Figure 18 data.  One-Way Analysis of Variance A n a l y s i s o f V a r i a n c e on H y d r o Source DF SS MS NaCl 1 41209 41209 Error 14 360638 25760 Total 15 401847  ANOVA analysis for Figure 19 data. MTB > Oneway 'I@exi280' SUBO Tukey 5 .  'temp';  One-Way Analysis of Variance Analysis Source temp Error Total  o f V a r i a n c e on I@exi280 DF SS MS 5 2 . 79 0 56 6 18.54 3 09 11 21 . 34  MTB > Oneway SUBC> Tukey  I@Exi29 7' 5 .  'temp'  One-Way Analysis of Variance Source temp Error Total  V a r i a n c e on I@Exi297 SS MS DF 0 . 145 5 0 .723 0 . 229 6 1 .372 11 2 .095  US=Cf/« mi £> * i t MTB > Oneway 'I§Emi355' SUBO Tukey 5 .  'temp';  One-Way Analysis of Variance A n a l y s i s o f V a r i a n c e on I@Emi355 Source DF SS MS temp 5 4.98 1.00 Error 6 10.93 1.82 Total 11 15.91  113  rH Ol  n r-  o OS  OS  w  w  o  rH  CTi CN  M  CM  ~H  rH  o  o  rH  o  *T T  O CN  O  O  O  to OS M  a  I  CO  I  tu  in  r-  Ol H  O H O U )  O'I  T  Cu !—t HH  o n  £. es < ;> fc O co  >H rJ  cs  TJ  in -c r-i  <  CO CN r H CN r H l-H  ex-  <u  s  1  O  O  o  P  Cd  rH n CO o CN  Ol o  O  CO  o  o  o  <T  H  T  i—I  rH o m  <r H  o CJ  f  fc o  SI  co  P  fa  i—i  T M  i—I  r-H  Ol >r  i-i  o  o  r H  CN  <  «T  o  CO rH  co  m o o  o o  u  rH  Cu  < fc  Cd  PS  m  o o  m m  rH O  O  rH O  O  CN  CN ID  fc o  DX)  rH  in  I  cs <  o  O  l Cu  n  OS  M CO  C3  O) CO CN r~ Oi n rH m  Oi  a H  O  CO'  m co  r/5  O O  Q O  )-  Cu  H  P  .o  o co in  CM rH  o m N o o o  Cu  o  rH  2  M  O O  P  13  > o  <  as  CO  >< Cu  Cd P  c 0")  a..  OS  Cu  o QS OS  rS fa"  114  r*  Cu  w p  Cd O OS  o co  CO •K  OS  Cu EH C u  o  EH CO  Cd  H  ANOVA analysis for Figure 22 data. MTB > Oneway 'ANS' SUBC> T u k e y 5.  'Eluted';  One-Way Analysis of Variance Analysis Source Eluted Error Total  o f V a r i a n c e on DF SS 4 2867618 5 1157 9 2868775  MTB > Oneway 'CPA' SUBO Tukey 5 .  ANS  MS 716904 231  F 3098.12  p 0.000  F 64.90  p 0.000  'Eluted';  One-Way Analysis of Variance Analysis Source Eluted Error Total  o f V a r i a n c e on CPA DF SS MS 4 1058546 264637 5 20388 4078 9 1078934  115  CN  in CTi  C<  W  O O O  cu M  E-t rH  P  O M  a  EH  CO  fa  r-  CO o rr rr  l£>  m  ta  KM  Cu M  rr CN  o  O  1—1  CO l  O O  •  o  o  fa Q  CTi  o  CO M  CO  •  EH HH  fa  o  C3 T3  oo  S CN  M  m  cn  CM CD i-  Kg  3  CN  rt rt rH  CN  o  o  OJ)  •  D.  a  1-  •  CO  o  1  fa  o !  "3  a  CO  es  < > O  <  I  u  CU  D O co  w Q  116  M  rH CU  Cd  pi o Cd  ANOVA analysis for surface hydrophobicity of dialyzed versus undialyzed control  One-Way Analysis of Variance Analysis Source treat Error ^r,"fZ1  o f V a r i a n c e o n ANS DF SS MS 1 21049 21049 27 3100 0 7 1143 2 23 3 310 5-5  F 1.33  p 0.137  F 2.37  p 0.135  MTB > O n e w a v ' c P A ' ' t r e a t SUBC;Tukey 5 . One-Way Analysis of Variance Analysis Source trea t To t a 1  o f V a r i a n c e on cFA DF SS MS 1 10 7 6 8 0 10 7 6 3 0 2S  1336 9 60  117  ANOVA analysis for Table 5 data. MTB  > ANOVA  ' 280'  =  file.  Analysis of Variance (Balanced Designs) Factor file  Type L e v e l s V a l u e s fixed 7 0  A n a l y s i s of Variance Source file Error Total  DF 6 7 13  1 '  2  3  4  f o r 280  SS 8.7411 0.4375 9.1786  MS 1.4568 0.0625'  118  . F 23.31  P 0.000  5  6  

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