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A study of some physical and chemical properties of egg white Beveridge, Herbert James Thomas 1973

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c.l A STUDY OF SOME PHYSICAL AND CHEMICAL PROPERTIES OF EGG WHITE HERBERT JAMES THOMAS BEVERIDGE B.S.A., University of British Columbia, 1968 M.Sc, University of British Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY .in the Department of Food Science We accept this thesis as conforming to the • required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1973 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Food Science The University of British Columbia Vancouver 8, Canada Date June 19, 1973 i i ABSTRACT A three-part investigation is described in which gel filtration was utilized to detect electrostatic interactions claimed to be important in determining functional properties of egg white; viscometry and ultracentrifugation were employed to detect changes which occur during aging of the egg; and beating of egg white for foam formation was studied utilizing bubble size analysis, gel electrophoresis and gel chromatography. A method for the gel filtration of whole egg white on columns of Sephadex G-150 was developed. Dilution of egg white 1:1 with 0.01 M phosphate buffer, pH 7 . 0 , containing 2$ (w/v) NaCI and 0.6$ (v/v) 2-mercaptoethanol caused rapid and complete dissolution of the thick white gel and resulted in a solution amenable to gel filtration. Elution was accomplished with 0.01 M phosphate buffer, pH 7 . 0 , containing 1$ NaCI and 0 .3$ 2-mercaptoethanol. Four distinct regions were discerned in the elution profile. Disc gel electrophoresis of the regions and the elution volume of the purified proteins were used to identify the components in the various regions. Region 1 contained high molecular weight material eluting at the V0 position where purified ovomucin eluted. Region 2 contained ovalbumin and conalbumin whereas regions 3 and k contained ovalbumin and lysozyme respectively, as major constituents. Lysozyme decreased in region h and increased in region 3 when the salt concentration was decreased to 0 .25$. A rotational viscometer and an ultracentrifuge were used to evalute the changes which occur in egg white and in ovomucin on aging. Egg white was found to be a time-dependent pseudoplastic fluid exhibiting decreasing apparent viscosity with shearing time. The time-dependent characteristic was fitted by an equation A = AQ - n.log t and the in i t i a l shear stress index (AQ) was found useful as an index of egg white thinning. Potassium bromate gave slightly lower values of A0 compared to an untreated control whereas KIO3 gave higher values of AQ. Sulfhydryl (-SH) levels were unaffected by KBrOj but KIO3 reduced them from 59 to 5^ uM/g protein. Blockage of up to 25$ of the -SH groups of egg white with mercuric chloride or p-chloromercuribenzoate resulted in reduction of rate of change of AD with incubation time, however, more extensive blockage with the same reagents caused increases in AQ suggestive of aggregation reactions. Ultracentrifugation of ovomucin isolated from -SH blocked egg white showed that the relative increase in a-ovomucin which occurs on aging untreated egg white was inhibited, but not completely, by blocking some -SH groups. The beating of egg white for foam formation was studied utilizing bubble size analysis, gel electrophoresis and gel chromato-graphy. Bubble size distributions were found to be fitted best by a lognormal distribution curve for a l l beating times tested up to 10 minutes and a l l aging times up to 20 minutes. The average bubble size increased on aging and the change was considerably faster for foam beaten 8 minutes than for foam beaten 2 minutes. In addition the polydispersity of the foam as indicated by the standard deviation of the distribution curve increased much more rapidly for 8 minute foam than for 2 minute foam. Quantitative data detailing these changes are presented. Disc gel electrophoresis of the material drained from the foam showed progressive depletion of lysozyme and the G^  and G3 globulins as beating time increased. After 8 minutes beating the globulins had essentially disappeared from the drained material. The depletion of lysozyme was confirmed qualitatively by an enzyme assay. Chromatography on Sephadex G-150 showed changes reflecting those observed by electrophoresis and also confirmed observations by other authors that ovomucin is transferred from egg white to the foam when egg white is beaten. Essentially a l l of the material eluting at the V0 position was removed in 2 minute beating with only a slight further decrease by 8 minute beating. V TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i i LIST OF FIGURES ix ACKNOWLEDGEMENTS x i i INTRODUCTION 1 CHAPTER I: SURVEY OF THE LITERATURE 2 Egg White proteins 2 A. Ovalbumin 2 B; Conalbumin 5 C. Ovomucoid 6 D. Lysozyme 6 E. Ovomucin 7 1. Isolation 8 2 . Solubility 8 3 . Heterogeneity 9 k. Composition 11 5. Molecular Weight 12 F. Globulins 13 G. Other Egg White Proteins ik Functional and Structural Properties of Egg White ik A. Structural Properties ik B. Foaming Properties 19 v i Page CHAPTER II: GEL FILTRATION OF EGG WHITE 22 INTRODUCTION 22 MATERIALS AND METHODS 25 RESULTS AND DISCUSSION 27 Effect of Gel Type 27 Effect of Column Length 3k Effect of Salt Concentration 3^ GENERAL DISCUSSION k'y CHAPTER III: EFFECT OF 3ULHYDRYL BLOCKING ON THE RHEOLOGICAL PROPERTIES OF EGG WHITE AND ULTRACENTRIFUGAL PROPERTIES OF OVOMUCIN ISOLATED FROM BLOCKED AGED WHITE U7 INTRODUCTION ^7 MATERIALS AND METHODS 50 Treatment with Iodate, Bromate and Cysteine 50 Treatment with HgClp and p-Chloromercuribenzoate 50 Sulfhydryl Determination 51 Preparation of Ovomucin 52 Analytical Ultracentrifugation 5 2 Viscosity Measurements 53 .RESULTS AND DISCUSSION 55 Viscosity as a Measure of Thinning 55 Effect of Bromate, Iodate and Cysteine 59 Effect of PCMB and HgCl2 6k GENERAL DISCUSSION 89 v i i Page CHAPTER IV: FOAMING OF EGG WHITE—THE BEATING PROCESS 92 INTRODUCTION 92 MATERIALS AND METHODS $k Foam Density and Stability 9I+ Bubble Size Analysis $h Electrophoresis 95 Gel Filtration 96 Other Determinations 96 RESULTS AND DISCUSSION 97 Bubble Size and Distribution 97 Electrophoresis llh Gel Fil t r a t i o n 119 GENERAL DISCUSSION 121 BIBLIOGRAPHY 123 v i i i LIST OF TABLES Table Page I Sulfhydryl levels and total solids in sulfhydryl blocking experiments. 78 II Effect of HgClg and PCMB on the sedimentation velocity of egg white. 82 III Chi-square values for fitting frequency distribution curves. 101 IV Effect of aging on bubble size parameters. 109 V Effect of beating on some of the attributes of egg white. I l l VI Effect of beating time on the lysozyme content of egg white drained from egg white beaten 2 and 8 minute s. 118 ix LIST OF FIGURES Figure Page la Analytical gel f i l t r a t i o n of egg white on Sepharose 2B. 28 lb Analytical gel f i l t r a t i o n of egg white on Sepharose 6B. 29 lc . Analytical gel f i l t r a t i o n of egg white on Sephadex G-100. 30 Id Analytical gel f i l t r a t i o n of egg white on Sephadex G-150. 31 le Analytical gel f i l t r a t i o n of egg white on Sephadex G-200. 32 2 Disc gel elctrophoresis of fractions collected as shown in Figure Id. 33 3 Analytical gel f i l t r a t i o n of egg white on Sephadex G-150. Effect of column length. 35 4a Gel f i l t r a t i o n of egg white on Sephadex G-150. Effect of salt concentration. 36 4b Gel f i l t r a t i o n of egg white on Sephadex G-150 Effect of salt concentration. 37 5 Disc gel electrophoresis of fractions collected as shown in Figure 4b . 40 6 Disc gel electrophoresis of fractions collected as shown in Figure 4 a . 4 l 7 Effect of centrifugation on the elution of egg white from Sephadex G-150. 43 8 Shear stress decay curves of egg white incubated at 37°C for 0 , 48, 72 and 120 hours. % 9 Equilibrium flow behavior curves for egg white incubated at 37°C for 0 , 48, 72 and 120 hours. 57 10 Effect of incubation time on the shear stress decay parameter A0. 58 Figure Page 11 Shear stress decay curves of control egg white and cysteine (CYS) treated egg white incubated 0 , 2k, kQ and 83 hours at 37°C. 60 12 Shear stress decay curves of egg white treated with potassium bromate. 6 l 13 Shear stress decay curves of egg white treated with potassium iodate. 62 lk The effect of treatment of egg white with cysteine, bromate and iodate on the intercept value A . 63 15 Effect of oxidizing agents on the -SH groups of egg white. 66 16 Effect of PCMB and HgCl2 on -SH groups of egg white. 67 17 Shear stress decay curves of egg white dialysed against 0 mg/ml HgCl2 in tris-KCl buffer. 68 18 Shear stress decay curves of egg white dialysed against 5 x IO"2 mg/ml HgClg in tris-KCl buffer. 69 19 Shear stress decay curves of egg white dialysed against 10 x 10~ 2 mg/ml HgCl2 in tris-KCl buffer. 70 20 Shear stress decay curves of egg white dialysed against 20 x IO-2 mg/ml HgCl2 in tris-KCl buffer. 71 21 Shear stress decay curves of egg white dialysed against 0 mg/ml PCMB in tris-KCl buffer. 72 22 Shear stress decay curves of egg white dialysed against 10 x 1 0 " 2 mg/ml PCMB in tris-KCl buffer. 73 23 Shear stress decay curves of egg white dialysed against 20 x 10~ 2 mg/ml PCMB in tris-KCl buffer. 7^ 2k Shear stress decay curves of egg white dialysed against 30 x IO-2 mg/ml PCMB in tris-KCl buffer. 75 25 The effect of blocking -SH groups of egg white with HgCl2 on the intercept value AQ. 77 26 The effect of blocking -SH groups of egg white with PCMB on the intercept value AQ. 79 XI Figure Page 27 Typical schlieren pattern of ifo egg white 60 minutes after reaching 59,600 rpm. 8 l 28a Ultracentrifuge pattern of ovomucin obtained 95 minutes after reaching 59>100 rPm—ovomucin from fresh egg white. 85 28b Ultracentrifuge pattern of ovomucin obtained 95 minutes after reaching 59>100 rpm—ovomucin from egg white aged 48 hours at 37°C. 86 29 Effect of blocking -SH groups on the relative amount of a-ovomucin (slow peak) in the ovomucin isolated from egg white after incubation at 37°C for 24 and 48 hours. 87 30 Effect of whipping time upon expansion. 98 31 Stability as affected by whipping time. 99 32 Photomicrograph showing effect of beating and aging on egg white foams. 102 33 Picture taken 20 minutes after beating for 2 minute s. 103 34 Effect of aging of foam on the frequency distribution of bubble size 2 minutes and 20 104 minutes after beating for 2 minutes. 35 Picture taken 2 minutes after beating 8 minutes. 106 36 Picture taken 10 minutes after beating 8 minutes. 107 37 Effect of aging of foam on the frequency distribution of bubble size 2 minutes and 10 minutes after beating for 8 minutes. 108 38 Effect of beating time on bubble diameter. 113 39 Dis&. gel electrophoresis of egg white and drainage from foamed egg white. ' l l 6 40 Densitometer tracing of disc gels shown in Figure 39. 117 41 Gel filtration of egg white (EW) and the drainage from 2 and 8 minute beating times in Sephadex G-150. 120 x i i ACKNOWLEDGEMENT The author wishes to express his sincere appreciation to Dr. S. Nakai, Associate Professor, Department of Food Science for his advice and encouragement throughout the course of this work and during the writing of this thesis. He is also thankful to the members of his graduate committee: Drs. J. F. Richards, M. A. Tung, and W. D. Powrie of the Department of Food Science; Dr. R. C. Fitzsimmons, Department of Poultry Science and Dr. R. J. Bose, Fisheries Research Board of Canada for their interest in and review of this thesis. The support of the National Research Council of Canada and the H. R. MacMillan Foundation through post-graduate scholarships is also acknowledged. INTRODUCTION Egg white may he considered a relatively concentrated solution of proteins dissolved in a dilute salt solution containing a small amount of carbohydrate. The proteins of egg white have been widely studied from a biochemical point of view and indeed some, notably ovalbumin, have achieved status as "standard" proteins for biochemical investigations. However, the functional and commercial significance of the proteins of egg white as they relate to commercial quality have received much less attention. Baker (h) in her review of egg white proteins has stated: "This topic is notable for the large number of papers and the small amount of useful information." While this statement perhaps overemphasizes the lack of knowledge in this fie l d , i t does serve to indicate the dirth of information available in this area. This thesis describes a three-part study of egg white and egg white proteins as related to functional properties and commercial quality. Gel chromatography was utilized to detect electrostatic interactions claimed to be of importance in determining functional properties; viscometry and ultracentrifugation were employed to detect changes which occur during aging of the egg and the effect of sulfhydryl blocking on these changes was assessed; and the beating of egg white for foam formation was studied utilizing bubble size analysis, gel electrophoresis and gel chromatography. 2 CHAPTER I: SURVEY OF THE LITERATURE Egg white is a clear material of yellowish tint which makes up about 60$ of the egg ( 7 0 , 7 5 ) . It contains 10 to 12$ solids of which about 92$ is protein, the remainder being made up of carbohydrate, salts and a trace of fat. The protein consists of 5 major and 7 to 9 minor constituents. The major constituents have been found to be inhomogeneous—existing in more than one form—and in addition there exists variations due to genetic influence. Thus egg white may be considered to be a complex solution of proteins in dilute salt, and the properties of egg white—particularly the functional properties—will depend almost exclusively upon its protein fraction. Since the work to be reported in this thesis is concerned mainly with the functional properties of egg white and the proteins of egg white, the review which follows is weighted heavily towards those proteins which have been indicated as contributing to the functional properties of egg white. These are generally considered to be ovalbumin, ovomucin, the globulins (55) a n ( i lysozyme ( l 4 ) . The proteins of egg white have been reviewed by Fevold (37) and Warner (87) and more recently by Parkinson ( 6 2 ) , Feeney (35) a n & Baker (k). Egg White Proteins A. Ovalbumin Ovalbumin is the most abundant protein of egg white comprising about 5^ 4$ of the total solids of egg white. It has been possible to obtain ovalbumin in pure form for many years and consequently its 3 composition and properties were studied extensively during the early history of protein chemistry. The results of these studies are reviewed by Fevold (37) and Warner (87) . It is a glycoprotein with a molecular weight in the region of 4-5,000 and isoelectric point of 4 .6 to 4.8 and is relatively easily denatured by heating, shaking or exposure to ultraviolet radiation or certain compounds such as urea. Crystalline ovalbumin is not homogeneous and may be resolved into two components (A]_ and Ag) by free boundary electrophoresis (51) or into three by starch gel electrophoresis (52) . A l l three have been isolated by Rhodes e_t al. who have shown that the molar ratio of phosphorous to protein was 1.99 for A]_ and 0.93 for Ag in close agreement with values of 2 and 1 obtained enzymatically (87) . Ovalbumin A3 presumably has no phosphate. The carbohydrate moiety of ovalbumin has a molecular weight of about 2,000 (20) and contains 4 . 5 - 5 . 0 residues D-mannose per molecule and 3.0 moles of 2-acetamido-2-deoxy-D-glucose (acetylglucosamine) covalently bound through an aspartic acid residue (44) . Ovalbumin is the classical example of a protein containing "masked" sulfhydryl (-SH) groups. In its native state, ovalbumin gives no reaction with reagents normally used to detect or react with -SH groups. Denaturation however gives rise to positive tests. The -SH and disulfide (SS) groups of ovalbumin have received considerable attention over the last few years. Early work gave rise to some confusion as to the actual number of -SH and SS groups present. Values ranging from 4 to 5 -SH and 1 SS bond per molecule (17) to 4 -SH and 2 SS per mole were reported (89) . However, recent work has shown k that ovalbumin contains 3 -SH reactive with p-chloromercuribenzoate at pH k.6 in the native proteins. Ovalbumin denatured with sodium dodecyl sulfate or guanidine hydrochloride has k -SH reactive with most common -SH reagents (9,2k , 36 ,38 , ^ k). One SS bond has been detected in ovalbumin in recent studies ( 9 5 38 ) . The only known source of -SH groups in egg white is ovalbumin. MacDonnell e_t a l . (5*0 have reported 0.44$ available -SH as cysteine in the native egg white and 0.62$ after guanidine denaturation (dry weight basis). This latter value is in excellent agreement with a value of 50.7 uM/g dry weight reported by Beveridge et_ al_. (9) who reported 79-7 uM/g dry weight SS groups. This value compares favourably with 84.5 uM/g SS which was computed from amino acid analysis. Recently, Smith and Back (77) have shown that ovalbumin is converted to a form (S-ovalbumin) less susceptible to heat denatur-ation during storage of eggs. The change in ovalbumin roughly paralled the deteriorative (thinning) changes which occurred in the eggs on storage. Further work by Smith and Back (78,79) showed the S-ovalbumin was indistinguishable from ovalbumin except by the resistance of S-ovalbumin to heat denaturation. Ovalbumin may be converted to S-ovalbumin' by heating a 5$ solution of ovalbumin at pH 9.9 for 16 hours at 55°0 . This preparation was considered to be 100$ S-ovalbumin after removal of a small amount of denatured protein on adjustment to pH k.7. The nature of the conversion of ovalbumin to S-ovalbumin was not known, however, the evidence for and possibility of intramolecular SS interchange was considered. 5 Recent work on ovalbumin has been concerned with elucidating the amino acid sequence of this protein (38,82) . Thompson et a l . (82) have reported the sequence of the plakalbumin peptide--a peptide obtained on treatment of ovalbumin with subtilisin--and the C-terminal sequence of this protein. Fothergill and Fothergill (38) also have reported the C-terminal sequence and have located the position of the single SS bond in ovalbumin. B. Conalbumin Conalbumin comprises about 13$ of the total solids of egg white (35) a n (i is notable on account of its ability to bind iron and render it nutritionally unavailable to micro-organisms. It is a homologue of transferrin, an iron binding globulin present in blood serum. The molecular weight has been variously reported from 70,000 to 87,000 (37)--a value of about 80,000 appears to be gaining acceptance. It has been postulated (37) that the molecule contains nine peptide chains. Conalbumin is capable of binding 2 moles of Fe ( i l l ) , Cu ( i i ) , or Zn (ii) per mole (in this order of affinity) to give metal complexes which are respectively rust-red or pink ( A max 4-70 nm), greenish-yellow ( X max kkO nm) or colorless (k ,lk). The protein is free of phosphorous, has no free -SH groups and has an isoelectric point of about 6 .6 . On binding of metal ions, conalbumin becomes very resistant to digestion by proteolytic enzymes or denatur-ation by heat or organic solvents, however, when no metals are bound, the protein is easily susceptible to heat denaturation. Carbohydrate has been found in the molecule—0.8$ hexose and l.kfo hexosamine (62) . 6 Electrophoretically, at least three co-dominant alleles coding variants of conalbumin have been found in the domestic fowl (4). Lush (52) and Feeney et al. (3*+) have observed two forms of conalbumin in starch gel electropherograms of egg white. C. Ovomucoid This protein is distinguished by its high content of carbohydrate, by its ability to inhibit trypsin and by its heterogeneity on isolation. This latter problem has been overcome somewhat by Davis et al (27). Ovomucoid contains 20 to 22$ carbohydrate of which 14-16$ is hexosamine (glucosamine), 4-6$ is mannose, 1.5$ is galactose and 0 .4-4.0$ is sialic acid. The protein is notable in that it has no tryptophan and is not coagulable by heat. Its molecular weight is about 28,000, its isoelectric point is 3 .9 to 4.3 and it comprises about 11$ of the total solids of egg white. D. Lysozyme Lysozyme is an enzyme which hydrolyses linkages involving W-acetyl-muramic acid. Its action in lysing the cells of the bacterium Micrococcus lysodeikticus is well known. Lysozyme is a basic protein of molecular weight about 17,000 and an isoelectric point of 10.5-11.0 (62), so that it migrates toward the cathode in conventional alkaline gel electrophoretic systems. It is upon this latter property that functional roles in egg white have been assigned to lysozyme. It is generally considered that lysozyme interacts electrostatically with other egg components and that the interaction product is responsible for the gain or loss of functional properties. Lysozyme has been shown to interact with ovomucin (4l) and 7 ovalbumin (59) among the proteins of egg white. The protein constitutes about 3 .5$ of "the total solids of egg white and has no free -SH group. Lysozyme is the egg white protein about whose structure we know the most. The primary amino acid sequence of lysozyme has been determined (16), and Blake et a l , (12) have worked out the structure of this enzyme utilizing x-ray techniques. In terms of functional properties lysozyme is considered to behave as a globulin and contribute to the foaminess of egg white (55)- The interaction of lysozyme with ovomucin has been suggested as a mechanism for the formation of thick gel structure in egg white, and the denaturation of a lysozyme-ovomucin interaction product has been postulated as a source of damage to the whipping properties of pasteurized egg white (ko). E. Ovomucin Ovomucin, a sulfated glycoprotein, is generally considered responsible for the physical structure and characteristics of thick white. It is consistently found that thick white contains about four times the ovomucin found in thin white (26) . In addition, ovomucin contains the influenza viral hemagglutination inhibitor activity found in egg white (37 5 74 ,87 ) . This fraction has, until recently, been very poorly characterized (25) , however in recent years a considerable amount of data has become available. Since this data does not appear in any of the reviews mentioned, this protein will be covered in somewhat more detail than were the proteins mentioned previously. 1. Isolation Ovomucin is generally considered to be that protein which is precipitated from egg white at low ionic strength and which is insoluble in dilute salt solutions. Three procedures have been outlined recently by Robinson and Monsey (68) , Donovan et a l . (28) and Dam (25) . Examination of these procedures reveals that they have in common the following steps: a) Reduction of the ionic strength of egg white by dilution (at least 1:4, egg white :distilled water) or dialysis, often at acid pH. b) Extraction of the crude ovomucin with dilute salt solutions (l - 2 $ NaCI or KCl) to remove proteins which co-precipitate--lysozyme and presumably other globulins. The gel-like material remaining after this extraction is usually designated purified ovomucin. Some authors prefer to remove lysozyme before precipitating the ovomucin (25 ,65) . It is characteristic of ovomucin prepared in this fashion that it becomes progressively more insoluble as the isolation proceeds. This difficulty has provided a major stumbling block in the characterization of ovomucin. Recently (91) a gel chromatographic procedure has appeared for isolation of ovomucin. 2. Solubility Robinson and Monsey (65) have shown that reduction of ovomucin may be accomplished by 2-mercaptoethanol in the presence of 9 8 M urea at pH 8 . 5 . Preparations reduced in this manner and stabilized by alkylation with iodoacetic acid were reported to be readily soluble in neutral and alkaline buffers. Donovan e_t al. (28) have reported that freeze-dried ovomucin could be dissolved in ammonia, to give a homogeneous, nearly clear, very viscous solution. Freeze-dried ovomucin was not soluble near neutral pH under non-denaturing conditions. The preparation was soluble in 6 M guanidine hydrochloride at pH 8.1 in the presence of 2-mercaptoethanol. The preparation of Dam (25) was apparently soluble in dilute NaOH at pH 9-5 and in phosphate buffer pH 7.k in the presence' of 3 M and 6 M urea. 3. Heterogeneity It has been observed by many authors that ovomucin isolated by dilution or dialysis was not a single component and may be contaminated by other proteins . In the early 1950's, Sharp et al. (7*0 and Lanni e_t al. (50) noted heterogeneity in ovomucin purified by salt extraction and in egg white itself. Utilizing free boundary electrophoresis, two and sometimes three components were observed with mobilities of -3-55 - 6 . 7 and -10 x 10~5 cm.2 sec."l v o l t ~ l . The - 3 .5 component contributed 60-90$ of the total refractive increment and the influenza virus hemagglutination inhibitor activity was found in the - 6 . 7 and -10 components but not in the -3 -5 component. Donovan et al,(28) have shown the presence of two components with mobilities of - 3 . 6 and - 7 . 6 x 10~5 cm.2 sec.-l volt-! 10 in their preparations of lysozyme free ovomucin obtained by gel filtration. Sedimentation analysis showed several components, prominent species had sedimentation coefficients 3 S, 19 S and greater than 30 S. Three were detected in ovomucin dissolved in 6 M guanidine hydrochloride and 2-mercaptoethanol with S2Qj W values of 6.4 (10$), 2.9 (5/0) and 1.4 (85/0) respectively. The bracketed figures refer to the approximate relative concentrations of the components. Robinson and Monsey (66,68) have shown the presence of two components in their reduced, alkylated preparation of ovomucin by ultracentrifugation and gel filtration in the presence of quanidine hydrochloride. Sedimentation coefficients of 2.7 (65$) and 5.8 (35$) were reported for the two components. Kato et al. (46,4-7) have detected two peaks in their free boundary electrophoretic pattern of their preparation of ovomucin solubilized with 2-mercaptoethanol. From the data shown above, i t is apparent that ovomucin consists of two or three components. In this connection i t is important to note that washing of the precipitate with dilute salt is a critical procedure, and should be continued until the optical density of the supernatant is very low. If this is done, two components are detectable in the ultracentrifuge as described by Robinson and Monsey (68), however, i f washing is inadequate, a third component having a low sedimentation coefficient in mercaptoethanol-guanidine hydrochloride buffers is observed (86). From this observation it would appear likely that ovomucin isolated by precipitation and washing contains two components. However, 11 Robinson and Monsey (67) have reported that an ovomucin-like protein is extracted from crude ovomucin precipitates by KC1. Dialysis of the KC1 extracts yields an insoluble protein fraction that consists apparently of lysozyme and an ovomucin fraction. The relationship of this fraction to the insoluble ovomucin remaining behind is not known. 4. Composition Ovomucin apparently contains two major components and two components have been isolated and characterized in part. Robinson and Monsey (68) have used CsCl gradients in the presence of 4 M guanidine hydrochloride to fractionate reduced alkylated ovomucin into two components. The more dense component—termed a-ovomucin — may be distinguished by its relatively high protein content and relatively low carbohydrate content. The lighter component—termed /?-ovomucin—may be distinguished by a high carbohydrate and low protein content. a -Ovomucin contains 69.5$ proteins, 7 .3$ hexosamine (6.7$ N-acetylglucosamine, 0.6$ N-acetylgalactosamine), 6.4$ hexose (1.8$ galactose, 4.6$ mannose), 1.0$ sialic acid and 0 .7$ sulfate. /?-Ovomucin contains 28 .1$ protein, 19.7$ hexosamine ( l l . 0 $ N-acetylglucosamine, 8.7$ ^ -acetylgalactosamine), 23.3$ hexose (19.2$ galactose, 4.1$ mannose), 13.8$ sialic acid and 2 .7$ sulfate. These two components may be distinguished in the ultracentrifuge in solvents containing guanidine hydrochloride but not in those containing urea. Reduced, alkylated /? -ovomucin sediments faster (5-7 S) than reduced, alkylated a-ovomucin (2.4 S) (69) . Kato and Sato (46) have used density gradient electro-phoresis to isolate the two peaks obtained in their free boundary 12 electrophoretic experiments. The fast peak (designated peak-F hy them) has been shown to be high in carbohydrate while the slow peak (peak-S) is relatively low in carbohydrate. Peak-F material contained 18.4$ hexose, 18.3$ hexosamine, 11.4$ sialic acid and 1.18$ sulfate. Peak-S material contained 6.8$ hexose, 6 .7$ hexosamine, 0 .8$ sialic acid and 0 .06$ sulfate. The amino acid composition of crude ovomucin has been given by Osuga and Feeney (60) , Donovan et al_. (28) and Robinson and Monsey (68) . Kato and Sato (46) reported the amino acid composition of their fast and slow components and Robinson and Monsey (68) report similar data for a-, and /?-ovomucin. Glutamic and aspartic acids were the major amino acids in a -ovomucin whereas serine and threonine were the major amino acids in the /3-ovomucin preparations. Similar results were reported by Kato and Sato (46) for peak-S and peak-F respectively. 5. Molecular Weight Lanni e_t a l . (49), in an early study of ovomucin obtained a molecular weight of 7.6 x 10^ from sedimentation-viscosity measurements. Donovan et a l , (28) gives a viscosity-average molecular weight of about 163,000 and a number average molecular weight of 110,000 - 20,000 for ovomucin in 6 M guanidine hydrochloride plus 0.2 M mercaptoethanol using viscosity and osmotic pressure techniques respectively. Robinson and Monsey (68) have estimated a weight average molecular weight of 210,000 for reduced a-ovomucin from sedimentation equilibrium data in guanidine hydrochloride and 2-mercaptoethanol. A l l preparations were large enough to be excluded from the pores of Sephadex G-200 and consequently were eluted at the void volume. Tomimatsu and Donovan (83) have reported values of 2h0 x 106 and ko x 10& at pH 6.2 and 7.9 respectively by light scattering techniques. In 6.5 M guanidine hydrochloride the value was 23 x 10^. From this data it is clear that the molecular weight of unreduced ovomucin is large and variable depending upon experimental conditions. The large, molecules are apparently made, up of smaller units, perhaps the monomer unit, of molecular weight in the region of 100,000 to 200,000. F. Globulins Originally, three globulins, labelled G]_, G2 and G3 were thought to occur in egg white, however Alderton e_t al. (l) have shown Gj_ to be lysozyme. Longworth et al. (51) a nd Feeney e_t al . (3*0 have partically purified globulin . (A^_ in Feeney's notation). Attempts at purification have yielded the following data for the globulins. G2 was found freely soluble in 0 .25$ NaCI but precipitated on removal of salt by dialysis and was insoluble in pure water over the pH range 5-5 to 7-5• The G2 preparations were generally contaminated with G3. The isoelectric point for these proteins was 6.0 and 5.6 for G2 and G2 respectively as determined from mobility values. The molecular weight of G2 was estimated as between 30,000 and 45,000 as determined by membrane filtration experiments. Apart from the two studies mentioned above, l i t t l e is known of the properties of G2 and G3 globulins. They have been implicated as important foaming proteins affecting the functional properties of egg white (55) hut l i t t l e is known of their role in this respect. Ik Most recent work has concerned the genetic polymorphism of these proteins. Gg globulins are controlled at, or coded from, a locus with at least two alleles, both of which are expressed in heterozygotes. G^  globulins are similarly controlled at a single locus. The detailed genetic variance of the globulins in the egg white of various species has been reviewed by Baker (k). G. Other Egg White Proteins In addition to the proteins noted, egg white contains a large number of proteins present in small quantities. Included in this group are the enzymes catalase, esterase, peptidase and glyco-sidases. These proteins are reviewed by Baker (k) and in general, l i t t l e is known of their properties. Recently, the kinetics and some of the properties of egg white catalase were described (6) and an attempt was made to isolate the active component (7 ). Additionally, this group of proteins also include an ovomacroglobulin, avidin—a biotin binding protein—and flavoprotein—a riboflavin binding protein. These proteins also are reviewed by Baker (k). While many of these proteins have interesting biochemical properties, they have not been demonstrated to contribute to the functional proterties of egg white and will not be dealt with further here. Functional and Structural Properties of Egg White A. Structural properties A factor of major importance in determining the acceptibility of eggs is the amount of thick white gel (75) . Many studies have been conducted to explain the basis of the general structure, and most of these have involved participation of ovomucin. Ovomucin 15 is found in the thick white in amounts 4 times that of thin white (25) . The gel breaks down continuously into thin white from the time of laying and this change may be retarded by lowering the temperature and controlling the pH of the white in an optimum range of 8.0 to 8.2. The reasons for the change and for the control by pH and temperature are s t i l l unknown. However, it is known (33) that components from the yolk, the shell or the shell membranes are not the agents responsible for thinning nor is bacterial contamination of the shell or its membranes. Thinning is an inherent property of white itself. One of the longest persisting theories relating to the structure of thick gel structure involves establishment of a complex between ovomucin fibers and lysozyme. It is known that the very basic lysozyme molecule will complex with acidic molecules (48). Hawthorne (4-2) has investigated the action of lysozyme on ovomucin in vitro and shown that a swollen ovomucin gel was changed to a stringy mass of small volume on addition of lysozyme. He suggested that the thinning reaction may be due to slow insolubilization of ovomucin rendering it unavailable for gel stabilization. Cotterill and Winter (21) examined the lysozyme-ovomucin interaction over the pH range 7.0 to 10.4 and observed a progressive decrease in interaction as the pH increased. A maximum interaction was observed at pH 7.0 and at pH 9.0 to 9-5 interaction had virtually disappeared. These latter pH values lie in the range attained by egg white during storage. These authors suggest that the ovomucin-lysozyme interaction is electrostatic in nature since consideration 16 of the respective isoelectric points of the two proteins indicates that less electrostatic interaction would be expected at higher pH values. A reversal of Hawthorne's proposal was suggested by these authors. They suggested that the electrostatic lysozyme-ovomucin interaction may be partly responsible for maintaining thick gel structure and that absence or reduction of this interaction as the pH of white rises on storage may be responsible for thinning. Brooks and Hale (ik) studied the mechanical properties of the thick white by measuring the displacement of a nickel sphere inserted into the gel by a magnetic field. They concluded that a simple network of ovomucin chains would not account for the mechanical properties of the thick egg white. These were better explained by assuming that chains of an ovomucin-lysozyme complex are cross-linked into a network. Recently Dam (26) has stated it is not necessary to invoke the use of another protein as a cross-linking agent. He suggested that changes in ovomucin alone may be sufficient to explain thinning of the gel structure of thick egg white during storage. A second theory relating to the thinning reaction requires only the participation of the protein ovomucin and arises from an observation of Hoover later confirmed by MacDonnell e_t a l . (53) that chemical reducing agents such as cysteine and thioglycollic acid cause rapid and extensive thinning of egg white. The suggestion is that reducing agents cause the thick white to break down by splitting the SS bonds of ovomucin. Deterioration was observed when reducing agents were added in quantities of less than 50 parts per million to broken out eggs and also when shell eggs were stored in low con-centrations of hydrogen sulfide and sulfur dioxide. The major difficulty with this theory lies in the lack of available reducing agent in egg white. The major protein of egg white, ovalbumin, contains -SH groups but they are not as reactive as the -SH groups of thioglycol. Feeney et al. (33) found no change in "native" -SH content of egg white during storage of shell eggs, however they suggested (53) that i f only 2$ of the -SH groups in ovalbumin were to become as active as the -SH of thioglycol, marked deterioration of the egg would result. This low level of change would not be detected by the methods used for -SH determination (33) . More recently, Smith and Back (77) have shown that ovalbumin undergoes a transition to a form more resistant to heat denaturation during storage of eggs. This change approximately parallels the change in gel structure of egg white during storage. It has been suggested to occur through an internal SS interchange reaction which results in no net change in -SH groups (78 ,79) . A relationship between these observations and the deteriorative changes which occur in egg white on storage have been suggested but no direct connection is available. It has been suggested recently by Donovan et al . ( 29 ) that the thinning reaction may be due to alkaline hydrolysis of the SS bonds of ovomucin. The possibility of disaggregation reactions as observed in ovomucin preparations by light scattering at pH 7.9 being responsible for thinning is discussed by Tomimatsu and Donovan (83) . Recent work in this field has taken advantage of the advances which have been made in our knowledge of the chemistry of ovomucin which has occurred over the past few years. The changes which occur in ovomucin during the thinning of shell eggs has been the subject of several investigations. Kato e_fc a l . (4-5,47) have shown that hexose and hexosamine in ovomucin isolated from thick white gel decreased to about one half the in i t i a l value while the sialic acid content decreased to one eighth after 20 days storage at 30°C. On the other hand, the carbohydrate content of ovomucin isolated from the thin white increased over the same storage period. In addition the fast moving component (peak-F in free boundary electrophoresis) decreased and finally disappeared, after 30 days storage, from ovomucin isolated from thick gel. On the other hand, peak-F increased in ovomucin isolated from the thin white fraction. It is known that peak-F contains a component high in carbohydrate content. These authors interpret their results in terms of a model for the thick white consisting of ovomucin molecules of both types held in a network by SS bonds. Lysozyme is also incorporated into the model. The thinning reaction is seen as a breakage of the SS bonds releasing the carbohydrate rich fragment into solution and leaving a gel residue consisting of the protein rich component. Robinson and Monsey (69) have also studied the change in composition of egg white during thinning. They followed the thinnin with a rotational viscometer. Egg white showed non-Newtonian behavior and the flow curves obtained were fitted by a form of the power law equation. They showed that as thinning progressed, the 19 flow behavior index approached a value of 1 and the egg white approached Newtonian behavior. In addition, shear-thinning of the egg white was observed. They suggest that the values of apparent viscosity obtained at a single rate of shear are useful for comparing the firmness of egg white. In general, carbohydrate analysis of ovomucin isolated from thick egg white showed slight decreases in hexose, hexosamine and sialic acid while a slight increase in protein was noted. The most dramatic change was observed in the ultracentrifuge patterns observed in the presence of 5 M guanidine hydrochloride with reduced ovomucin isolated from thick white. The area of the a -ovomucin peak increased from 71.3$ to 93 .1$ over 4-7 hours incubation at 37°C while the ^-ovomucin peak area decreased from 28.6$ to 6.8$ over the same incubation period. B. Foaming Properties In spite of the importance of foam formation by egg white to its utilization in food products, the property has not been studied extensively. Early work in the field concerned itself mainly with the influence of physical and chemical factors on egg white foams. Barmore (8) found that foam density decreased as beating time was increased, that foam stability as measured by the amount of liquid draining from the foam, decreased rapidly as beating time increased, and that bubble size increased as the foam aged. Furthermore, a decrease in bubble size was claimed with increased beating time, and an increase in foam stability was noted when organic acids were added to egg white prior to foaming. 20 A few studies are available in which investigation of the roles of individual components of egg white on functional properties were undertaken. An early observation by Forsythe and Berquist (39) that the major portion of the ovomucin is concentrated in the foam was confirmed by MacDonnell e_t a l . (55) who showed that the viscosity and stability of the foam (as indicated by drip tests) was dependent upon this fraction. These latter authors used whipping tests and angel-cake baking tests to determine the contribution of some of the components of egg white to its functional properties. When ovomucin and globulin were removed from egg white, whip time increased and angel cake volume decreased. Cake volume may be restored and whipping time decreased by adding back the globulin fraction. Adding back the ovomucin fraction shortened whipping time but had no effect on cake volume. -When extra globulins were added to egg white increases in cake volume above that obtained with the control were noted. Although an angel cake could be made from ovalbumin alone, the resulting cake was coarse in texture. MacDonnell et a l . (55) concluded from these observations that the important constituents were the globulins (including lysozyme) ovomucin and ovalbumin. It was felt that the globulin fraction was in large part responsible for good foaming while ovomucin appeared to stabilize the foam. Ovalbumin and the other egg white proteins were considered to contribute heat denaturable bulk to support the structure of cakes such as the angel cake. Nakamura and Sato (56,57 s58) have also studied egg white foaminess. In general they reached similar conclusions as MacDonnell et al. regarding the role of the egg white proteins. Ovomucin was found responsible for foam stability and the globulin were considered good foaming proteins. In addition these authors noted a decrease in the globulin fraction in repeatedly whipped e white. 22 CHAPTER II: GEL FILTRATION OF EGG WHITE INTRODUCTION In modern-day protein chemistry it is frequently desirable to know the molecular weight, the molecular weight distribution and the composition distribution of a proteinaceous material. Gel filtration, a chromatographic procedure which separates materials on the basis of molecular size, has become a standard laboratory technique for the determination of these parameters. The chromatographic medium for this technique consists of grains of a porous gel swollen in solvent, usually an aqueous solvent. Large molecules are completely excluded from the porous gel grains and are eluted from the gel bed at the void volume. Smaller molecules, in a specific size range depending upon the particular gel chosen, are able to penetrate some gel pores and emerge later—the exact position depending upon the size of the molecules. The result of a gel filtration experiment then consists of a tracing of some detector response as a function of elution volume, which is related to molecular size, and a collection of fractions containing the separated material. Gel filtration has been widely applied in food science research notably in the dairy science area. The contributions of gel filtration to this area have been recently reviewed by Yaguchi and Rose ( 9 0 ) . The technique has been useful for the separation and/or analysis of skimmilk, casein, casein micelles, whey proteins and some milk enzymes. In addition, the effect of various treatments, 23 and temperature induced aggregations and disaggregations of caseins and whey proteins have been studied. The molecular weight of the major components of egg white ranges from about 163,000 for reduced ovomucin (68) to about 15,000 for lysozyme (35). This suggests that gel filtration would be useful for separation of the major components of egg white and determination of the relative distribution of the major components. In addition, by choosing appropriate conditions, it should be possible to detect aggregations or interactions which occur in the egg white since interacting species should tend to co-chromatograph. Gel filtration has not been widely applied to egg white. Young and Gardner (91) used Sepharose kB for the isolation of egg white ovomucin and reported variable results. From two to five peaks were obtained by these authors and, except for ovomucin which was eluted at the void volume, l i t t l e or no separation of egg white components was'demonstrated. Ball and Cotterill (7) have used Sephadex G-200 to fractionate the globulin fraction of egg white. It has been known for some time that compounds containing free sulfhydryl (-SH) groups can cause extensive thinning of egg white (53), This effect presumably is due to reduction of disulfide (SS) bonds in the glycoprotein ovomucin—thought to be associated with the thick gel structure of egg white. In view of these considerations i t was considered possible that gel filtration in the presence of a -SH compound such as 2-mercaptoethanol may yield valuable inform-ation about the distribution of a l l of the macromolecular components of egg white. This chapter then describes the 2k gel filtration of whole egg white under reducing conditions and the distribution of the egg white components. MATERIALS AND METHODS 25 Columns and gel materials used in this study were obtained from Pharmacia Fine Chemicals and the gel beds were prepared and packed as recommended by the manufacturer. Elution was accomplished with 0.01 M phosphate buffer pH 7 . 0 , containing NaCI as required and 0.3$ (v/v) 2-mercaptoethanol. The 2-mercaptoethanol was added to the buffer system and the pH checked immediately before use to avoid loss of reducing power which occurred after the buffer had stood at room temperature for several days. ; Eggs were obtained on the morning of lay from the University farm. Samples of fresh egg white were prepared by dilution of egg white 1:1 with 0.02 M phosphate buffer containing 0 .6$ (v/v) 2-mercaptoethanol and NaCI at twice the concentration to be used in the elution buffer. Ten ml of the diluted egg white was removed, 100 mg of sucrose was dissolved into the egg white and a volume (usually k.0 ml) of this solution was applied to the top of the gel bed by layering the dense sucrose solution on top of the gel and under the buffer above the bed. This method was found to give good reproducible sample application. The elution profile was obtained by reading the optical density at 280 nm of each fraction. Fractions were collected with an Isco Golden Retriever fraction collector equipped with drop counting apparatus. Fraction size was 100 drops (3.1 ml). Pooled fractions obtained from the column were concentrated by pervaporization and analysed for protein components by disc gel electrophoresis after dialysing against 0.01 M phosphate buffer, pH 7.0 containing 1$ NaCI. Disc gel electrophoresis was done as described by Zweig and Whitaker (92) using buffer system A (p. 159), an alkaline buffer system recommended for serum proteins. Lysozyme was detected by setting up the electrophoresis run a second time and reversing the polarity of the electrodes. Ovalbumin, conalbumin and lysozyme were obtained from Sigma Chemical Company. Ovomucin was isolated from egg white by dilution l:k with water followed by washing of the precipitate with 2$ NaCI until the optical density at 280 nm of the washings was less than 0.05.- The resulting gel-like precipitate was slowly soluble in 2% NaCI containing 0.3$ (v/v) 2-mercaptoethanol. 27 RESULTS AND DISCUSSION Effect of Gel Type The effect of gel porosity on the elution pattern of egg white is shown in Figures la-e. The void volume as determined with blue dextran is shown on these profiles by an arrow with the symbol V . As can be seen from the figures, the Sepharose gels afford l i t t l e or no separation of egg white components. Furthermore, it is apparent that reduced ovomucin is eluted with the other egg white proteins at or near the V^ . position. This is in contrast to "native" ovomucin which has been shown by Young and Gardner (91) to elute at the void volume of a Sepharose kB column. Sepharose k~B is intermediate in porosity between Sepharose 2B and 6B (63). The Sephadex gels gave very much improved separation—five to six peaks were obtained with Sephadex G-150. Disc gel electrophoresis of some of the peaks collected as shown in Figure Id are in Figure 2. Peak 1 contained material remaining at the origin—behavior characteristic of ovomucin preparations reported in the literature. The other major band is presumably "line 18" material (7). Peak 2 contained mainly globulin-like material plus some conalbumin. Peak 3 contained conalbumin, ovalbumin and globulins and peak k contained mainly ovalbumin. Peak 5 contained lysozyme and so is not shown in this figure and peak 6 contained no detectable protein component. It is known that 2-mercaptoethanol absorbs in the ultraviolet region of the spectrum and i t was considered that this peak 6 represented the 2-mercaptoethanol in the sample as an excess of that present in 28 2.0 • FRACTION NUMBER Figure l a . Analytical gel filtration of egg white on Sepharose 2B. Figures la-e. Analytical gel filtration of egg white on gels of various types. Column size 2 .5 x 38 cm. Buffer is 0 . 0 1 M phosphate pH 7 . 0 , 1$ NaCI, 0 . 3 $ (v/v) 2-mercaptoethanol. Fraction size is 3 . 1 " i i . Sample size is 2 .5 ml. V0 = void volume determined with blue dextran. 29 Figure lb. Analytical gel filtration of egg white on Sepharose 6B. 3b Figure l c . Analytical gel filtration of egg white on Sephadex G-100. 31 FRACTION NUMBER Figure Id. Analytical gel f i l t r a t i o n of egg white on Sephadex G-150. 0, C, 0 V and L mark the elution position of ovomucin, conalbumin, ovalbumin and lysozyme respectively. 32 Figure le. Analytical gel filtration of egg white on Sephadex G-200. 33 EW 5 4 3 2 1 Figure 2. Disc gel electrophoresis of fractions collected as shown in Figure Id. EW = egg white treated under the same conditions as the fractions but not chromatographed. Band a is conalbumin; b is the "globulin" region; c is the ovalbumin region; the bands may be regarded as ovalbumin A^ _, A2 and A3 in decreasing electrophoretic mobility ( 1 8 ) . 3h the buffer system. The letters 0, C, 0V and L represent the positions--measured at maximum peak height—of reduced ovomucin, conalbumin, ovalbumin and lysozyme as determined by chromatography of the individual components. The elution position of reduced ovomucin coincides with the void volume as determined with blue dextran. The four regions corresponding to these four components can be discerned in the elution profile. Effect of Column Length It is commonly considered that increasing column length will increase resolution of components in chromatographic separations. The effect of lengthening the column to 78 cm is shown in Figure 3 . Some improvement was obtained in the separation of the ovomucin and lysozyme peaks but otherwise no improvement was obtained. In view of the fact that columns such as this took up to four times as long to run and gave endless problems with decreasing flow rates due to bed compactation, long columns were abandoned in favour of the shorter ones for future work. Effect of Salt Concentration Since it is known that lysozyme interacts with other protein components of egg white under conditions of reduced ionic strength--notably with the ovomucin component (21 ,26) , it was considered possible that interactions involving lysozyme could be demonstrated using co-chromatography as a criterion of interaction. This technique has two major advantages over other techniques which have been used to demonstrate interactions. First, instead of only two components being present in the reaction mixture (an ovomucin 35 FRACTION NUMBER Figure 3. Analytical gel filtration of egg white on Sephadex G-150. Column length is 2.5 x 78 cm. Conditions as in Figures la-e. 36 oo CM 4 & 20 40 FRACTION NUMBER Figure 4a . Gel filtration of egg white on Sephadex G-150. Conditions—0.25$ NaCI, 0.01 M phosphate pH 7 . 0 , 0.3$ (v/v) 2-mercapto-ethanol. Fraction size = 3-1 ml. Sample size = 4.0 ml. Flow rate = 20 ml./hr. 10 30 50 FRACTION NUMBER Figure k~b. Gel filtration of egg white on Sephadex G-150. Conditions—1.0$ NaCI, .0.01 M phosphate pH 7.0, 0.3$ (v/v) 2-mercapto-ethanol. Fraction size = 3.1 ml. Sample size = 4.0 ml. Flow rate = 20 ml/hr. 38 preparation plus lysozyme)—the most commonly used experimental procedure—all of the components normally present in egg white will he available for interaction. This may allow assessment of possible competition among components for lysozyme binding. Secondly, because of the nature of Sephadex gels}relatively high protein concentration may be used. Other procedures have involved the use of dilute protein solutions (about 1$), considerably more dilute than egg white. Since it is possible that interactions are different in high protein concentrations than in low, the ability to use high concentrations of protein constitutes a distinct advantage for this technique. Since lysozyme accounts for only 3-5$ (35) of the total solids of egg white and since the lysozyme peak is small compared to the other major peaks, i t was considered necessary to increase the amount of lysozyme applied to the column so that any changes^in this peak would be clear and unequivocal. This was accomplished by using a sample volume of 4.0 ml. The effect of reducing the salt concentration from 1$ to 0.25$ on the elution profile obtained when egg white was chromatographed on Sephadex G-150 is shown in Figures 4a and b. Two things are immediately apparent from these figures. First, the resolution of the various components has been impaired by the larger sample volume as expected. Secondly, the lysozyme peak has been markedly reduced in the 0.25$ salt pattern as compared to the 1$ salt pattern. Exactly where the lysozyme may have eluted is not clear from these patterns. The elution position of the various components was determined by disc gel electrophoresis of concentrated fractions pooled according to the bars and numbers on the patterns. The disc gel electropherograms obtained from the pattern in Figure 4b (l$ NaCl) is shown in Figure 5' These patterns are for the most part very similar to those shown previously in Figure 2. In general, region 1 contained aggregated or high molecular weight material which did not penetrate the pores of the gel as well as some other material including what is presumed to be "line 18" material reported by Lush (52). Region 2 contained most of the components found in egg white but was enriched with conalbumin, globulins and other components. Region 3 is clearly enriched in ovalbumin and in region 4 only lysozyme was detected. No protein stainable with amido black was detected in region 5-This peak was previously ascribed to mercaptoethanol. The disc gel electrophoretic patterns obtained from the pattern in Figure 4a (0.25$ NaCl) is shown in Figure 6. In most respects these electropherograms are similar to those of Figure 5-However, it is apparent that lysozyme instead of being detected in only a single fraction—fraction 4 — i s now also detectable in fraction 3. A small amount of lysozyme was also detected in fraction 5. From this i t would appear that lysozyme has interacted with other proteins of egg white and co-chromatographed with them. The lysozyme-ovomucin interaction reported by Hawthorne (42) has particularly attracted attention since upon this interaction has been based one of the theories pertaining to the rigidity of the thick gel structure of egg white, and an accompaning theory of the storage thinning of egg white. In particular it is proposed ko Figure 5« Disc gel electrophoresis of fractions collected as shown in Figure 4b. EW = egg white treated under the same conditions as the fractions but not chromatographed. Figure 6. Else gel electrophoresis of fractions collected as shown in Figure ha. 42 that an interaction between lysozyme and ovomucin is responsible for the gel characteristics and that as the pH of egg albumen rises during aging the strength of the interaction decreases and the gel thins. It seems apparent from the gel filtration data reported here that lysozyme does not interact with any egg white components in 1$ salt. At 0.25$ salt—ionic strength conditions about 70$ that of egg white (reported to be about 0.1 by Donovan e_t al_. (29))—lysozyme shows no tendency to interact with high molecular weight ovomucin. Instead, it interacts with materials eluted in the ovalbumin region, perhaps with ovalbumin itself. Further evidence that lysozyme does not interact with ovomucin in egg white is to be found in Figure 7. This figure shows the effect of centrifugation at 78,500g in a Beckman L2 - 6 5 B preparative ultracentrifuge on the elution profile of egg white from Sephadex G-150. It is known that centrifugation at high speed is capable of separating the gel and liquid phases of egg white (47). If lysozyme and ovomucin interact in native egg white to form a cross-linked gel network, then it is possible that the gel phase would show enrichment in both the ovomucin and lysozyme components. As can be seen from this figure, the only discernible difference in the gel and liquid phases lies in the material eluted at the void volume which is increased in the gel phase and reduced in the liquid phase. This material probably represents the ovomucin content of the gel phase. It has been consistently observed in the literature that thick white contains about four times as much ovomucin as thin 43 2.0-2 0 4 0 6 0 F R A C T I O N NUMBER F i g u r e 7. E f f e c t o f c e n t r i f u g a t i o n o n t h e e l u t i o n o f e g g w h i t e f r o m S e p h a d e x G-150. C o n d i t i o n s a s i n F i g u r e s l a - e . A = s u p e r n a t a n t , B = e g g w h i t e , C = g e l p h a s e . S a m p l e s i z e = 4.0 m l . white ( 2 6 ) . The material in the liquid phase which elutes at the void volume is probably "line 18" material (52) and is in agreement with patterns reported by Young and Gardner ( 9 1 ) . GENERAL DISCUSSION It is clear from the foregoing data that reduction of the ovomucin gel of egg white with 2-mercaptoethanol results in a solution of proteins amenable to gel filtration techniques. Of the gel filtration media commercially available from Pharmacia Fine Chemicals, Sephadex G-150 was the gel of choice. Furthermore, doubling the column length improved the resolution of components only marginally but increased the technical difficulties considerably due to flow rate problems and compactation of the bed. Operation of these longer columns in an upward flow manner eased but did not eliminate these problems. Consequently, the use of shorter columns is recommended for most purposes. Lysozyme has been shown to interact and co-chromatograph with other egg white components at relatively low ionic strength. The component with which lysozyme interacts was not identified but was shown to elute from Sephadex G-150 in the ovalbumin region of the pattern. It has been shown that lysozyme and ovalbumin interact at low ionic strength (59) so that it is tempting to suggest ovalbumin as the interacting partner. However, i t is possible that some other component that elutes in the same region as ovalbumin is interacting with lysozyme. Robinson and Monsey (67) have shown the presence of an ovomucin-like component that is extracted along with lysozyme from crude ovomucin preparations with KC1. This component (67) also interacts with lysozyme at low ionic strength, however, when reduced and alkylated, the component was eluted at the void volume of a Sephadex G-200 column. This makes i t unlikely to he the interacting component in the ovalbumin region, but does indicate the possibility of an unknown interacting species other than ovalbumin. The possibility that 2-mercaptoethanol interfers with the lysozyme-ovomucin interaction appears unlikely since Dam ( 2 6 ) has shown the interaction to be unaffected by this compound. Further evidence which suggests the ovomucin and lysozyme do not interact in the native egg system was obtained by detecting the distribution of macromolecular components in the gel and liquid phases. The failure to detect any increase in concentration of lysozyme in the gel phase in spite of the fact that ovomucin was increased tends to support this suggestion. Dam ( 2 6 ) has suggested that changes in ovomucin may be sufficient to explain thinning of the gel structure in egg white during storage and that it is unnecessary to invoke the use of another protein as a cross-linking agent. A l l of the observations made in this study support this view and suggest that while lysozyme may interact with other egg white components such as ovalbumin, it does not interact with ovomucin under the conditions which exist in the egg. ^7 CHAPTER III: EFFECT OF SULHYDRYL BLOCKING CN THE RHEOLOGICAL PROPERTIES OF EGG WHITE AND ULTRACENTRIFUGAL PROPERTIES OF OVOMUCIN  ISOLATED FROM BLOCKED AGED WHITE. INTRODUCTION The rigidity and amount of the thick albumen fraction of egg white is an important factor in determining the acceptability or grade score of eggs. During the storage of shell eggs, the thick white undergoes a thinning reaction resulting in lowered grade scores. In spite of intensive study in recent years ( 5 , 2 6 , 6 9 ) , biochemical changes which might cause the thinning have not been elucidated. It has been known for some time that reducing agents—notably thiol compounds—are capable of causing rapid dissipation of the ovomucin gel of thick egg white ( 5 3 ) . In addition, broken out eggs or shell eggs exposed to these compounds rapidly simulate old, badly deteriorated eggs. Observations such as these have led to the proposal that naturally present reducing agents thin egg white by splitting the disulfide (SS) bonds of ovomucin, but, it has not been possible to show significant amounts of reducing substances in egg white (30). Ovalbumin, the major protein of egg white, contains sulfhydryl (-SH) groups but these groups are not as reactive as those of thioglycol. However, it was noted by MacDonnell et al_. (53) that " i f only 2$ of the -SH groups in ovalbumin were to become as active as the -SH of thioglycol, marked deterioration of the egg would result." 48 Recent workers in this field have concentrated upon ovomucin and upon-the changes which ovomucin undergoes on aging of egg white. Robinson and Monsey (68) have shown that reduced, alkylated ovomucin consists of two components detectable by gel filtration or ultra-centrifugation in the presence of 5 M guanidine hydrochloride. On aging of thick egg white a shift in the relative concentration of these two components occurs-- a-ovomucin increasing from 71.3$ of the total schlieren pattern area to 93 .1$ while /?-ovomucin decreased from 28.6$ to 6.8$ over 47 hours of incubation at 37°C. Kato e_t al_. (45,46,47) obtained similar results by free boundary electrophoresis of ovomucin reduced with 2-mercaptoethanol. These later authors have proposed a mechanism for the thinning reaction involving breakage of SS bonds. The viscous properties of egg white have also been used to evaluate the thinning reaction (4-5,69) and have been studied extensively by Tung et a l . (84,85) who report non-Newtonian behavior. Albumen was found to exhibit time and shear rate dependent thinning and to be pseudoplastic in its equilibrium flow behavior--the data being fitted by a form of the well known power law. Tung e_t al_. (84) were unable to show any change in the power law parameters determined as equilibrium flow properties on aged egg white. However, the stress decay (shear thinning) parameters showed significant changes on aging. Robinson and Monsey (69) have fitted their flow behavior data by a form of the power law and have reported that the power law parameters change with thinning—the apparent viscosity decreasing and the flow properties approaching that of a Newtonian fluid. They suggest "the values of apparent viscosity obtained at a single rate of shear are useful for comparing the firmness of egg white." The present chapter describes the effect of -SH blocking or oxidation on the thinning reaction as detected by rheological and ultracentrifugal measurements. MATERIALS AND METHODS 50 Eggs were obtained on the day of lay from the University farm, broken open and the thick white obtained and bulked as described by Robinson and Monsey ( 6 9 ) . A l l glassware, blender cups and other equipment used were autoclaved and a l l operations were carried out in a laminar flow cabinet. The thick white was blended in a Sorvall Omni-mixer for 20 sec at speed setting 2 . 5 . Thinning was induced by incubation of the thick white in sterile flasks at 37°C. The reaction was stopped by immersing the flasks, in crushed ice. The pH of the white obtained was 8 . 6 to 8 . 7 and remained constant throughout the experimental period regardless of the treatments applied. Treatment with Iodate, Bromate and Cysteine Potassium iodate (O.963 g), 0.751 g potassium bromate and 0.180 g L-cysteine (Schwarz/Mann Lot Wo. W1024) were each dissolved in 15 ml distilled water. Ten ml of each solution was added to hOO ml of egg white and mixed thoroughly by inversion. Swirling of white was avoided since it leads to entanglement of the fibers of thick white and difficulties in obtaining homogeneous samples for later testing. The control consisted of a similar lot of egg white treated with 10 .0 ml water. The treated white was dispensed in 100 ml lots into sealed flasks and incubated. Treatment with HgCl9 and p-Chloromercuribenzoate Addition of either HgCl2 or p-chloromercuribenzoate (PCMB) directly to egg white as concentrated solutions caused immediate localized coagulation of the egg white. This necessitated the addition of the compounds carefully from dilute solution which was accomplished by dialysis. The solution outside the dialysis bag consisted of tris-KC1 buffer (0.01 M t r i s , 0 .09 M KCl) pH 8 . 6 . The ionic strength of this solution was about 0.1—similar to that present in egg white ( 2 9 ) . Mercuric chloride (l.O g) was dissolved in 50 ml water and 5 j 10 or 20 ml of this solution was added to 2,000 ml lots of dialysis buffer yielding solutions containing 55 10 or 20 x 10 mg/ml HgClg. Prepared egg white (500 ml) was placed in sterile dialysis bags and dialysed for 18 hours against the buffer at k°C. On removal from the dialysis bag, the egg white was mixed carefully by inversion then dispensed in 100 ml lots into sealed flasks and incubated. PCMB treatment was done similarly except that 2.0 g were dissolved in 50 ml water (0.1 N NaOH added as required to dissolve PCMB) and 5? 10 and 15 ml were placed in the dialysis buffer giving concentrations of 10, 20, or 30 x 10"^ mg/ml. Sulfhydryl Determination These determinations followed the procedure outlined by Beveridge et a l . ( 9 ) for egg white. Biuret reagent was prepared by dissolving 6.0 g sodium potassium tartrate in about 300 ml water. Sodium hydroxide (300 ml, 10$) was added and the solution made to 1 l i t e r . Ellman's reagent ( 15) was made by dissolving 3 9 - 7 nig 5 , 5 ' dithiobis-2-nitrobenzoic acid (DTNB) in 10.0 ml of buffer (0.01 M PO^, pH 7.0, 1$ NaCl). Dilution of egg white was accomplished by mixing thoroughly in a centrifuge tube 1 to 1.5 g egg white and 5 - 0 ml egg white 52 diluter (0.01 M phosphate, 1$ NaCI, pH 7 . 0 ) . The solution was centrifuged at 12,000g for 5 minutes to remove small pieces of ovomucin gel and obtain a clear solution containing 1 to 1.5$ protein. To 0.1 ml protein solution was added 2.9 ml 0.1 M PO^  buffer, pH 8.0 containing 0.5$ sodium dodecyl sulfate (SDS), followed by 0.02 ml DTNB. Optical densities were measured in a Beckman DB spectrophatometer at kl2 nm and the extinction coefficient given by Ellman (31), 1.36 x 10^ was used for calculation of -SH. The protein concentration of pipetted protein solution was determined by the biuret method standardized against protein nitrogen values obtained by Kjeldahl (2) analysis. Egg white protein was assumed to contain 16$ nitrogen and results were calculated as uM -SH per g protein. Preparation of Ovomucin Ovomucin was prepared according to the procedure of Robinson and Monsey (68) except that 2$ (w/v) NaCI replaced 2$ KC1. Analytical Ultracentrifugation Sedimentation velocity measurements were carried out using schlieren optics. Egg white was diluted approximately 1:10 in such a way that the final protein concentration was 1$ (biuret) with 0.01 M tris buffer, 0.2 M NaCI, pH 8 . 6 . Schlieren patterns for ovomucin were obtained by dissolving, with gentle stirring, 20 mg isolated, purified ovomucin in 2 ml of a solution containing 0.01 M PO^, 5 M guanidine HC1 (Schwarz/Mann Ultra Pure ),.0.1 M NaCI and 2.3$ (v/v) 2-mercaptoethanol, pH 7.5- Solution was complete in 30 minutes. The samples were run in a double sector cell at 595000 rpm in a Beckman L2 - 6 5 B preparative ultacentrifuge equipped with a schlieren optics accessory. Patterns obtained were printed on photographic paper, the enlarged peaks were cut out and weighted to obtain the relative amounts of the two components. This procedure was done for six prints of two pictures to obtain a total of 12 measurements for each experimental condition. No attempt was made to correct for the Johnson-Ogston effect. Relative viscosity measurements were carried out in a capillary viscometer having a flow time of 217 sec for water at 20°C. Densities were determined pycn ome trically. Viscosity Measurements Viscosity measurements were carried out at 1 . 0 - 0.5°C with a Haake rotational viscometer using an MV1 spindle providing a maximum shear rate of 1370 per sec, the output of which was fed to a strip chart recorder as described by Tung e_fc al_. (85). Each sample was first tested for the time dependent characteristic at a constant shear rate of 1370 per sec. After the shear stress curve had reached an apparent equilibrium value—about five minutes—the equilibrium flow curves were obtained by decreasing the shear rate over a series of steps and then increasing the shear rate stepwise to the original value. Data were collected on duplicate samples at each experimental condition, pooled and treated as a single sample. Shear stress decay data was sampled by the method of Cramer and Marchello ( 2 2 ) . The shear stress range between the in i t i a l maximum value and the apparent equilibrium value was divided into 5h ten equal logarithmic intervals and the corresponding shear stresses and times were recorded. The data were fitted by a function of the form ( 8 5 ) A = AQ - n.log t (l) where t is time in seconds, A is the shear stress in dynes•cm"2, AQ is a constant which reflects the in i t i a l shear stresses as shear begins (84), n, is a constant reflecting rate of structural breakdown. This model was originally suggested by Weltmann ( 8 8 ) for stress relaxation in thixotropic systems. Equilibrium flow data were fitted to a form of the power law B = K-Xn (2) where B is the shear stress in dynes•cm-2, X is the shear rate in sec--*- and n and K are the flow behavior index and the consistency index respectively ( 8 5 ) . Fitting of the data was accomplished by a least squares method and testing of the linear regressions for difference in slope and intercept was accomplished by a covariance method ( 8 0 ) . 55 RESULTS AND DISCUSSION Viscosity as a_ Measure of Thinning Preliminary work suggested that obtaining flow behavior curves for thick egg white from which apparent viscosity values could be obtained for comparison purposes was difficult or impossible due to the rheodestructive nature of the thick albumen gel structure (84,85). Consequently, shear stress decay and equilibrium flow behavior were investigated and evaluated as measures of the thinning reaction. The results of a typical experiment are shown in Figures 8 and 9 . Figure 8 shows the changes in shear stress decay behavior which occur in thick egg white aged at 37°C for 0 , 48, 72 and 120 hours.t The levels of the four curves differ significantly and curve 1 has a significantly different slope ( P $ 0 . 0 l ) . It is apparent that the level of the lines decreases in a regular fashion as a function of incubation time. Figure 9 shows the comparable curves for equilibrium flow behavior. Lines B and C do not differ but both lines A and D differ from each other and from lines B and C ( P $ 0 . 0 l ) . The lines do not change in a regular fashion, however, a tendency exists for the consistency index (K in the power law equation) to decrease and for the flow behavior index (n in the power law equation) to increase, approaching 1 as aging continues. This, in general, is in agreement with data reported by Robinson and Monsey ( 6 9 ) . These tendencies, as well as the eratic, irregular nature of the changes observed, have been confirmed in other trials ( 8 6 ) . Figure 10 is a plot of the intercept value AQ in equation 1 56 Figure 8. Shear stress decay curves of egg white incubated at 37°C for 0, 48, 72 and 120 hours. 57 T= Holding T ime - hours —i , i , i 2.0 2.5 3.0 log SHEAR RATE Figure 9- Equilibrium flow behavior curves for egg white incubated at 37°C for 0, 48, 72 and 120 hours. 58 Figure 10. Effect of incubation time on the shear stress decay parameter AQ„ 59 as a function of incubation time. A is a reflection of the in i t i a l o value of shear stress and would be expected to reflect the amount of thick gel structure originally present in the sample. On this basis, AQ should be a useful index to use as a measure of the thinning reaction. The value of AQ in Figure 10 drops smoothly as expected from the lines of Figure 8 suggesting the use of this index as a measure of thick gel structure for comparison of various treatments. Effect of Bromate, Iodate and Cysteine The effects of addition of the oxidizing agents iodate and bromate and of the -SH compound cysteine are shown in Figures 1 1 , 1 2 , 13 and ik. Figure 11 shows the shear stress decay curves obtained for water treated control thick white and cysteine treated thick white. The levels of the control lines decrease in regular fashion, the levels are significantly different, however, the slopes are not (P^O.Ol). This is in agreement with the data obtained previously on untreated egg white. The effect of cysteine addition is shown by the sharp change in level and slope of the treated samples. The cysteine treatment lines differ from the control lines in both level and slope but differ from each other only in level (P-^O.Ol). Figure 12 shows the effect of bromate treatment on the shear stress decay behavior of thick egg white. The lines differ significantly in level and line k differs in slope (P^O.Ol). Figure 13 shows the effect of iodate treatment on the shear stress decay behavior of thick white. The lines do not differ in 6o 'Figure 11. Shear stress decay curves of control egg white and cysteine (CYS) treated egg white incubated 0, 2k, k8 and 83 hours at 37°C. Figure 12. Shear stress decay curves of egg white treated with potassium bromate. Conditions as for Figure 11. 62 Figure 13. Shear stress decay curves of egg white treated with potassium iodate. Conditions as for Figure 11. 63 240 200 160-120 2 0 60 100 HOLDING TIME -- hours Figure 14. The effect of treatment of egg white with cysteine, bromate and iodate on the intercept value A . The bars represent - 1 standard error associated with the intercept. • Control; • Iodate; • Bromate; O.Cysteine. slope and lines 2 and 3 do not differ in level. The effect of the treatments were compared by plotting the intercept values Ao obtained for each line as previously suggested. The results are shown in Figure ik. The cysteine points are clearly different from either the control or oxidized samples, the low values indicating l i t t l e or no gel structure present. This is in agreement with reported data ( 5 3 ) . The line for the bromate treatment lies somewhat below the control line, the 83 hour point notably being different from the control. The points making up the iodate line after 2k hours are significantly (P^O.Ol) higher than the control. Analysis of -SH groups for these treatments are shown in Figure 1 5 . Iodate treatment lowered the -SH content of egg white about Qfo whereas bromate did not affect the levels as compared to the control. The results of this experiment must be considered incon-clusive, however, examination of the stress decay behavior curve (Figure 1 2 , line k) from which the 83 hour bromate point was obtained shows that the slope was significantly different from the other flow behavior lines. This anomalous behavior may be the cause of the low value rather than any loss in gel structure. Effect of PCMB and HgClg It is known that mercury compounds react with the -SH groups of egg white and ovalbumin under mild conditions (2k ,5k). However, direct mixing of the compounds PCMB and HgClg into egg white caused irreversible precipitation of the proteins. This difficulty was overcome by dialysing the mercury compound into the protein solution. The effect of dialysing egg white against various concentrations of mercury compound for 18 hours at k°C is shown in Figure 1 6 . The ratio of egg white to external solution was l:k. Unfortunately, complete blockage of -SH was impractical because of gelation reactions which began after about 8 0 $ of the -SH had been blocked. However, extensive blocking is possible without gross visable changes occurring in the egg white. This figure formed the basis for the mercury compound concentrations chosen in the section of materials and methods. The effect of blocking -SH groups with HgCl2 or PCMB on the shear stress decay behavior of thick egg white is shown in Figures 17 to 2k. In general, the disturbance of the normal decreasing pattern of the lines can be seen in these figures. This includes the patterns labelled controls—the egg white in these two cases (Figures 17 and 21 ) having been dialysed against tris-KCl buffer. In the figures representing the treatments with mercury compound, the general pattern is that the lines first rise to a maximum level in 2k hours and show a falling pattern thereafter from this level. Comparison of the intercept values (AQ) of the. HgCLj blocking experiment is shown in Figure 2 5 , and the -SH levels and total solids of the treated and untreated egg white for both the HgCl2 and PCMB experiments are shown in Table I. The disruption of the normal thinning pattern observed previously is clearly shown. To facilitate comparison, the control thinning pattern shown in Figure ik has been reproduced on the figure. 70 50 • control • bromate A iodate 40 80 Holding Time -hours Figure 15. Effect of oxidizing agents on the -SH groups of egg white. 67 * I I " » 5 15 25 MERCURY COMPOUND IN OUTSIDE S O L U T I O N - mg/ml x 10~2 Figure l 6 . Effect of dialysing egg white against PCMB and HgCl2 at pH 8.6 for 18 hours on -SH content of the white. 68 Figure 17. Shear stress decay curves of egg white dialysed against 0 mg/ml HgCl2 in tris-KCl buffer. The holding times and correlation coefficients are as follows: line 1; 0 hours, 0 . 8 l , line 2; 2k hours, 0.89, line 3; k8 hours, O.96, line k; 72 hours, 0.9 1 *. 69 Figure 18. Shear stress decay curves of egg white dialysed against 5 x IO-2 mg/ml HgCl2 in tris-KCl buffer. The holding times and correlation coefficients are as follows: line l j 0 hours, 0.94, line 2; 2k hours, 0.97, line 3; k8 hours, 0.92, line k; 72 hours, 0.97. cc H (/) 100 < LU X C/> 1 : I 1.0 2.0 log SHEARING T IME-sec . Figure 19. Shear stress decay curves of egg white dialysed against 10 x 10"2 mg/ml H g C l 2 in tris-KCl buffer. The holding times and correlation coefficients are as follows: line l j 0 hours, 0.9*+, line 2; 2k hours, 0.97, line 3; k8 hours, 0.97, line k; 72 hours, 0.83. 71 1.0 2.0 log SHEARING TIME sec. •Figure 20. Shear stress decay curves of egg white dialysed against 20 x 10~2 rag/ml HgC]_2 in tris-KCl "buffer. The holding times and correlation coefficients are as follows: line 1; 0 hours, 0.°A, line 2; 24 hours, 0.92, line 3; 48 hours, 0.93, line 4, 72 hours, 0.97. 72 1.0 2.0 log SHEARING TIME sec Figure 21. Shear stress decay curves of egg white dialysed against 0 mg/ml PCMB in tris-KCl buffer. The holding times and correlation coefficients are as follows: line 1; 0 hours, O.98, line 2; 2k hours, O.98, line 3; k8 hours, 0.97, line k; 72 hours, 0.97. 73 < 100 UJ X , (0 : I I 1.0 2.0 log S H E A R I N G T I M E - s e c •Figure 22. Shear stress decay curves of egg white dialysed against 10 x IO"2 mg/ml PCMB in tris-KCl buffer. The holding times and correlation coefficients are as follows: line 1; 0 hours, 0.90, line 2; 2k hours, 0.97, line 3; k8 hours, 0.99, line k; 72 hours, O.98. Figure 23. Shear stress decay curves of egg white dialysed against 20 x IO"2 mg/ml PCMB in tris-KCl buffer. The holding times and correlation coefficients are as follows: line 1; 0 hours, 0.97> line 2; 2k hours, 0.88, line 3; kQ hours, O.65, line k; 72 hours, 0.95. 75 rJ5 2J0 log SHEARING T I M E - s e c . Figure 2k. Shear stress decay curves of egg white dialysed against 30 x 10-2 mg/ml PCMB in tris-KCl buffer. The holding times and correlation coefficients are as follows: line 1; 0 hours, 0.98, line 2; 2k hours, 0.99, line 3; kQ hours, O.kO, line k; 72 hours, 0.90. Two points may be noted from Figure 2 5 . First, the control, dialysed against tris-KCl buffer showed a decrease of about 1 5 $ in -SH groups (Table I) and failed to show the typical thinning pattern. Secondly, more extensive blockage of -SH groups caused a large increase in the AQ value followed by a steady decline. The intercept values for the PCMB blocking experiment are shown in Figure 2 6 . Again the dialysed control showed a decrease of about 15$ in -SH groups and an altered thinning pattern and again more extensive blockage was associated with a large increase in A0 followed by a steady decline. In addition to the altered thinning pattern shown by the dialysed control, blockage of 3 2 $ of the -SH groups of egg white (10 x IO-2 mg/ml PCMB treatment) showed almost no thinning reaction in 72 hours at 37°C. The variation observed in ini t i a l AQ values cannot be ascribed to differences in total solids among the treatments since, while there was a lowering of the total solids values on dialysis, there was essentially no differences between PCMB treatments. In addition, the HgC^ treatments which showed a drop of 1 . 7 $ in total solids when dialysed did not exhibit wide variation in initi a l AQ values. It seems likely that this init i a l variation exhibited by the PCMB treatments is due to the handling of the white which accompanied the dialysis step. An attempt was made to determine the reason for the marked increase in AQ caused by relatively extensive blocking of -SH groups. Since it is known that the mercuric cation has a very high affinity for -SH groups and is bifunctional (8l) it was considered ^ ^ J L n M M B M B B I M H H M M M X i • • • • M M • • • • • M B M i M M M M M M I A M I M M M I M M M M M M M M M M l M M M B B M M B M j 0 40 80 HOLDING TIME hours Figure 25. The effect of blocking -SH groups of egg white with HgCl2 on the intercept value AQ. The bars represent ± 1 standard error associated with the intercept. • Undialysed control; • Dialysed control; • 5 x IO"2 mg/ml HgCl2; A 10 x 10~2 mg/ml HgCl2; A 20 x IO"2 mg/ml HgCl2 . 78 Table I. Sulfhydryl levels and total solids in sulfhydryl blocking experiments. Sample Sulfhydryl Total uM/g Protein % of Original Solids (jo Untreated (HgCl2) egg white 6 0 . 4 — 1 1 . 9 Dialysed (HgCl2) egg white 5 1 . 2 85 1 0 . 4 5 x 1 0 - 2 mg/ml HgClg 4 5 . 6 75 1 0 . 5 10 x 1 0 - 2 mg/mi HgCl2 3 ^ . 2 57 1 0 . 2 20 x 1 0 - 2 mg/ml HgCl2 1 7 . 2 28 1 0 . 2 Untreated (PCMB) egg white 6 2 . 5 — 1 2 . 2 Dialysed (PCMB) egg white 5 3 . 0 85 1 1 . 5 10 x 1 0 - 2 mg/ml PCMB 4 3 . 0 68 1 1 . 5 20 x IO"2 mg/ml PCMB 3 8 . 0 6 l 1 1 . 5 30 x IO"2 mg/ml PCMB 3 0 . 6 49 1 1 . 7 7 9 380 340 300 260-220 180 0 40 80 HOLDING TIME - hours F i g u r e 26. The e f f e c t o f b l o c k i n g - S H g r o u p s o f e g g w h i t e w i t h PCMB o n t h e i n t e r c e p t v a l u e A 0 . The b a r s r e p r e s e n t - 1 s t a n d a r d e r r o r a s s o c i a t e d w i t h t h e i n t e r c e p t . • U n d i a l y s e d c o n t r o l ; • D i a l y s e d c o n t r o l ; • 10 x I O " 2 m g / m l P C M B ; O 20 x 10-2 m g / m i P C M B ; A 30 x 10-2 m g / m l P C M B . possible that dimerization or polymerization of ovalbumin through -SH groups cross-linked by divalent mercury was responsible for the increased apparent gel structure. Since PCMB is monofunctional the increase in apparent gel structure was unexpected, however, both reagents are capable of precipitating egg white proteins under appropriate conditions so that non-specific aggregation reactions cannot be eliminated as a possible cause. Steinberg and Sperling (8l) were able to demonstrate extensive aggregation of ribonuclease treated with mercuric chloride in the ultracentrifuge. A typical ultracentrifuge pattern of egg white is shown in Figure 27. This figure shows the pattern obtained after treatment of egg white with 20 x 10~2 mg/ml HgCLg by dialysis (-SH is 17.2 uM/g protein) 60 minutes after reaching f u l l speed. As can be seen in the photograph no evidence of aggregation was demonstrated. Calculation of the sedimentation coefficient of the peak yielded a value of 3 . 7 . A value such as this would be expected from a molecule of about 4-0,000 molecular weight (19) . This molecular weight is in agreement with that of ovalbumin, the major protein of egg white, and suggests that aggregation or polymerization reactions are not occurring in the presence of HgCl2. The sedimentation coefficients of egg white determined after treatment with both HgClg and PCMB are shown in Table II. It is apparent from the values shown in this table that aggregation or polymerization could not be detected for any of the treatments used and that the treatment coefficients do not differ from coefficients obtained for untreated egg white. Figure 2 7 . Typical schlieren pattern of 1$ egg white 60 minutes after reaching 59*600 rpm. Temperature is 20°C, phase plate angle 68 Table II. Effect of HgCl2 and PCMB on the sedimentation velocity of egg white proteins. Sample 0 Hours 2k Hours kQ Hours at 37°C at 370c at 37°C No treatment ' 3 . 8 l Dialysis 3-71 3 . 7 1 HgCl2 (5 x 10-2mg/ml) 3.68 3 .87 3.84 HgCl2 (20 x 10-2mg/ml) 3 . 7 1 3.68 3.88 PCMB (10 x 10-2mg/ml) 3 . 8 1 3-71 3.83 PCMB ( 3 0 x 10-2mg/ml) 3-71 3 . 8 0 3.79 83 In view of the failure of the ultracentrifuge to demonstrate any aggregation of the egg white, the reason for the sharp increase in AQ values after 2k hours incubation is difficult to explain. However, it is possible that a mercury cross-linked gel structure existed in the concentrated protein solution that was disrupted when the solution was diluted for ultracentrifugal analysis. At least three explainations may be entertained in order to explain the effects of the PCMB and HgClg -SH blocking on viscometric data. First, since it is known that mercury binding to -SH groups is reversible ( 8 l ) , it is possible that mercury induced, i n i t i a l aggregation after dialysis is followed by rearrangement and diffusion of mercury throughout the egg white resulting in an overall weaker gel structure. Thinning then, would be inhibited by -SH blocking as suggested by the dialysed control lines of Figures 25 and 2 6 and by the 10 mg/ml PCMB line in Figure 2 6 . Secondly, it is possible that thinning is either not inhibited or only slowed by -SH blocking. In this case the lines representing high blocking levels in Figures 25 and 26 represent ini t i a l aggregation of the proteins followed by natural thinning reactions which account for the decreasing AQ values. If this interpretation is accepted then the dialysed controls and the 10 mg/ml PCMB lines represent a balance between aggregation and thinning reactions in which l i t t l e or no net change is observed. Thirdly, a combination of both of the above interpretations could explain the data. 8k It has been observed by Robinson and Monsey ( 6 8 , 6 9 ) that shifts in the relative amounts of the two components— a-ovomucin and /?-ovomucin—that make up the ovomucin complex of egg white occurs as egg white ages and thins. The two components may be detected in isolated ovomucin in the ultracentrifuge under structure breaking (5 M guanidine-HCl) and reducing conditions. The results of analytical ultracentrifugation of ovomucin isolated from fresh and aged egg white are shown in Figures 28a and b. As can be seen in the figures the shift in the relative amounts of the two components are clearly demonstrated and, qualitatively at least, is in agreement with the observations of Robinson and Monsey ( 6 9 ) . For fresh white ovomucin, S2Q w values of k .81 for the fast (/? -ovomucin) component and 3.*+3 for the slow ( <* -ovomucin) component were obtained. These values are lower than those obtained by Robinson and Monsey ( 6 8 ) . Furthermore, with aged white ovomucin, S2Q w values of 6 . 3 3 for the fast component and 3 . 0 2 for the slow component were obtained. The differences observed in the fast component can be seen in the two photographs of Figures 28a and b. The reason for the disagreement with the S values reported by Robinson and Monsey ( 6 8 ) is not known, however, the conditions under which the determinations were made are considerably different and direct comparison may not be possible. Components in ovomucin with S2Qj W values of 6.k and 2 . 9 have been reported by Donovan e_t al> ( 2 8 ) . The relative amount of the slow peak in untreated, aged and -SH blocked egg white is shown in Figure 2 9 . The vertical bars 85 Figure 2 8 a . Ultracentrifuge pattern of ovomucin obtained 95 minutes after reaching 5 9 , 1 0 0 rpm--ovomucin from fresh egg white. Conditions: temperature 20°C, 5 M guanidine-HC1, 2 . 3 $ (v/v) 2-mercapto-ethanol, u 0.1, pH 7 . 5 . phase plate angle 7 5 ° . Figure 28b. Ultracentrifuge pattern of ovomucin obtained 95 minutes after reaching 59,100 rpm—ovomucin from egg white aged 48 hours at 37°C. Conditions: as in Figure 28a. 87 24 48 HOLDING TIME - hours Figure 29. Effect of blocking -SH groups on the relative amount of a-ovomucin (slow peak) in the ovomucin isolated from egg white after incubation at 37°C for 2h and k& hours. • Undialysed control; •Dialysed control; O 5 x IO"2 mg/ml HgCl2; • 20 x 10~2 mg/ml HgCl2J A Literature values. on the points represent 95$ confidence intervals computed on the basis of the multiple photographic measurements made. The values obtained by Robinson and Monsey (69) are also.shown on the figure. As can be seen from the figure the values obtained by Robinson and Monsey and the values obtained for untreated egg white in this study lie in the same region of the graph and show reasonable agreement. Further, in a l l cases shown, reduction in -SH level results in a slowing of the rate of change of the relative areas of the two peaks. PCMB treated egg white gave lines falling in the same region of the graph as the dialysed and HgCl2 treated white. It seems clear from this figure that blockage of some -SH groups inhibits but does not completely stop the changes in ovomucin associated with thinning reactions. 89 GENERAL DISCUSSION The use of apparent viscosity as an index of liquefaction of egg white has been suggested by Robinson and Monsey (69). However, in this study, i t was found that the time and shear rate dependent thinning exhibited by egg white made single point deter-minations at one shear rate impossible. This difficulty was overcome by evaluation of the time dependent shear thinning behavior using an emperical equation. In this way the rotating cylinder viscometer has provided a means for comparing changes of apparent viscosity which occur during the thinning of egg white. For this purpose it was found convenient to use the intercept value obtained in equation 1 , A , as an index of thick gel breakdown. That this index can be used as a measure of thinning has been demonstrated in Figures 10 and ik. However, use of this index has two major drawbacks. First, a large amount of data must be collected and fitted by the equation to obtain just one value of AQ. This necessitates the use of automatic, programable computing machines to do the calculations. Secondly, the index is sensitive to disturbances caused by gelation or aggregation reactions such as those which occurred when relatively extensive blocking of -SH with PCMB or HgCl.2 was obtained. The nature of the aggregation or polymerization reactions which were observed during the course of this investigation, were not determined. Ultracentrifugation indicated that it was not due to dimerization, aggregation or polymerization through -SH groups cross-linked by mercuric ions as has been observed in reduced ribonuclease ( 8 l ) . PCMB is well known for its high specificity for -SH groups in proteins (72) and in addition it is monovalent. In spite of this, apparent aggregation was observed viscometrically in PCMB treated egg white. It has been shown that the molecule p-chloromercuribenzene-sulfonate (closely related to PCMB) binds to crystals of sperm-whale myoglobin in such a way that it lies between two myoglobin molecules at a point in the unit cell where they lie rather close together, and apparently binds to both molecules ( 1 3 ) . Also, it has been shown that mercuric salts can bind to sites other than -SH groups in proteins such as lysozyme which contain no free -SH. Since egg white is a concentrated solution of proteins, it is possible that binding of reagent to sites other than -SH is responsible for the observed aggregation. The effect of -SH blocking on thinning reactions may be assessed by making two observations. First, whenever -SH groups are decreased and complications due to aggregation are minimal, viscosity data suggests a disruption of normal thinning patterns and a decrease in the rate of thinning. Data which particularly suggests this includes the iodate treatment, the 10 mg/ml PCMB treatment, and the two dialysed controls in which a 15$ drop in -SH content was observed. In this connection, experiments carried out later suggest that the KC1 was responsible for the loss in -SH, possibly due to a heavy metal contaminant. The nature of the 91 agent was not determined, however, heavy metal contaminants normally present in reagent grade chemicals have been shown to be responsible for a loss of -SH in myosin preparations ( 1 5 ) . The second observation that may be made is that the rate at which ovomucin undergoes changes during thinning is reduced when -SH groups are blocked. Taken together these two observations suggest that the -SH groups of egg white play a role in the reduction of SS bonds in ovomucin which results in the well known thinning phenomenon. Since ovalbumin is the only known source of -SH in egg white ( 3 0 , 3 5 ) ? ovalbumin seems likely to be involved in the reactions leading to thinning. The suggestion that ovalbumin is in some way involved in the thinning reaction has already been made by Smith and Back ( 7 7 j 7 8 , 7 9 ) . These authors have found that ovalbumin undergoes a transition on heating and on storage in the egg that results in a more stable form of the protein. Shifting of the SS bond in ovalbumin by SS interchange has been suggested as a mechanism for this transition. If such an interchange does occur in the ovalbumin molecule, it is possible that ovalbumin molecules in the region of the SS bonds of ovomucin interchange with ovomucin instead of effecting the internal conversion of ovalbumin and thinning results. 92 CHAPTER IV: FOAMING OF EGG WHITE—THE BEATING PROCESS INTRODUCTION Utilization of egg white in food preparations depends, to a large extent, upon its foaming power and the stability of the foams produced. Several studies have been initiated to determine the factors important to the stability and foaming power of egg white. MacDonnell e_t a l . (55) studied the contribution of specific protein fractions to the functional properties of egg white. The constituents found to be important were the globulins (including lysozyme), the ovomucin and the ovalbumin. It was felt that the globulin' fraction was in large part responsible for good foaming while ovomucin appeared to stabilize the foam. The ovalbumin was considered to contribute heat coagulable bulk which supported the structure of angel cakes. Nakamura and Sato ( 5 6 , 5 7 ) have also indicated that ovomucin is responsible for foam stability and that the globulins are good foamers. In addition, these authors have shown a decrease in the globulin fraction to occur on repeated whipping of egg white drained from foams. Lysozyme has been implicated as playing a role in egg white foaminess by Sauter and Montoure ( 7 3 ) . Garibaldi et al.(ho) have suggested that heat denaturation of a lysozyme-ovomucin complex during pasteurization of egg white may explain the damage to whipping properties which occurs. Early work in this field concerned itself mainly with the influence of chemical and physical factors on egg white foams ( 8 ) . Generally it was determined that foam density decreased with beating 93 time, that foam stability, as measured by drainage from the foam, decreased with beating time and that bubble size increased on aging of the foam. This latter property was also influenced by beating time. However, l i t t l e quantitative data, particularly with reference to bubble size analysis, is available from the literature. The object of this study was to obtain quantitative data on bubble sizes and size distributions and to attempt to relate these with losses of macromolecular components in the material drained from the foam. MATERIALS AND METHODS 9^ All chemicals used in this study were reagent grade and were used without further purification. Eggs were obtained from the University farm on the morning of lay, broken open and separated, the yolks being discarded. The white was blended by placing in a Sorval Omni-mixer and flicking the switch at setting 3 for 3 to 5 times. This procedure was found to just shear the fibrous envelope into pieces but did not cause liquefaction. The white was then placed in a beaker and stirred on a magnetic stirrer at slow speed for 30 to 60 minutes to obtain a reasonably homogeneous mix. The pH of the white was 8 .5 . Foam Density and Stability Funnels of known weight and volume and graduated cylinders of known weight were used for. this determination. Foam density was obtained from the weight of a known volume of foam. Foam stability was considered inversely related to the amount of liquid draining from the faom and was obtained by use of the graduated cylinder. Foams were obtained from 50 ml egg white beaten in a Sunbeam Mixmaster at setting 12. Bubble Size Analysis Foam was placed in a small shallow petri dish (Falcon Plastics No. 3001 35 x 10 mm) and illuminated from below on a standard microscope stage. Photomicrographs were taken using the low power . objective and 10X eyepiece giving a magnification factor of about 38 X. Photographs were also taken of a 2 mm microscope comparator 95 under the same conditions as the foam. The microscope lighting system was left off except when a picture was being taken in order to minimize heating effect from the lamp, and the foam was covered with the petri dish l i d held slightly above the foam surface to minimize the effect of evaporation. The camera was a 35 ™ Asahi Pentax Spotmatic equipped with a microscope adaptor and a "through the lens" metering system. The metering system was not used to judge exposure since it was found best to under expose the film (Kodak Tri X (ASA 125) at l / 3 0 to l / 6 0 of a second). This resulted in some variation in darkness of the negatives but did not appear to affect measurements of bubble size. The bubble and comparator negatives were printed on number h photographic paper from the same batch and manufacturer to a size of 5 x 7 inches. This gave an overall magnification of 6 I . 5 X. The . photographs were developed and dried in a single operation and allowed to come to equilibrium with atmospheric moisture for at least one week before measurements were made to minimize shrinkage or swelling effect in the photographs. Bubble diameters were measured using the enlarged comparator as a ruler. Calculation of distribution parameters were performed on the University computer ( 7 1 ) . Electrophoresis Disc gel electrophoresis was done as described by Zweig and Whitaker ( 9 2 ) using buffer system A (p. 1 5 9 ) , an alkaline buffer system recommended for serum proteins. Lysozyme was detected by setting up the electrophoresis run a second time and reversing the 96 polarity of the electrodes. Samples were prepared by adding 2 9 . 0 ml 1 $ NaCI to 1 . 0 ml egg white and mixing carefully. Exactly 0 . 0 2 ml of this solution was pipetted onto the electrophoresis gel and subjected to electrophoresis at 3 ma per tube for !§• to 2 hours. Staining was done for 10 minutes in 1 $ amido black in 1 0 $ acetic acid. Densiometric scans were performed on a Chromoscan instrument produced by Joyce, Loebl and Co. Ltd. Gateshead, England. Quantitation of some areas of the scans was obtained by carefully tracing the scan onto graph paper, cutting out the area and weighing. Gel Filtration This was performed as described previously ( 1 0 ) . Other Determinations The enzyme assay for lysozyme was done as described by Rhodes et a l . (64). The substrate was Micrococcus lysodeikticus cells produced by Difco Laboratories. Sulfhydryl (-SH) groups were determined by a procedure outlined by Beveridge et a l . ( 9 ) . Protein was determined in diluted egg white by procedures described previously. Relative viscosity was determined using an Ostwald pipette with the flow time for distilled water between two marks being assigned a value of 1 . 0 . Solids in the egg white were determined by drying white for 24 hours at 104°C. RESULTS AND DISCUSSION 97 As shown in Figures 30 and 31 , the egg white used in this study behaved in a typical fashion and gave density and stability data similar to that obtained by Barmore (8 ) . As whipping time was increased, foam density decreased and stability as measured by liquid draining from the foam decreased rapidly with increased whipping time. It is clear from this data that 2 minutes whipping gave relatively stable foams whereas 6 and 8 minutes caused overwhipping and produced unstable foams of low density. On this basis, 2 and 8 minute whipping times were chosen in most of the subsequent experiments. Bubble Size and Distribution The availability of a computer program (71) for performing •Chi-square "goodness of f i t " tests made the possibility of fitting the observed bubble size frequency distribution by some known theoretical frequency distribution a realistic possibility. The frequency distributions available for fitting included: a) Normal distribution Poisson distribution c) Binomial distribution a) Negative binomial distribution e) Gamma distribution f) Lognormal distribution g) Exponential distribution It has been shown (6l)' that bubble sizes in thick-walled foams follow a single log-normal distribution so it was considered 9 8 Figure 3 0 . Effect of whipping time upon expansion. Each point is the average of two determinations. 99 100 possible that this distribution could be fitted to the data obtained for bubble sizes in spite of the fact that egg white foams cannot be considered as thick walled. The results of the goodness of f i t tests are given in Table III. This table gives the Chi-square value obtained for the "goodness of f i t " tests at several whipping times and after aging of the foam for up to 20 minutes. The lognormal distribution gave significant Chi-square values in a l l but two tests, and in these two series, the lognormal distribution represented the best f i t of a l l the distributions tested. Furthermore these Chi-square values just missed the required probability value. Further inspection of Table III reveals that the lognormal distribution is not the only one to which a significant f i t was obtained. The gamma distribution also yielded a significant f i t in some cases, most notably after extended beating of the white. However, in a l l cases but two the lognormal distribution gave smaller Chi-square values with larger probabilities than did the gamma distribution. One instance of f i t to the normal distribution was observed for extensively beaten egg white. On the basis of this table it was concluded that, of the distributions tested, the lognormal distribution offered the best f i t to the observed data and was of the widest applicability. The bubble size frequency distribution of egg white foamed 2 minutes is shown qualitatively in the photomicrographs of Figures 32 and 3 3 . It was from photographs such as these that bubble sizes were assessed. The actual size distributions and the distributions as modelled by the lognormal frequency curve are shown in Figure 3*+. Table III. Chi-square values for fitting frequency distribution curves. :ating Aging Distribution Tested : (min .) Time (min .) Normal Poisson Binomial Negative Binomial Gamma i r O g n o r m a l Exponential 2 826 186 244 464 31.4 4 i . i 18.5 (s) NA 6 350 76.2 143 263 478 15.3 (s) 2.96 (s) 197 10 392 146 177 279 367 26.6 18.2 269 15 308 131 74.4 237 108 52.2 18.4 210 20 182 75.7 116 170 266 28.1 7.31 (s) 88.1 4 2 182 24.3 55.1 106 21.6 10.1 (s) 3.63 (s) NA 6 0.5 182 23.6 104 227 188 8.61 (s) 10.6 (s) 112 8 2 168 12.6 (S) 128 197 67.2 8.18 ( s ) ' 10.2 (s) NA 6 112 13.5 51.4 130 234 13.3 (s) 4.77 (s) 18.5 10 112 44.0 6o.6 96.6 NA 10.7 (S) 2.85 (s) 11.9 15 65.6 126 NA 2'. 73 (s) 14.8 10 2 224 21.3 167 NA 242 4.32 (s) 4.21 (s) 97-9 (s) The observed distribution (P$ 0 . 0 5 ) . is not significantly different from the theoretical distribution NA D i s t r i b u t i O E L not applicable due either to very high Chi-square values or incompatible parameters. Figure 3 2 . Photomicrograph showing effect of beating and aging on egg white foams. Picture taken 2 minutes after beating for 2 minutes. Magnification 6 1 . 5 X. Figure 33. Picture taken 20 minutes after beating for 2 minutes. Magnification 61.5 X. Figure 34. Effect of aging of foam on the frequency distribution of bubble size 2 minutes and 20 minutes after beating for 2 minutes. Class size interval is 20 microns in both cases. The arrows represent the arithmetic mean of the distribution. o 105 The model fits the data very well and shows the same trends as the actual data which should make it useful for comparative purposes. Foam formed by beating egg white for 2 minutes does not show any marked change in distribution on aging and this is reflected in the relatively small change in the arithmetic mean values exhibited by the data. However, there is a shift to larger sized bubbles in the foam aged 20 minutes. In addition, the formation of "polyhedral" foam as liquid drains from between the bubble lamella is easily seen in Figure 33 • The bubble size frequency distribution of egg white foam 2 and 10 minutes after beating 8 minutes is shown qualitatively in the photomicrographs of Figures 35 and 36 and quantitatively, including the lognormal model in Figure 3 7 - The effect of over-beating is easily seen in both the frequency distribution and the photomicrographs. The most notable effect is the shift to large bubble sizes—particularly the formation of very large bubbles--which is reflected in the shift of the arithmetic mean values from 9^«1 microns to 191 microns over the 8 minute time span. In addition the dryness or stiffness of the foam is evident in Figure 35 as dark areas in which no bubbles can be seen. This is caused by small elevations or depressions in the foam which lie out of the plane of focus of the microscope. The non-spherical nature of the aged foam is evident in Figure 36 but l i t t l e of the thin-walled "polyhedral" foam can be seen in the photomicrograph. The effect of beating time on the changes in the distribution parameters which occur on aging is shown in more detail in Table IV. Figure 3 5 . Picture taken 2 minutes after beating 8 minutes. Magnification - 6 l . 5 X. 107 Figure 3 6 . Picture taken 10 minutes after beating 8 minutes. Magnification 6 1 . 5 X. S I Z E C L A S S MIDPOINTS - microns Figure 37. Effect of aging of foam on the frequency distribution of bubble size 2 minutes and 10 minutes after beating for 8 minutes. Class size interval is 15.6 and kk microns for 2 minutes and 10 minutes distributions respectively. o co 109 Table IV. Effect of aging on bubble size parameters Beating Aging Average Lognormal Distribution Parameters Time Time Bubble (min.) (min.) Size (u) Mean s 2 103 1.9582 + .0153 0.2238 6 109 1.9716 + .0252 0.2403 10 108 1.9735 + .0224 0.2260 15 116 2.0030 ± .0251 0.2247 20 130 2.0348 + .0371 0.2551 2 94.1 1.9265 + .0310 0.2053 6 177 2.1693 + .0515 0.2779 10 194 2.1384 + .0666 0.3595 15 200 2.0841 ± .0965 0.4120 20 387 2.3953 + .1526 0.4356 The range shown for the mean represents the 95$ confidence interval. s = standard deviation 110 The rate of growth of average bubble size for a stable (2 minute) foam may be contrasted with the very rapid growth of bubble size exhibited by the unstable (8 minute) foam. In addition, the rate of growth of polydispersity in the two foams as suggested by the geometric standard deviations may be contrasted. It is apparent from the results reported that the instability of egg white foams caused by overheating is associated with a rapid increase in average bubble size coupled with a marked increase in the polydispersity of bubble sizes in the foam, notably the growth of very large bubbles. Several factors affecting foam stability have been considered by Bikerman ( l l ) . The pressure inside a small bubble is greater than the internal pressure of a large bubble ( l l , 4 l ) resulting in diffusion of gas from the small bubble to the larger one. Thus foams are inherently unstable systems—large bubbles growing larger at the expense of the smaller bubbles. Secondly the viscosity of the material between bubble lamella will affect stability since high viscosities will slow drainage from between the bubbles. The third factor affecting bubble stability involves the nature of the material forming the air-liquid interface. Materials forming strong interfacial films are able to withstand extensive drainage of interfacial material without breakage of the interfacial films and coalescence of the bubbles. Some attributes of the material drained from 2 and 8 minute foams are compared with the attributes of the original egg white in Table V. The total solids of the drained material decreased slightly with whipping time which would be expected since some solids must be suspended in the foam. The decrease had essentially been Table V. Effect of beating on some of the attributes of egg white. Factor Original 2_ Minute 8 Minute Egg White Drainage Drainage <fo Solids 1 0 . 9 1 0 . 6 1 0 . 5 Relative viscosity 3 . 6 0 1 . 3 0 1 . 1 0 -SH content uM/gm. protein 6 l 6 l 59 112 completed within two minutes beating and l i t t l e or no further decrease in solids content was observed after 8 minutes. The -SH content of the protein did not change suggesting that ovalbumin in the drip had gone through the whipping process unchanged. Ovalbumin is the only known source of -SH groups in egg white ( 3 5 ) ' It is apparent that most of the viscosity decrease occurs within 2 minutes whipping although longer whipping times further decrease the relative viscosity. In general, these results are in agreement with those reported by MacDonnell e_t aL. (55) for repeatedly whipped white, although these authors did not show the lower solids content of whipped white. If the viscosity of the material draining from the foam is taken as a measure of the viscosity of the material between bubble lamella, it appears that the small change in relative viscosity which occurs between the 2 and 8 minute drips cannot account for the marked loss of stability and rapid bubble growth observed in egg white beaten 8 minutes. The viscosity of egg white has been associated with the protein component ovomucin ( 5 5 , 5 6 ) which has also been suggested ( 5 5 , 5 6 , 5 7 ) as a foam stabilizer. Presumably the ovomucin is insolubilized in the bubble lamella enhancing stability of the foam by retarding coalescence of the bubbles and diffusion of gas from bubble to bubble. Gas diffusion is dependent upon several factors such as the nature of the gas ( 3 ) and the thickness and composition of the lamella and the material between the bubbles ( l l ) . It has been reported that increased beating times yield bubbles of progressively smaller size ( 8 ) . This could lead to instability because the distance between bubbles 2.1 2 6 10 BEATING TIME- MINUTES Figure 38. Effect of beating time on bubble diameter. Diameters measured from photographs taken 2 minutes after cessation of beating. Bars represent plus or minus 1 standard deviation. Points having different letter are different (P^O.Ol). Arithmetic mean values are given in brackets. 114 would be reduced resulting in somewhat faster diffusion rates. In addition the available ovomucin would be spread over increasingly-greater surface areas potentially limiting its ability to stabilize the foam. The effect of beating time on average bubble size is shown in Figure 3 8 . While there is a trend to smaller bubble sizes with increased beating times, the change is not great. Furthermore, the geometrical standard deviations shown in the figure suggest that l i t t l e change in the polydispersity of the foam occurs. It is possible however, that very small bubbles undetectable by the photomicrographic technique used occur in the foam beaten for extended periods. Electrophoresis The very rapid growth of bubble size evident in 8 minute foams is almost certainly due to bubble coalescence. Diffusion of gas from bubble to bubble is likely an important factor influencing the growth of bubble size in 2 minute foam since here the bubble lamella are sufficiently stable to allow the formation of "polyhedral" foam as liquid drains from between bubbles (see Figure 33) • This difference suggests that a significant change has occurred in the nature of the material forming the bubble lamella and may be detectable by examination of the macromolecular components in the material drained from the foam. The effect of whipping time on the distribution of protein components between the foam and the liquid drained from the foam and 115 the liquid drained from the foam in 1 hour as determined by disc gel electrophoresis is shown in Figure 3 9 - A densitometric scan of these three gels is shown in Figure ko. The pattern obtained for whole egg albumin closely resembles those obtained by Chang et a l . ( l 8 ) . The effect of whipping can be discerned in two areas of the electro-pherograms. First, there is progressive depletion of bands 8 and 9 until depletion is essentially complete after 8 minutes beating. In addition, depletion of band 7 is suggested by the densitometry results. It is possible, however, that the apparent depletion of band 7 is due to the lowered baseline caused by the depletion of bands 8 and 9 a nd is not due to actual removal of material. Proteins running in this region of disc gel electropherograms have been tentatively identified as globulins by Chang et a l . ( l 8 ) . In particular it was suggested that the bands labelled 8 and 9 in this study may be globulins A]_ and A2 as noted by Feeney et_ a l . (3*0. The second notable change in the electrophoretic pattern which occurs on beating involves lysozyme which also undergoes progressive depletion with increased beating time. The extent of lysozyme depletion as determined from the electrophoretic patterns is shown in Table VI, which also shows the results of enzymatic determinations. As can be seen in the table, the decrease in lysozyme was confirmed although quantitative agreement between the two methods was not obtained. The reason for this disagreement is not known although it may be related to inaccuracies in the densitometry and to the high dilutions ( 1 : 1 0 0) used for the enzyme assays. • mm III i l l 8 2 EW Figure 39. Disc gel electrophoresis of egg white and drainage from foamed egg white. EW = original egg white, 2 = drainage from egg white foamed 2 minutes, 8 = drainage from egg white foamed 8 minutes. 117a Figure ko. Densitometer tracing of disk gels shown in Figure 3 9 . Small numbers represent band numbers, large numbers are 2 and 8 minutes whipping time respectively. EW = original egg white, 0 = origin. Table VI. Effect of beating time on the lysozyme content of egg white drained from egg white beaten 2 and 8 minutes. Original 2_ Minute 8 Minute Egg White Drainage Drainage Lysozyme by densitometry 1 0 0 $ 79$ 67$ Lysozyme by enzyme assay 1 0 0 $ 9 3 $ 84$ 119 Gel Filtration The results of gel filtration through Sephadex G-150 of egg white and the drip from 2 and 8 minute foams is shown in Figure kl. It has been shown ( i o ) that, for this system, peak 1 contains mainly ovomucin, peak 2 some macromolecular material that may include the "line 1 8 " material reported by Lush ( 5 2 ) , peak 3 contains conalbumin and globulins, peak h ovalbumin and some other material presumably globulins and peak 5 contains lysozyme. It has been reported ( 3 9 , 5 5 ) that material drained from egg white foams contained less ovomucin than was present in the original egg white. This is confirmed in the present study by the dramatic drop in material eluted at the void volume. It is clear from this figure that practically a l l of the ovomucin in egg white is transferred, presumably into the bubble lamella, within two minutes beating. In addition i t appears that the macromolecular components in region 2 of the chromatogram do not undergo any change on foaming either for 2 or 8 minutes. The other changes observed in the pattern may be related to those observed in the disc gel electropherograms. The decrease in the peak 3 region may be accounted for by the electrophoretically observed globulin decrease, and the decrease in peak 5 (lysozyme) by the previously observed lysozyme decrease. In these chromatograms the difference .in the lysozyme peak between the 2 and the 8 minute samples was not easily discernable and no attempt has been made to distinguish them. 120 oo CM 30 50 70 FRACTION N U M B E R Figure kl. Gel filtration of egg white (EW) and the drainage from 2 and 8 minute beating times in Sephadex G-150. Conditions: 0.01 M phosphate, pH 7 . 0 , 1$ NaCI, 0.3$ (v/v) 2-mercaptoethanol. Fraction size in 3.1 ml. Sample size 2.5 ml. Column is 2.5 x 78 cm. Small numbers represent peak numbers, large numbers are 2 and 8 minute whipping times. EW = original egg white. 121 GENERAL DISCUSSION The finding that bubble size distributions of egg white foams may be fitted by a lognormal distribution is similar to results reported for thick-walled foams ( 6 l ) . If a dispersion is attained by some process such as milling or grinding, the dispersion often appears to be governed by the lognormal distribution ( ^ 3 ) . It has been suggested (ll) that foams are formed by incorporation of air into a system followed by successive splitting of the resulting bubbles into smaller and smaller sizes. This may be considered analogous to a grinding process so that fitting of a lognormal curve to the resulting frequence distribution is not unreasonable. It has been shown by Nakamura and Sato ( 5 6 ) that globulins were decreased in the material draining from repeatedly whipped egg white, and lysozyme has been implicated as a factor affecting the foam volume and stability by Sauter and Montoure ( 7 3 ) , and ovomucin has long been considered as a foam stabilizer ( 5 5 ) . It seems clear from the results reported here that a l l three components are progres-sively and selectively transferred into the foam on whipping. Globulins (including lysozyme) have been reported to be good foaming proteins ( 5 5 , 5 6 , 5 7 ) and the foaming power of egg white is dependent upon this fraction. Thus egg white may be considered as a globulin foam stabilized by ovomucin being insolubilized in the bubble lamella and contributing its viscosity to the inter-lamella fluid. 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