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' w .: .1 ' u 1 1 m r A»: r ‘ :4. “HT-4 t - 1 ‘ a 32’ .1146“: “"93" mm A 1- “$1411 LBIIRARE lllllllllllllllllllllllllllllllllllllllllllllllllllllll 31293 0090 This is to certify that the dissertation entitled EFFECT OF PH, SALT TYPE AND DENATURANTS ON THE DENATURATION PROPERTIES, STORAGE MODULUS, SECONDARY STRUCTURE AND MICROSTRUCTURE OF HEN EGG S-OVALBUMIN HEAT-INDUCED GELS presented by Thomas Joseph Herald has been accepted towards fulfillment of the requirements for Ph .D. degree in Food Science Major professor Date /0 ’30! 9! MS U is an Affirmative Action/Equal Opportunity Institution 042771 I“ ‘1 22.533113? Michigan State University L J PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE MAR llivzooz I —_—ll_— MSU Is An Affirmative Action/Equal Opportunity Institution chS—ni EFFECT OF PH, SALT TYPE AND DENATURANTS ON THE DENATURATION PROPERTIES, STORAGE HODULUS, SECONDARY STRUCTURE AND .NICROSTRUCTURE OP HEN EGG S-OVALBUNIN HEAT-INDUCED GELS BY THOMAS JOSEPH HERALD A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPY Department of Food Science and Human Nutrition 1991 ABSTRACT EFFECT OF PH, SALT TYPE AND DENATURANTS ON THE DENATURATION PROPERTIES, STORAGE HODULUS, SECONDARY STRUCTURE AND MICROSTRUCTURE OF HEN EGG S-OVALBUHIN HEAT-INDUCED GELS BY THOMAS JOSEPH HERALD Changes in thermal properties, storage modulus, secondary structure and microstructure of S-ovalbumin as a function of pH (3.0, 7.0 or 9.0), salt type (NaCl, NaI or Na2804), guanidine hydrochloride (0.5, 1.0 or 2.01!) or B- mercaptoethanol (0.5, 1.0, 2.0 and 3.0%) were studied using differential scanning calorimetry (DSC), dynamic rheological testing, Fourier transform infrared spectroscopy (FT-IR) and electron microscopy. The DSC onset temperature of ovalbumin denaturation (To) occurred 5.8°C - 7.5°C prior to structure development (Ta) as measured by storage modulus. Enthalpy, To, and TB increased as pH increased. Denaturation temperature decreased in the order of the Hoffmeister series, whereas 'I'8 decreased in reverse order. .Activation energies of ovalbumin denaturation and structure development were dependent on pH and salt type. increasing GuHCl and B-ME decreased the denaturation temperature 10°C and 9°C, respectively. Enthalpy did not differ (P > 0.05) at any of the GuHCl concentrations but decreased with increased B-ME (62.4-44.6 kJ/mol) . Onset temperature for ovalbumin structure formation decreased and storage moduli increased with increased concentrations of GuHCl and B-ME. Solutions of hen egg S-ovalbumin at pD 3.0, 7.0 or 9.0 were heated at temperatures between 30°C to 90°C to study changes in secondary structure by FT-IR. Second derivative infrared spectra of native ovalbumin at pD 7.0 and 9.0 revealed protein absorption bands for B-sheet at 1626 cm'l, BIO-helix at 1638 cm’l, a-helix at 1656 cm“, and turn at 1682 cm'l. The B-sheet absorption band was not observed for S- ovalbumin at pD 3.0. The quantity of a-helix and B-sheet structure decreased as heating temperature was increased. Changes in microstructure of heat-induced hen egg S- ovalbumin gels influenced by pH, salt type and denaturants (GuHCl and B-ME) were studied using low temperature scanning electron microscopy (LTSEM) and chemical fixation procedures with seaming electron microscopy (SEM) . The LTSEM of 8% S- ovalbumin gels were honey comb in appearance, while S- ovalbumin gels prepared by chemical fixation exhibited a microstructure of grape-like clusters. S-ovalbumin gels prepared at pH 3.0 or in 0.5M Na2804 exhibited the smallest pore size as viewed using LTSEM. Using SEM the largest void volumes exhibited by the S-ovalbumin gels were prepared at pH 3.0 or 0.5M NaI. This dissertation is dedicated to my wife, Christine, and children, Jacob and Sarah iv ACKNOWLEDGEMENTS The author wishes to extend his genuine gratitude to his advisor, Dr. Denise Smith, for her guidance and financial support during my graduate program. .Appreciation. is also» extended. to Dr. Stan Flegler, Department of Electron Optics, Dr. Jack Holland, Department of Biochemistry, Drs. James Steffe and Mary Zabik, Department of Food Science and Human Nutrition, for participating on the guidance committee. The author is most grateful for constant encouragement and support provided by his family, especially his parents Geraldine and Edward Herald. TABLE OF CONTENTS LIST OF TABLES...................... ..... . ...... ..........ix LIST OF FIGURES....... .................... ...... ..... ....xii Chapter Page I. INTRODUCTION....................... ..... ............1 II. REVIEW OF LITERATURE.... ............ ................4 III. Properties of Ovalbumin...............................4 S-ovalbumin...........................................6 The Effect of Heat on 0va1bumin.......................7 The Effect of pH on 0va1bumin........................11 The Effect of Neutral Salts on 0valbumin.............13 The Effect of Denaturants on 0valbumin...............16 Differential Scanning Calorimetry....................18 Differential Scanning Calorimetry of Egg Proteins....20 Rheological Properties of 0va1bumin..................23 Determination of Activation Energy...................25 Fourier Transform Infrared Spectroscopy..............28 Secondary Structure of Proteins......................33 Scanning Electron Hicroscopy.........................39 Study 1. Denaturation and Structure Development of S-Ovalbumin as Influenced by pH and salt TypeOOOO0.00000IOOOOOOOOOOOO0.0...0.044 IntrOductionO00.000.000.000...0.0..0.0.0.000000000000045 materials andHethOdSOOOOOOOOOO00.0.00...0.0.0.0...00.48 Materials.......................................48 Electrophoresis.................................48 Differential Scanning Calorimetry...............49 Thermodyanmic Calculations......................51 Dynamic Rheological Testing.....................51 Statistics......................................53 Results and Discussion.................................53 Proximate Analysis..............................53 Effect of pH on Thermal Denaturation............54 Effect of pH on Structure Development...........58 Effect of Salt Type on Thermal Denaturation.....63 vi TABLE OF CONTENTS (Cont'd) Effect of Salt Type on Structure Development....66 Effect of Concentration on Thermal Denaturation and Structure Development.......................68 Conclusions.......... ...... ......... ......... . ..... .....73 IV. Study 2. Denaturation and Structure Development of S-Ovalbumin as Influenced by Guanidine Hydrochloride and fl-Mercaptoethanol..........75 Introduction...........................................76 Materials and Methods..................................79 Materials.......................................79 Differential Scanning Calorimetry...............80 Thermodynamic Calculations......................81 Dynamic Rheological Testing.....................82 Statistics......................................83 Results and Discussion................................84 Effect of Guanidine Hydrochloride on Thermal Denaturation ...................................84 Effect of Guanidine Hydrochloride on Structure Development.....................................89 Effect of B-Mercaptoethanol on Thermal Denaturation....................................91 Effect of B-Mercaptoethanol on Structure Development.....................................95 Comparing differential scanning calorimetry to dynamic rheological testing..................98 Conclusions...........................................99 V. Study 3. Changes in Secondary Structure of S-Ovalbumin During Heating and Perturbation.............100 Introduction.........................................101 Materials and Methods................................104 Results and Discussion...............................106 Secondary Structure of Native S-Ovalbumin......106 Effect of pD and Heating on Secondary Structure ....................................108 Effect of Denaturants on Secondary Structure .....................................117 Conclusions....................................117 VI. Study 4. Changes in Microstructure of S-Ovalbumin due to Perturbation with pH, Salt Type and Denaturants..........................120 Introduction........................................121 Materials and Methods...............................122 Materials......................................122 Electron Micrscopy.............................123 Low Temperature Scanning Electron Microscopy...123 Chemical Fixation..............................124 vii TABLE OF CONTENTS (Cont'd) Results and Discussion.........................126 Low Temperature Scanning Electron Microscopy...126 Chemical Fixation Comparison of Chemical Fixation Techniques................ ....... ...........129 Effect of pH on Microstructure..............132 Effect of Salt Type on Microstructure.......135 Effect of Guandine Hydrochloride on Microstructure..............................137 Effect of B-Mercaptoethanol on Microstructure..............................139 Conclusions........................................139 VII. Summary and Conclusions.............................141 Study 1........................................141 Study 2........................................142 Study 3........................................142 Study 4........................................143 VIII. Recommendation for Further ResearChOOOOOOO0.00.00.00.000.0.0.0.00000000000144 IX. List of References............... ............ .......146 viii Table 10 11 12 LISTS OF TABLES Page Frequency range for characteristic absorption bands of secondary amides in the crystalline state...‘OOOOOOOOOOOOOOOOOOOO0.0.0.0.0000...000......30 Estimate protein conformation by three independent methods............................ ...... 31 Characteristic IR frequencies and assigments for amide I band components for 19 globular proteins in D20 salutionOOOOOO0.0000000000000...000......0.0.033 Secondary structure contents calculated from circular dichrosim spectra of native, heat-denatured, and cooled ovalbumin.................39 Influence of pH on thermal denaturation of 8% (w/v) S-ovalbumin in 0.5M NaCl using differential scanning calorimetry............... ..... 54 Influence of pH on the thermodynamic properties of 8% (w/v) S-ovalbumin in 0.5M NaCl.................57 Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 3.0 in 0.5M NaCl during isothermal heating for 15 min.................61 Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaCl during isothermal heating for 15 min.................61 Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 9.0 in 0.5M NaCl during isothermal heating form 15 min................62 Influence of pH on structure development of 8% (w/v) S-ovalbumin in 0.5M NaCl during heating...0.0.0.0....0.0.0.000...OOOOOOOOOOO...00.0.62 Influence of salt type on thermal denaturation of 8% (w/v) S-ovalbumin at pH 7.0 using differential scanning calorimetryooO...OOOIOIOOOOOOOOOOOOOIOO0.0.065 Effect of salt type on the thermodynamic properties of 8% (w/v) S-ovalbumin at pH 7000OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 66 in 13 14 15 16 17 18 19 20 21 22 23 LIST OF TABLES (Cont'd) Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaI during isothermal heating for 15 min....70 Influence of on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M Na2804 during isothermal heating for 15 min................7o Influence of salt type on structure development of 8% (w/v) S-ovalbumin at pH 7.0 during heating OOOOOOOOOOOOOOOOOOOO0.0...0.0.71 Influence of S-ovalbumin concentration at pH 7.0 in 0.5M NaCl on thermal denaturation using differential scanning calorimetry... .......... 71 Effect of S-ovalbumin concentration at pH 7.0 in 0.5M NaCl on thermodynamic properties............72 Influence of temperature on structure development of 4% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaCl during isothermal heating form 15 min...............72 Effect of S-ovalbumin concentration at pH 7.0 in 0.5M NaCl on structural properties during heating.0......0.000000000000IIOOOOOOO...0.0... ..... 73 Influence of guanidine hydrochloride (GuHCl) on thermal denaturation of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaCl using differential scanning calorimetry.........................................85 Effect of guanidine hydrochloride (GuHCl) on the thermodynmic properties of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M"aCIOOOOOOOOOOOO0.0.0.0.000000000087 Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M guanidine hydrochloride (GuHCl) and 0.5M NaCl during isothermal heating for 15 min......................90 Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 1.0M guanidine hydrochloride (GuHCl) and 0.5M NaCl during isothermal heating for 15 min.....90 24 25 26 27 28 29 30 31 32 TABLES (Cont'd) Influence of temperataure on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 2.0M guanidine hydrochloride (GuHCl)and 0.5M NaCl during isothermal heating for 15 min.....92 Influence of guanidine hydrochloride (GuHCl) on structural properties of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaC1 during heating..................92 Influence of B-mercaptoethanol (B-ME) on thermal denaturation of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaC1 using differential scanning calorimetry.... ..... . ....... ..... ..... . ............. 94 Effect of B-mercaptoethanol (B-ME) on the thermodynamic properties of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaC1..................95 Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5% B-mercaptoethanol (B-ME) and 0.5M NaC1 during isothermal heating for 15 min ..... ... ......... 96 Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 1.0% B-mercaptoethanol (B-ME) and 0.5M NaC1 during isothermal heating for 15 min.................96 Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 2.0% B-mercaptoethanol (B-ME) and 0.5M NaC1 during isothermal heating for 15 min.................97 Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 3.0% B-mercaptoethanol (B-ME) and 0.5M NaC1 during isothermal heating for 15 min......... ....... 97 Influence of B-mercaptoethanol (B-ME) on structural properties of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaCl during heating.................99 xi LIST OF FIGURES Figures Page 10 11 Amino acid sequence of ovalbumin.............. ........ 5 A proposed model for the thermal denaturation and aggregation behavior of ovalbumin molecules...........9 Differential scanning calorimetry thermogram of egg white at pH 7.0 heated at 10°C/min...............21 Infrared spectra of bovine serum albumin (salt-free, pD 6.7) heated from 25°C to 90°C then COOIed to 25°C..OIOOOOOOOOOOOOOOOOO0.00.00...0.00.0035 Circular dichrosim spectra of native ovalbumin and ovalbumin denatured by heat or guanidine hYdrOChlorideeeeeeee ..... .....OOOOOOOOOOOOOO0.0.0....38 Effect of pH on the differential scanning calorimetry thermogram of 8% S-ovalbumin in 0.5M NaC1 heating at 10°C/min............... ..... 59 Subtracted second derivative Fourier transform infrared spectroscopy absorption spectra of 1. 75% S-ovalbumin in 020 (pD 3. 0) after different heat treatments for 30 min...............................109 Subtracted second derivative Fourier transform infrared spectroscopy absorption spectra of 1.75% S-ovalbumin in D2 0 (pD 7. 0) after different heat treatments for 30 min...............................110 Subtracted second dervative Fourier transform infrared spectroscopy absorption spectra of 1.75% S-ovalbumin in 020 (pD 9. 0) after different heat treatments for 30 min...............................111 Subtracted second dervative Fourier transform infrared spectroscopy absorption spectra of 1.75% S-ovalbumin in 0'20 and 2. 0M guanidine hydrochloride (pD 7. 0) at 30°C2 and 80°C for min...................118 Influence of pH on the microstructure of 8% (w/v) S-ovalbumin gel in 0.5M NaC1 heated at 80°C for 30 min using low temperature scanning electron microscopy A. pH 3. 0 B. pH 7. 0 C. pH 9. 0...........................................127 xii 12. 13 14 15 16 17 18 LIST OF FIGURES (Cont'd) Influence of salt type on the microstructure of 8% (w/v) S-ovalbumin gel at pH 7.0 heated at 80°C for 30 min using low temperature scanning electron microscropy A. NaI B. NaC1 C. Na2804......128 Influence of denaturants on the microstructure of 8% (w/v) S-ovalbumin gel at pH 7.0 heated at 80°C for 30 min using low temperature scanning electron microscopy A. 2.0M guanidine hydrochloride B. 3.0% B-mercaptoethanol.................... ..... ..130 Comparison of scanning electron microscopy chemical fixation techniques for 8% (w/v) S-ovalbumin gels formed at pH 7.0 in 0.5M NaC1 by heating at 80°C for 30min. A. gluteraldehyde B. osmium tetroxide C. osmium-thiocarbohydrazide-osmium (0T0) D. osmium-tannic acid uranyl acetate (0TU)..........131 Microstructure of 8% (w/v) S-ovalbumin gel in 0.5M NaC1 heated at 80°C for 30 min prepared using the osmium-tannic acid-uranyl acetate procedure A. pH 3.0 B. pH 7.0 C. pH 9.0...........133 Microstructure of 8% (w/v) S-ovalbumin gel at pH 7.0 heated at 80°C for 30 min prepared using the osmium-tannic acid-uranyl acetate procedure A. 0.5M NaI B. 0.5M Na2804........................136 Microstructure of 8% (w/v) S-ovalbumin gel at pH 7.0 in 0.5M NaC1 heated at 80°C for 30 min prepared using the osmium-tannic acid-uranyl acetate procedure A. 1.0M guanidine hydrochloride B. 2.0M B-mercaptoethanol...........................138 Microstructure of 8% (w/v) S-ovalbumin gel at pH 7.0 in 0.5M NaC1 heated at 80°C for 30 min prepared using the osmium—tannic acid-uranyl acetate procedure A. 1.0% B-mercaptoethanol B. 2.0% B-mercaptoethanol C. 3.0% B-mercaptoethanol...........................140 xiii I. INTRODUCTION Hen eggs contain a variety of proteins that contribute unique functional and nutritional properties to food systems. When cooked, eggs form a gel that provides a matrix for holding water, flavors, ingredients and provide texture, a critical attribute in such foods as custards. Ovalbumin is the major egg white protein and is predominately responsible for egg white gelation. Ovalbumin gels are made of a three dimensional matrix of cross-linked polypeptides. Gelation of ovalbumin is an orderly aggregation of protein. The protein may be partially or completely denatured by heating or denaturants (Hermafisson, 1979). Aggregation of the unfolded ovalbumin protein form strands that interact to produce a network. Bonds giving structure to the network may be electrostatic (Beveridge et a1., 1980), hydrophobic (Hayakawa and Nakai, 1985), hydrogen, covalent (Beveridge et a1., 1984), van der Waals forces (Hatta et a1., 1986) or a combination thereof. From a nutritional standpoint, egg proteins are considered the best source of high quality protein and are used as a standard for protein quality determinations. Eggs contain all of the essential fatty acids, large quantity of unsaturated fatty acids, many of the minerals needed in our diet and all vitamins except vitamin C (Froning, 1988). 2 The present study used a multitechnique evaluation to determine changes in secondary structure, rheological properties and microstructural characteristics of ovalbumin during heat-induced gelation. It is hypothesized that perturbing the ovalbumin system by changing pH (3.0, 7.0, and 9.0), salt type (0.5M Na2804, 0.5M NaC1 and 0.5M NaI) or by addition of chemical denaturants (0.5, 1.0, and 2.0M guanidine hydrochloride and 0.5, 1.0, 2.0 and 3.0% B-mercaptoethanol) will provide a better understanding of the conditions needed to produce ovalbumin gels with the desired functions. Many studies have investigated the gelation behavior of ovalbumin (van Kleef et a1., 1978: Egelandsdal, 1980; Clark and Lee-Tuffnell, 1986; Hatta et a1., 1986) although none have attempted to relate changes in secondary structure to heat-induced gel properties. The objectives of the present research include heating and perturbating ovalbumin (1) to monitor the denaturation temperature and quantify the enthalpy changes, (2) to characterize and quantify storage modulus of ovalbumin, (3) to monitor the changes in secondary structure and (4) to observe changes in the microstructure. This dissertation contains 5 chapters. The first chapter is a review of the literature for the entire dissertation. Chapters 2 through 5 are the four studies which present the dissertation research. Each study was 3 organized using the format of the gourpal of Food Science and contains the following sections: Introduction, Literature Review, Materials and Methods, Results and Discussion. Chapters 7 and 8 are the overall conclusions of the research and recommendations for future research, respectively. The final chapter contains the references for the entire dissertation. II. LITERATURE REVIEW Properties of Ovalbumin Ovalbumin is a phosphoglycoprotein and makes up 54% of the total proteins in the albumen. Ovalbumin has an isoelectric point (pI) of 4.5 and a molecular weight of 45,000 (Powrie and Nakai, 1986). The complete amino acid sequence of ovalbumin with 385 residues (Fig. 1) was determined by Nisbet et al. (1981). The N-terminal amino acid is acetylated glycine and the C-terminal amino acid is proline. Stein et a1. (1990) and Wright et a1. (1990) have determined the crystal structure of ovalbumin. Ovalbumin contains a carbohydrate moiety attached to asparagine residue 292 (Lee and Montgomery, 1962; Nisbet et a1., 1981). The carbohydrate moiety has a molecular weight between 1560 and 1580 daltons (Lee et a1., 1964; Montgomery et a1., 1965) and consists of a core of two N-acetylglucosamine units and four mannose units. Ovalbumin exists in three forms, A1, A2, and A3, which differ in phosphorous content. A1 has 2 phosphates per molecule, A2 has 1 phosphate per molecule and A3 does not contain phosphate (Longsworthe et a1., 1940; Cann, 1949; Perlman, 1952). The relative proportion of A1, A2, and A3 in ovalbumin is about 85:12:3, respectively. Phosphate groups are attached to serine residues 68 and 344 (Nisbet et a1., 1981). Ovalbumin has been reported to contain four S to 15 AcGly-Ser-Il e-Gly-Al a-Al a-Ser-Met-Gl u-PheI-Phe-ASp-Val -Phe- 20 25 Lys- Glu-Leu-Lys- Val- His-His- Ala-Asn-Glu-Asn-Ile- Phe-Tyr{§:§- [ 119 | an: -------- Gln-Cys- Gly---------------------Gln- Cys- -Val-----Ala- 360 Glu-Ala-Gly-Val- -Asp- -Ala-Ala-Ser-Val -Ser-Glu-Glu-Phe-Arg-Ala- 370 375 Asp-His-Pro-Phe- -Leu-Phe-C/=-Ile-Lys-His- -Ile- Ala-Thr-ASn-Ala- 300 38! Val- Leu-Phe-Phe- Gly-Arg C1 s'r‘lal-Ser-Pro rig. 1-Anino acid sequence of ovalbumin. Taken from Nisbet et ll. (1981). . 6 sulfhydryl groups and one disulfide group. Three sulfhydryl groups were observed in native ovalbumin and a fourth was observed after denaturation (Fernandez-Diez, 1964). Optical rotary dispersion studies have indicated that ovalbumin secondary structure may be composed of 25% a-helix and 25% B-sheet (Cho, 1970). Kato and Takagi (1988) using circular dichroism (CD) determined the secondary structure of 0.1% ovalbumin solution (67 mM sodium phosphate buffer, pH 7.0 and 0.1M NaC1) to be 49% a-helix and 13% B-sheet. s-Ovalbunin Smith (1964) and Smith and Back (1965) have shown that ovalbumin was converted to S-ovalbumin, a more heat-stable protein, during storage of eggs and ovalbumin solutions. In the latter case, the rate of conversion to S-ovalbumin increased with pH and temperature. Freeze dried ovalbumin stored in the cold for 20 years showed partial conversion to an intermediate form, but not to S-ovalbumin (Smith and Back, 1968). There are two additional ionizable carboxyl groups in S-ovalbumin that are reactive compared to ovalbumin (Nakamura et a1. 1980). Kato et a1. (1986) reported that one of the carboxly groups was involved in electrostatic cross-linkage with a positively charged amino acid residue in the interior of the molecule, leading to the formation of S-ovalbumin. S-ovalbumin has a slightly lower Imolecular weight and was more resistant to heat denaturation 7 and to denaturation with urea and guanidine hydrochloride (GuHCl) (Smith and Back, 1965). Smith and Back (1964) suggested sulfhydryl-disulfide interaction may be involved in the ovalbumin to S-ovalbumin transition. However, this hypothesis was rejected by analysis of peptides from tryptic and peptic digests, that showed no differences between cystine peptides from either protein (Smith and Back, 1968). Donovan and Mapes (1976) studied the conversion of ovalbumin to S-ovalbumin in eggs using differential scanning calorimetry (DSC). The denaturation temperature (Td) of ovalbumin and S-ovalbumin was 84.5°C and 92.5°C, respectively at pH 9.0 and a heating rate of 10°C/min. Shitamori et al. (1984) reported that heat-induced gel strength of S-ovalbumin was less than ovalbumin. Kint and Tomimatsu (1979) using Raman spectroscopy observed that 3% to 4% of a-helix changed to B-sheet configuration upon conversion from ovalbumin to S-ovalbumin. The Effect of Heat on Ovalbumin Heat treatment causes denaturation and aggregation of proteins and Ferry (1948) described this mechanism as a two- step process: native----> denatured protein ---->aggregated protein (long chains) (associated network) 8 Heat denaturation is the process of native protein conformation changing to an unfolded conformation. Aggregation is the intermolecular protein interactions leading to higher molecular weight molecules. Kato and Takagi (1988) described that heat-induced gelation of ovalbumin is a thermally irreverisble process as measured by circular dichrosim (CD). Knowing the rate of reaction between denaturation and aggregation help to determine gel characteristics (Gossett et a1., 1984). Ferry (1948) reported a gel network occurs if the aggregation step takes place slower than the denaturation step giving the denatured protein time to orient before aggregation. Gels of this nature are higher in elasticity. If denaturation and aggregation occur simultaneously a less elastic gel results. Tombs (1974) proposed globular protein form gels as a result of aggregation forming a "string of beads" followed by interactions of the beads to form a mesh. Koseki et a1. (1989) developed a model (Fig. 2) showing high molecular weight ovalbumin linear aggregates or "string of beads" based on sedimentation analysis, viscometry and transmission microscopy. Kato et al. (1983) reported that ovalbumin aggregates were formed by hydrophobic interaction due to the presence of surface hydrophobicity of heat-denaturatured ovalbumin. rig. 2-A proposed model for the thermal denaturation and aggregation behavior of ovalbumin molecules. Shadows and 0 indicate hydrophobic areas exposed by thermal denaturation and sinus charges on the surface of ovalbumin molecules, respectively. Taken from 001 et al. (1987). 10 presence of surface hydrophobicity of heat-denaturatured ovalbumin. Protein solubility decreases with time and temperature of heat treatment. Evidence of conformational changes in protein structure as measured by CD has been related to changes in protein solubility (Neucera and Cherry, 1982). Protein functionality has been associated with protein solubility. Kato et al. (1981) reported protein functionality can improve with denaturation as long as a decrease in solubility does not occur. Surface tension decreases with denaturation as exposure of hydrophobic molecule become exposed to the surface of the protein. Surface hydrophobicity increases as denaturation precedes increasing emulsion and foam stability (Kilara and Sharkasi, 1986). The increase in surface hydrophobicity due to denaturation causes proteins to be amphiphilic. The amphiphillic nature adsorbed at the interface between oil and water or air causes reduction of surface free energy that facilitates emulsification and foaming (Kato et a1., 1981). Polyacrylamide gel electrophoresis (PAGE) showed no change in ovalbumin solubility with pasteurization temperatures of 60°C, 64°C or 68°C for 3.5 min (Herald, 1987). Heat stability of ovalbumin varies with pH. Less than 1.0% of ovalbumin was denatured at pH 6.5-7.0 when 11 heated at 65°C for 30 min (Li-Chan and Nakai, 1989). Hegg et al. (1979) found that ovalbumin had maximum thermal stability between pH 6.0 and 10.0. The addition of 170 mM NaCl did not affect denaturation temperature while 17 mM CaC12 caused a 2-3°C decrease in denaturation temperature. Shimada and Matsushita (1980b) used absorbance at 340 nm to determine the effect of protein concentration on coagulation by observing turbidity changes. Ma and Holme (1982) reported that turbidity is related to an increase in hydrophobic residue exposure. Effects of pH on Ovalbumin In theory, as solution pH approaches the pI of ovalbumin, the charge on the protein is neutralized. This results in attractive forces and protein-protein interactions with minimal protein unfolding (Seideman et al., 1963; Arntfield et al., 1989). Koseki et al. (1990) reported that ovalbumin was unstable and susceptible to denaturation at pH below the pI. The researcher reported that ovalbumin existed in a "molten globular state". The molten-globule state exists when a molecule is in a compact globular (native state) state, but the tertiary structure slowly fluctuates. At high pH, most egg albumen proteins are negatively charged and the ability of proteins to cross-link through sulfhydryl-disulfide exchange is enhanced, possibly 12 contributing to the structural integrity of gels (Shimada and Matsushita, 1980). At high pH the high net charge may inhibit thermal coagulation due to repulsive forces, whereas high protein concentration may overcome electrostatic forces to form networks through hydrophobic interactions (Shimada and Matsushita, 1981). Slosberg et al. (1948) reported a greater heat stability for egg albumen at pH 6.5 than at 8.5 using whip time and angel cake volume to evaluate stability. Seideman et al. (1963) concurred using gelation scores as stability criteria. Koseki et al. (1990) used DSC to monitor denaturation temperature (Td) of ovalbumin. The researchers reported that as pH was decreased, the endothermic peak and cooperativity declined suggesting changes in ovalbumin conformation. Arntfield and Murray (1981) reported that acidic pH reduced Ta and enthalpy of ovalbumin as measured by DSC. The researchers suggested a weakening of intermolecular interactions due to an increase in positive charges was responsible for the decrease in Ta. The decrease in enthalpy was due to an increase in the stability of the hydrophobic interactions. .Holt et a1. (1984) reported that maximum strength and elasticity of ovalbumin gels were observed at pH 9.0 whereas no gel structure was evident at acid pH. At pH 9.0, 13 the ability of the proteins to crosslink through sulfhydryl-disulfide contributed to gel formation. Beveridge et al. (1980) measured the effect of pH between 5.5 to 9.0 on egg gel firmness using a shear press. They suggested the major mechanism responsible for ovalbumin gel strength was sulfhdryl-disulfide interchange reactions at alkaline pH. As pH decreased to 6.0, these reactions were inhibited because concentration of S" was reduced. Arntfield et a1. (1990 a) suggested 10% ovalbumin in 150 mM NaCl at pH 3.0 was denatured as indicated by Ta and enthalpy. Enthalpy decreased from 15 J/g to 3.3 J/g at pH 7.0 and 3.0, respectively. Thermal denaturation temperature decreased from 83.9°C to 63.9°C at pH 7.0 and 3.0, respectively. Effects of Neutral Salts on ovalbumin Salts of the lyotropic (Hofmeister) series include F‘, CH3COO", $04,, Cl", Br', C104“ 1' and SCN". Chloride salts at the same ionic strength exhibit neither salting-in or Baiting-out properties (Damodaran and Kinsella, 1982) and therefore, does not affect protein conformation. Protein solubility in NaC1 solutions can be regarded as a reference point for comparisons with solubility in other salt sollrtions of equal ionic strength (Kakalis and Regenstein, 1986 ) . The chaotropic (structure-disrupting) thiocyanate, iodide and chlorate reduce the free energy of entropy 14 associated with the exposure of apolar residues. Chaotropic anions can reduce interprotein hydrophobic interactions and disrupt or unfold the compact native structure of proteins (Preston, 1989). Increasing concentrations of nonchaotropic anions, such as F’, increase the free energy of entropy associated with the exposure of apolar residues and increase inter- and intraprotein hydrophobic interactions and stabilize native protein structure (Preston, 1989). Salts in the lyotropic series have been used to probe hydrophobic interactions in network formation. At ionic concentrations sufficient to minimize electrostatic interactions, changes in protein properties due to anion concentration and salt type can be attributed to hydrophobic interactions (von Hippel and Schleich, 1969; Melander and Horvath, 1977). Hydrophobic interactions between amino acid side chains are recognized as the major force responsible for the stability of protein structure. The driving force for such interactions arise from the energetically ‘unfavorable effect hydrophobic interactions have on the structure of water molecules (Damodaran and Kinsella, 1982). The reseachers suggested that chaotropic ions altered the waster structure, decreasing hydrophobic interactions of the prtrtein molecules. This action disrupted the compact native structure of the protein, favoring unfolding and destabilize water structure. Preston (1989) observed that increasing Cl' :from 0.05 to 0.1M increased the free energy of entropy 15 associated with exposure of apolar residues. The author suggested increased interprotein and intraprotein hydrophobic interactions tended to stabilize native protein structure. Protein structure can be manipulated by decreasing hydrophobic interaction by changing solvent conditions using chaotropic ions (Tanford, 1979). Babajimopoulos et al. (1983) observed supression of electrostatic repulsive interaction between soy protein molecules at 0.5M ionic strength. Decreased electrostatic interaction allowed association and formation of hydrogen bonds and other interactions on the surface of native protein molecules. There is an optimum salt concentration for network formation. It has been suggested that low salt concentrations aid in protein solubilization before heating and provide a cross-link in the network (Kohnhorst and Mangino, 1985; Mulvihill and Kinsella, 1988). At high levels of salt concentration the net repulsive charge on the protein is masked; any further salt addition promotes aggregation. Maximum gel strength for B-lactoglobulin is between 75-300 mM NaCl (Mulvihill and Kinsella, 1988), while maximum gel strength for ovalbumin is between 50-100 mM NaCl (Egelandsdal, 1984; Holt et al., 1984; Hayakawa and Nakamura, 1986). Salts inhibit interactions between water molecules and hydrophilic groups in protein molecules. Bull and Bresse (1970) observed that ions of higher order lyotropic series 16 can dehydrate proteins enhancing protein-protein interaction. Arntfield et al. (1990 b) observed that the storage modulus (G') of ovalbumin in 0.5M NaSCN increased compared to other anionic salts because of the high negative charge on the protein causing unfolding and subsequent cross- linking. Inclusion of 0.5M Nazso4 decreased 6' suggesting increased intramolecular hydrophobic interactions. The hydrophobic interaction decreased the tendency for ovalbumin to unfold and interact to form a cross-linked structure. Effect of Denaturants on Ovalbumin Guanidine hydrochloride weakens hydrophobic interactions and inhibits hydrogen bonds and ionic attractions in proteins (Nandi and Robinson, 1972). Pace (1975) used optical rotation to follow changes in conformation of proteins in 6.0-8.0M GuHCl at room temperature. Globular proteins were randomly coiled with the addition of 6.0M GuHCl without residual ordered structure. High concentrations of GuHCl eliminated protein electrostatic interactions (Bismuto and Irace, 1988). Strambini and Gonnelli (1986) worked with liver alcohol dehydrogenase and hypothesized that at predenaturational concentrations, GuHCl penetrates the protein interior. The GuHCl decreased intramolecular interactions that resulted in an increased fluidity in the interior region of the 17 macromolecule. The researchers suggested structural organization was destroyed and decreased denaturation cooperativity. Heertje and van Kleff (1986) observed the effect of urea on ovalbumin solutions to distinguish between formation of covalent and non-covalent cross-links during gelation. Ovalbumin gels prepared at pH 10.0 and in urea formed networks by protein unfolding, whereas at pH 5.0, the network formed by aggregation as the protein conformation was stabilized by hydrogen bonds and hydrophobic interactions. (Clark et a1., 1981 a). Ovalbumin in 6.0M GuHCl and 0.1M B-mercaptoethanol (B-ME) was completely unfolded as determined by intrinsic viscosity (Ansari et al., 1972; Ahmad and Salahuddin, 1974). The inclusion of 6.0M GuHCl increased protein solubility and prevented the formation of strong ovalbumin gels by reducing interactions between polypeptide chains (Egelandsdal, 1984). Katsuta and Kinsella (1990) observed that 10% B-lactoglobulin formed gels in 6.0M urea and 6.0M GuHCl. The G' of B-lactoglobulin increased with time at 25°C indicating gel formation. Thiol groups and disulfide bonds play an important role in the heat-induced gelation of whey proteins (Hillier et al., 1980; Zirbel and Kinsella, 1989). Covalent cross-linking of protein molecules by disulfide bonds can be induced by thiol oxidation or by a thiol-induced disulfide interchange reaction that were 18 enhanced at alkaline pH. Matsudomi et al. (1991) observed a decrease in gel hardness with bovine serum albumin (BSA) and B-lactoglobulin with increasing concentrations of N-ethylmaleimide (0-50 mM) confirming that thiol disulfide links were important in gel formation. Hirose et al. (1986) observed the addition of up to 70 mM B-ME induced egg white gelation at room temperature. Gel hardness increased until a concentration of 70 mM B-ME then remained constant suggesting that the cleavage of the S-S bond might induce association of egg white protein molecules assisting to stabilizing the protein network. Differential Scanning Calorimetry Differential scanning calorimetry is a practical technique for studying thermal behavior of food proteins. The DSC can be used to simulate denaturation of proteins during cooking by providing comparable thermal conditions (Wright, 1982). The effects of variables such as storage, pH or stabilizing treatments on the thermolability of proteins can be monitored using DSC (Donovan et al., 1975). Differential scanning calorimetry is used to measure the heat absorbed or liberated as a material changes in state (Skoog, 1985). In this technique, a sample and an inert reference are maintained at the same temperature while the temperature of both are gradually increased. Thermally induced changes occurring in the sample are recorded as a 19 differential heat flow displayed as a peak in a thermogram. Intergration of heat flow with respect to temperature yields a value for the enthalpy change associated with the process (Skoog, 1985). Donovan (1984) suggested the heat absorbed by the protein can be of two kinds. The first is the heat capacity (Cp) that produces vibrational and rotational motion of the molecules. The energy is stored in molecules pushing it to the next energy level. The second is heat that is absorbed by the molecule when it undergoes denaturation. Based on Ferry's (1948) investigation, unfolding of a single domain protein is a two-state phenomenon in which half the molecules are folded while the other half are unfolded in a protein solution. This indicated that folding is a cooperative phenomenon, in which disruption of the folded structure leads to complete unfolding of the molecule. Therefore, the stability of each part of the protein was dependent upon the stability of all the parts. Partially folded protein must be unstable relative to being folded or unfolded (Creighton, 1984). The unfolding step was usually highly cooperative and was seen as an endothermic peak in a DSC thermogram (Kitabatake et al., 1990). Denaturation enthalpy is associated with the amount of heat absorbed by the protein molecule that breaks hydrogen bonds and leads to unfolding of the protein (Privalov and 20 Khechinashvili, 1974). The denaturation enthalpy can be calculated from DSC data (Delben and Crescenzi, 1969; Delben et al., 1969). The thermogram for protein denaturation is endothermic. Arntfield and Murray, (1981) reported that differences in protein Td and enthalpy were due to hydrophobic interactions and intramolecular chemical bonds. Differential Scanning Calorimetry of Egg Proteins Three transitions (Fig. 3) were identifed in the DSC thermogram of egg white at pH 7.0 corresponding to denaturation of conalbumin, lysozyme and ovalbumin (65°C, 73°C and 85°C, respectively) (Donovan et a1, 1975). Analysis of isolated egg-white proteins indicated that other minor protein constituents (ovomucoid, globulin and avidin) also contributed to the total thermogram, but were masked by the ovalbumin transition. Agreement was obtained between the observed enthalpy of denaturation for total egg white and that calculated from the enthalpies and relative amounts of the individual proteins (Donovan et al., 1975). Ovomucin, comprises about 5% of egg white proteins, exists in a random coil configuration in the native state and exhibits no denaturation transition when analyzed by DSC (Donovan et al., 1975). Arntfield et al. (1989) observed Td for 10% ovalbumin solution of 71.2°C and 83.8°C at pH 3.0 and pH 5.0, 21 50 p.20! ’5 <———- Endothermic heat flow 1 1 1 1 1 1 1 1 4O 50 60 7O 80 90 IOO HO Temperature (’6) Fig. 3-Differential scanning calorimetry thermogram of egg white at pH 7.0 heated at 10°C/min. Adapted from Donovan et 81. (1975) . 22 respectively. No significant differences in Td of 10% ovalbumin at pH 5.0 or pH 9.0 were reported. The Td for 10% ovalbumin in 0.5M NaCl and 0.5M Na2804 were 89.6°C and 92.2°C, respectively. Ovalbumin displayed optimum thermal stability in the pH range 6-10 with a Td of approximately 79°C and a denaturation enthalpy of 3.64 cal/g (Donovan et al., 1975). The.Ta decreased to 62°C at pH 3.0 and 73°C at pH 11.0. Because of charge shielding by cations (Na or Ca) to ovalbumin solutions the temperature at which aggregation occurred is effected. Addition of 0.17M NaC1 had little effect on Td, compared to 17 mM CaC12 that resulted in a 2-3°C decrease in Td at pH values above the pI. Comparison of Td (measured with DSC at the pI) with the observed aggregation temperature (temperature at which 10% ovalbumin aggregated evaluated by a decrease in absorbance at 280 nm) indicated that precipitation, rather than gelation, took place when the temperature of aggregation was significantly lower than that of denaturation (Hegg et al., 1979). The reseachers suggested that by manipulating pH and salt concentration to reduce protein-protein interaction, gelation rather than aggregation was predominate. Thermograms of ovalbumin and S-ovalbumin at pH 7.0 exhibited T6 of 84.5°C and 92.0°C, respectively heated at 10°C/min. Therefore, the quality of stored or processed egg white can be monitored using DSC (Donovan and Mapes, 1976). 23 Factors such as concentration and heating rate have been reported to influence Td and denaturation enthalpy. Increasing soy protein solution from 6% to 70% increased the Ta 80°C (Kitabatake et al., 1989). Not all proteins have exhibited different Td as a function of concentration. Heating rate has been shown to influenced Td and enthalpy in myoglobin and whey protein solutions (Hagerdal and Martens, 1976; Ruegg et al., 1977). In these cases, Td and enthalpy decreased as the heating rate decreased. Rheological Properties of ovalbumin Food systems exhibit solid (elasticity) and fluid (viscosity) properties, know as viscoelasticity. A key difference in the properties of elastic Versus viscous material is the response to an applied stress (force). For an elastic material, the amount of strain (deformation) is proportional to the applied stress. For a viscous material, the rate of strain is proportional to the applied stress (Ngo and Taranto, 1986). Dynamic testing is a rheological method to measure viscous and elastic properties of a fluid. Dynamic testing is based on different fundamental responses of the loss modulus (6"), a measure of energy dissipated as heat due to viscous flow within the sample and storage modulus (G'), a measure of energy stored due to elastic deformation of the sample (Beveridge et al., 1984 b). In dynamic testing, 24 oscillatory movements and a pre—determined frequency between oscillation are used. Dynamic testing does not alter the structure of the material (Ferry, 1970). Continuous measurement of rheological properties during heating gives insight into structure development in gel-forming proteins. This information contributes to an understanding of the mechanism of protein gelation and provides the basis for selecting adequate conditions for using proteins as texture building components in heat-processed foods (Tung, 1978). van Kleef et al. (1978) used the rubbery elastic theory (Ferry, 1970) to describe gel structure in terms of the number of cross-links per molecule. The researchers reported 10% ovalbumin had 3.5 cross-links per molecule and a G' of 2,240 Pa whereas 30% ovalbumin exhibited 11.8 cross links/molecule and a G' of 133,000 Pa. Higher 6' values are expected for gels made at a solution pH near the pI of ovalbumin since attractive ionic interaction will be greatest at that pH (van Kleff, 1986). Egg protein gels are viscoelastic. When an egg protein is heated, it changes from a fluid to a gel with solid-like properties. van Kleef (1986) reported factors such as pH and ionic strength influenced G'. The G' for 25% ovalbumin solutions were higher at pH 6.1 than at pH 10. The researcher suggested that as ovalbumin moved away from it's pI the interaction between molecules decreased as was 25 measured by a decrease in G'. Beveridge et al. (1985b) observed the Td of egg white from DSC data and G' from small amplitude oscillatory testing coincided (approximately 80°C). They concluded the formation of three-dimensional network responsible for typical gel structures of egg white did not form until ovalbumin was denatured. Arntfield et al. (1990b) reported the G' of 10% ovalbumin decreased with increased concentration of NaCl or Na2804 (from 0.1 to 0.5M). Temperature of structure development (TB) as determined by changes in G' decreased as pH decreased. The reseachers attributed the decrease in G' to an increase in hydrophobic interaction. Determination of Activation Energy Time temperature superpositioning (TTS) was developed for use in the polymer industry. It has been demonstrated in polymers (Ferry, 1980) that viscoelastic data collected at one temperature can be superimposed upon data obtained at a different temperature by shifting of curves. The superposition principle is that the processes involved in molecular relaxation or rearrangements occur at greater rates at higher temperatures. The time over which these processes occur can be reduced by conducting the measurement at elevated temperatures and transposing the data to lower temperatures. Thus, viscoelastic changes that occur quickly at higher temperatures can be made to appear as if they 26 occurred at longer times simply by shifting the data with respect to time (Ferry, 1980; Sichina, 1988). The degree of horizontal shifting (i.e., time) required to superimpose a given set of data upon a reference can be mathematically described as a function of temperature. Two models are commonly used in time temperature superpositioning. The first of these relations is the Williams-Landel-Ferry (WLF) equation. The WLF equation is used to describe time-temperature behavior of polymers in the glass transition region (the change in an amorphorous region of a partially crystalline polymer from a viscous condition to a brittle one). The other model uses the Arrhenius relationship that measures a time dependent process at several temperatures. The Arrhenius equation is as follows: (1) log a1. = -Ea R (T-To) Where, Ea is the activation energy associated with the relaxation transition, R is the gas constant (R = 8.314 J/mol K), T is the measurement temperature, To is the reference temperature and a.r is the time base shift factor. Patel et al. (1990) developed a method for calculating denaturation Ea of proteins from a DSC 27 endotherm. This method is unique as the calculation is based on ramping temperature (increasing the temperature at constant rate) rather than an isothermal temperature over a specified time. The equation, reduced to linear form is: (2) 1n (dc/dt) = 1n Ko - Ea/RT + n [1n (1-a)] Here, the reaction rate (da/dt) at any temperature T is calculated as the ratio of peak height to total area, and the fraction of denatured protein (a) is calculated as the ratio of partial area to total peak area. Values of K0, Ea and n were obtained by multilinear regression using ln (da/dt) as dependent variable and 1/T and 1n (l-a) as two independent variables. Various methods have been used to determine activation energies resulting in different values for egg albumen. Donovan and Mapes (1976) using curve fitting analysis determine the Ea to be 73.3 kJ/mol, Goldsmith and Toledo (1985) used NMR data to calculate Ea in the range of 183.7 to 188.4 kJ/mol and Harte (1989) determined the denaturation Ea for 5% egg white protein (0.50M NaC1, pH7) was 158 kJ/mole using a time temperature history model. Time temperature superposition can be used in the food industry to assist in predicting the quality of gels in foods. The ability to predict the effects of time and temperature is becoming increasingly important as lower cost 28 proteins are being substituted into food systems. Time temperature superposition supplies a means to monitor and mathematically compare performance of protein under different processing conditions. Fourier Transform Infrared Spectroscopy Fourier transform infrared spectroscopy (FTIR) is used for the qualitative and quantitative estimation of protein secondary structure (Surewicz and Mantsch, 1988). FTIR provides structural information about the effect of environmental conditions on bond types such as C=0, N-H and S-S. Fourier transform infrared spectrometers are nondispersive and differ from the conventional dispersive infrared instruments (IR). Conventional IR instruments uses a grating or prism to disperse a collimated beam onto a slit that blocks out all but the desired frequeny range from reaching the detector. The entire spectrum is scanned one interval at a time by continuously changing the grating angle based on the incident light beam. The FTIR instrument uses an interferometer that simultaneously collects data from the entire spectrum (Byler and Susi, 1986). The FTIR has higher resolution, sensitivity, signal-to-noise ratio (S/N) and frequency accuracy compared to the dispersive IR instrument (Susi and Byler, 1986). 29 Krimm and Bandekar (1986) reported that bond types exhibited discrete vibrations and their changes can be monitored during molecular geometry variations. Therefore, different folding patterns of the polypetide backbone are assigned discrete frequencies within the mid-IR range that describe protein secondary structure. In the mid IR region nine absorption bands characterize polypeptides and proteins. These are termed the Amide A, Amide B, and Amide I-VII bands (Table 1). The Amide A and Amide B vibrations (in solid sample) are associated with N-H stretching vibrations of hydrogen bonded groups. The Amide I band of proteins represents the vibrations of amide carbonyl groups coupled to the in-plane N-H bending and C-N stretching modes (Byler and Susi, 1986;-Surewicz and Mantsch, 1988; Havel, 1989). The Amide II band is not well established for the polypeptide backbone but includes both C-N stretching and N-H bending. The Amide III band of polypeptides produce a weak IR signal. Amide III band includes C-C stretching, C=0 stretching, C-N stretching, and N-H bending. The Amide III band produces a weak IR signal that can be detected by Raman spectroscopy. The amide IV vibration is complex and involves bending of the OCN angle. Little information is available on Amide V, VI and VII (Susi, 1969). Table 2 compares Raman, FTIR and x-ray analysis for eStimating protein secondary structure. Immunoglobulin G 30 Table 1-Frequency Range for characteristic absorption bands of secondary amides in the crystalline state (based on model compounds) In-plane modes Out-of-plane modes Amide A, s 3300 cm'1 Amide v, 640-800 cm‘1 Amide B, s 3100 cm"1 Amide VI, 537-606 cm'1 Amide I, 1620-1700 cm‘1 Amide VII, s 200 cm"1 Amide II, 1430-1575 cm‘1 Amide III, 1229-1301 cm‘1 Amide IV, 625-767 cm'1 1 Adapted from Susi (1969) Table 2 compares Raman, FTIR and x-ray analysis for estimating protein secondary structure. 'Immunoglobulin G exhibited variation between FTIR and X-ray studies for all structures. B-lactoglobulin A showed a 10% difference in % other between Raman and x-ray studies. The overall results of the studied showed little variation between the three methods. Protein bands assignments were established based upon model system studies using proteins with a large proportion of helix, sheet, turns or unordered structure. Hemoglobin is 80% helix (Susi and Byler, 1983), whereas immunoglobulin G (IgG) and concanavalin A are classified as all B-structure but x-ray studies (Dong et al., 1990). Unordered structure 31 Table 2-Estimated protein conformation by three independent methods 1 Protein % a-Helix % B-Structure % Other2 R3 FT- x5- R FT- x- R FT- x- IR4 ray IR ray IR ray— Bovine serum 39 47 - 32 28 - 29 25 - albumin Carbonic 11 13 16 51 49 45 38 38 39 anhydrase Immuno- 8 9 3 67 76 67 27 15 30 globulin G a- 31 33 - 36 41 - 33 26 - Lactalbumin B- Lacto 10 10 7 54 50 47 36 40 46 globulin A Lysozyme 43 41 45 25 21 19 32 38 36 Ribo- 21 21 22 50 50 46 31 29 32 nuclease A Cytochrome - 51 49 - 34 10 - 15 41 C ..- 01th Taken from Byler and Susi (1988). turns and undefined segments Raman spectroscopy Fourier transform infrared spectroscopy x-ray crystallography 32 contains neither a-helix, B-sheet, nor turns (Richardson, 1981). Crystallography studies of a-lactalbumin (Acharya et al., 1989) support the assigned band at 1640 cm'1 to 310- helix (Byler and Susi, 1986; Halloway and Mantsch, 1988). Table 3 shows band assignments based up the studies of 17 proteins (Byler and Susi, 1988). Aqueous analysis of protein structure using FTIR is difficult because of highly absorbing bands such as the 0-H stretch of water (1645 cmfl) tend to obscure the weaker conformation sensitive amide I band. Subtraction of this water band, although possible in principle, is associated with numerous problems. First, because of protein water interaction the shape of the water band is changed so adequate substraction is not always achieved. Second, signal-noise ratio is decreased in the difference spectra if a strong water band is subtracted. Finally, no criteria has been established for determining the scaling factor applied to the water spectrum that is subtracted from the solution spectrum (Surewicz and Mantsch, 1988). Deuterium oxide has been substituted for water in protein solution preparation (Susi and Byler, 1983) to avoid the problem of highly absorbing water bands. Clinger et al. (1986) concluded that D20 solutions produce more consistent results than water. If D20 is used as a solvent, one must ensure that complete H--> 0 exchange has taken place in 33 Table 3-Characteristic infrared frequencies and assignments for amide I band components (1700-1620 cm' 1 ) for 19 globular proteins in D20 solution1 Mean frequency (cm-1) Assignment 1623 i 3 B-structure 1630 i 4 B-structure 1637 r 3 310-helix 1645 i 4 unordered 1653 i 4 a-helix 1670 i 2 turns 1675 i 2 turns 1683 i 2 turns 1 Taken from Byler and Susi (1988). the backbone amide groups. This is done by following the decrease in the intensity of the amide II band near 1550 cm."1 as progressive deuteration shifts it to lower frequencies (approximately 1450 cm71) (Timasheff et al. 1967; Susi et al. 1967). Mathematical procedures, termed resolution-enhancement techniques, are required to resolve the component absorption bands (a-helix, B-sheet, turns and unordered structure). Derivative spectroscopy and Fourier self-deconvolution are two such resolution-enhancement methods (Prestrelski et al., 1991 a). Secondary Structure of Proteins Byler and Purcell (1989) investigated effects of heat on the secondary structure of B-lactoglobulin, bovine serum albumin (BSA) and a-lactalbumin. All three proteins changed 34 conformation during heating as compared to their native structure at 30°C. All proteins exhibited decreased a-helix and B-sheet contents as observed by changes in peak intensity, although, a-lactalbumin retained more native structure than B-lactoglobulin or BSA. Clark et al. (1981 b) used IR spectroscopy to study the secondary structure of a 10% BSA solution (pD 6.7, no added electrolytes) heated from 25°C to 90°C then cooled to 25°C (Fig. 4). A peak at 1650 cm'1 and a shoulder at 1620 cm"1 representing a-helix and B-sheet, respectively were present during heating. Peak intensity of the shoulder at 1620 cm'1 decreased when cooled to 25°C. Surewicz and Mantsch (1988) examined the conformation of native B-lactoglobulin in aqueous solution at neutral pH and that of the protein denatured by alkaline solution or acidic methanol. The native structure contained mostly B-structure. In the spectrum of the alkali-denatured protein, all the fine structure had disappeared and the only 1 was aSsigned to an unordered broad band around 1640 cm" structure. The spectrum of B-lactoglobulin in acidic methanol depicted a strong band at 1647 cm'1 and weaker bands at 1687 cm71.and 1618 cm’l. Chen et al. (1990) observed changes in secondary zsPc 75°C 25': 0 cm“ 1:00 1200 L L A 1000 1600 Pig. 4-Infrared spectra of bovine serum albumin (salt-free, pD 6.7) heated from 25°C to 90°C then cooled to 25°C. Taken from Clark et al. (1981). 36 structure of soy 118 globulin during heating and shearing. During heating of 118 globulin from 25° to 90°C a—helix increased, random coil decreased and B-sheet did not change. During shearing (25°C and 384 sec'l) random coil increased, B-sheet decreased and a-helix remained the same as compared to the non-sheared sample. Raman spectroscopy is another method to study conformational changes in thermally denatured proteins. The Ramam spectrum of whole egg white is similar to that of ovalbumin because ovotransferrin, the other major egg albumen, is a "poor Raman scatterer" (Painter and Koenig, 1976). Thermal denaturation of egg albumen reveals an amide III line at 1236 cm'1 and a shift in the amide I line from 1667 to 1672 cmfl. These changes indicate formation of regions of antiparallel B-sheet between ovalbumin molecules. Thermal denaturation of isolated ovalbumin and ovotransferrin caused formation of intermolecular B-sheet structures when monitored using Raman spectroscopy. The formation of intermolecular disulfide bridges was suggested to play only a secondary role since no new disulfide bridge formation was indicated at 500 cm'1 in the Raman spectrum upon heating. Circular Dichroism (CD) is an optical technique used to monitor unequal absorption of left and right circularly polarized light with chiral molecules (Yang et al., 1986). 37 The conformation of the protein (asymmetric and periodic arrangement of peptide units in space) gives rise to characteristic CD spectra (Alder et al. 1973). Chin et al. (1987) suggested that CD was complementary to other methods for evaluating the secondary structure of proteins. From proteins of known three-dimensional structure it was found that the helical structure gives minima in CD spectra at 222 nm and 208 nm and the B-sheet structure gives a minimum at 218 nm (Johnson, 1988). A negative CD value at 222 nm is characteristic of order structure. Therefore, the presence of disorder structure can be determned by checking for positive ellipticity at 222nm. Figure 5 shows CD spectra of native and denatured ovalbumin heated to 80°C. Native ovalbumin shows a minimum and a shoulder at 222 nm and 210 nm, respectively. Secondary structure of native and heat denatured ovalbumin has been calculated from (Table 4) CD spectra. Kato and Takagi (1988) reported that when ovalbumin was heated to 80°C, the amplitude at 222 nm decreased and the shoulder at 210 nm increased. Therefore, changes in CD spectra of ovalbumin during heat denaturation suggest that the B-sheet structure increased. The increase in B-sheet was due to the irreversible decrease in helical structure. Kato and Takagi (1988) investigated the effects of salt concentration (20 to 160 mM NaCl, pH 7.0) on secondary 38 ’33 O E .. 2 ‘32 O a _ O 3 6 E F x [—1 O H 200 220 240 A (nm) Fig. s-Circular dichroism spectra of native ovalbumin and ovalbumin denatured by heat or guanidine hydrochloride. Taken from Doi et al. (1987). 39 Table 4-Secondary structure of native, heat-denatured and cooled ovalbumin calculated from CD spectra1 a t s 0 se 0 dar s u t ovalbumin helix B-sheet turn unordered native 0.49 0.13 0.14 0.24 heat-denatured 0.16 0.36 0.15 0.33 cooled 0.14 0.46 0.09 0.31 1 Taken from Doi et al., 1987. structure of ovalbumin. They reported that increasing salt concentration decreased the amount of o-helix and B-sheet. Egelandsdal (1986) reported that for salt free solutions (<10 mg/mL) of ovalbumin the a-helix content was independent of protein concentration. Clark and Lee-Tuffnell (1986) reported that the a-helixical content of BSA decreased at the expense of B-structure with increasing protein concentration up to 20 mg/mL (no salt added, pH 8.0). Scanning Electron Microscopy Scanning electron microscopy (SEM) is a tool for evaluating the microstructure of protein gels at nanomeric distances (Clark et al., 1981). The greatest advantage of SEM is the large depth of focus that is about 300 times that of the light microscope (Lewis, 1979). Microstructure can provide information on the effect of attractive and repulsive molecular forces on the formation of the protein 40 gel network (Heertje and Van Kleef, 1986). Microstructural information can assist in interpreting textural characteristics of food systems (Stanley and Tung, 1976). Clark et al. (1981) concluded that the electron microscope can contribute to structural investigation of globular protein gels by giving a qualitative impression of prevailing distributions of pore size, strand thickness and shape. Information about how much unfolding has occurred during gel formation is not detected using an electron microscope. van Kleef (1986) reported that 20% ovalbumin gels prepared at pH 5.0 showed a granular inhomogeneous microstructure. van Kleef (1986) suggested that at pH 5.0 network formation occurred via folded aggregates of globular protein chains. Clumps of aggregated protein were observed but without the fibrillar and sheetlike structures observed for ovalbumin/urea gels at pH 10.0 Ovalbumin gel structure at pH 5.0 is composed of individual protein aggregates (with a diameter of about 0.1 um). These aggregates form large particles (of about 0.3 um diameter), which eventually form the network (van Kleef, 1986). At optimal pH, a fine, uniform gel matrix was formed with high gel strength. Pores are small and water-binding was improved as free water was entrapped. The key to formation of a gel with fine structure and optimum water-binding properties is the balancing of attractive and 41 repulsive forces (Ferry, 1948). The effect of charge on microstructure is related to the net charge of the proteins in solution (Hermansson and Lucisano, 1982). Proteins tend to aggregate when heated at their pI, forming a coarse network with large pores, low gel strength and minimal water binding. Beyond the optimal pH, repulsive forces may be too strong so that fewer protein interactions are possible, and a weaker gel results (Egelandsdal, 1980). Beveridge et al. (1980) attributed increased firmness of ovalbumin gel at pH 9.0 to sulfhydryl-disulfide exchange that is accelerated at alkaline conditions. Woodward and Cotterill (1986) evaluated the microstructure of 11% egg white gels at pH 5.0, 6.0 and 9.0 in 1.0M NaC1. At pH 5.0 and 6.0 the microstructure was in spherical clusters that were aggregated leaving large irregular void spaces. The egg white gel at pH 9.0 exhibited a uniform structure with pores being small and evenly distributed throughout the gel matrix. Heertje and van Kleef (1986) reported that ovalbumin gels prepared at pH 10.0 in urea solution (6.0M or 8.0M) showed a uniform, homogeneous microstructure. The reseachers used NMR measurements to show protein unfolding at pH 10.0 verus partially unfolding followed by aggregation at pH 5.0. The studied concluded that complete ovalbumin unfolding at pH 10.0 occurred before network formation. Harte (1989) reported that 5% ovalbumin gels adjusted 42 to pH 6.0 and 7.0 before heat setting produced non-homogeneous, grape-like, aggregated protein clusters. It was suggested by Montejano et al. (1984) the spherical particles in micrographs of low pH ovalbumin gels may be caused by random aggregation that resulted in non-homogenous structure. At pH 7.0, Harte (1989) and Egelandsdal (1980) reported a smooth ovalbumin network and attributed it to the balancing of attractive and repulsive forces. The addition of salt shielded repulsive charges, thus promoting protein aggregation (Hegg et al., 1979; Hatta et al., 1986). Low temperature scanning electron microscopy (LTSEM) is a superior method for determining ultrastructure of food products (Freeman and Shelton, 1991). The LTSEM procedure is less time-consuming, decreases induced artefacts associated with chemicals and reduces speciment shrinkage (Sargent, 1988). Harte (1989) used LTSEM for examining 5% ovalbumin gels at pH 5.0 in 3.0% NaCl. The ovalbumin gel ultrastructure showed an increase in the quantity of aggregated protein under these conditions. The reseacher observed differences between the LTSEM and a chemical fixation method (osmium-thiocarbohydraxide-osmium). The researcher reported that ovalbumin gels prepared by LTSEM exhibited a web-like appearance whereas the gels prepared by the chemical fixation method showed a grape-like cluster appearance. other researchers (Sargent, 1988; Freeman and 43 Shelton, 1991) have used LTSEM for viewing a variety of food mircostructures. Arntfield et al. (1990 a) used light microscopy to follow the formation of two dimensional network of ovalbumin at pH 8.5 in 150 mM NaC1. The investigators observed that as the concentration of ovalbumin increased from 5.5% to 15%, the intensity of the network also increased. Arntfield et a1. (1990 b) reported that 10% ovalbumin gel at pH 8.5 in 0.5M NaCl formed well cross-linked strands, while 10% ovalbumin at pH 8.5 in 0.5M Na2804 exhibited little evidence . of cross-linking. The authors also reported that increased G' and destabilizing salts correlated with decreased hydrophobic interaction in heat-induced ovalbumin gels. Sone et al. (1983) reported 11% whey protein concentrate gels in 0.3% Na2304 showed higher G' and denser microstructure than gels containing 0.3% CaClz. The researchers suggested the dense microstructure was due to strong protein-water interaction, leading to less randomness and elastic gels. The Na2804 increases hydrophobic interaction stabilizing the protein molecule. Therefore, there is more protein-water interaction enhancing gel elasticity. 111. Study 1 Denaturation and Structure Development of Ovalbumin as Influenced by pH and Salt Type ABSTRACT Changes in the denaturation and gelation properties of ovalbumin as a function of pH (3.0, 7.0, and 9.0) or salt type (NaC1, NaI, Na2804) were studied using differential scanning calorimetry (DSC) and dynamic rheological testing. The DSC onset temperature of ovalbumin denaturation (To) occurred 5.8°C - 7.5°C prior to structure development (T8) as measured by storage modulus. Enthalpy, To and T8 increased as pH increased. Denaturation temperature decreased in the order of the Hoffmeister series, whereas Ts decreased in reverse order. Activation energies of ovalbumin denaturation and structure development were dependent on pH and salt type. 44 45 INTRODUCTION Ovalbumin is the major egg white protein responsible for egg white gelation. Ovalbumin is a phosphoglycoprotein making up 54% of total proteins in the albumen. Ovalbumin has a molecular weight of 45,000 daltons and it's isoelectric point (pI) is 4.5. The complete amino acid sequence of ovalbumin includes 385 residues that have been determined by Nisbet et al. (1981). Gelation is an important functional property of egg white proteins. Protein gels provide a matrix for holding water, flavors, ingredients and provide texture. Protein gels are made of a three-dimensional matrix of cross-linked polypeptides. Bonds giving structure to ovalbumin network may be electrostatic (Egelansdal, 1980) hydrophobic (Hayakawa and Nakai, 1985), hydrogen (Hata et al., 1986) covalent (Beveridge et al., 1984), van der Waals forces (Hatta et a1., 1986) or a combination thereof. At pH's below the pI ovalbumin dissociates and unfolds at a lower temperature compared to pH 7.0 and 9.0 (Arntfield, 1989). Koseki et a1. (1990) observed that the native compact globular molecule of ovalbumin remained intact while the tertiary structure fluctuated at pH conditions below the pI. At high pH ovalbumin is negatively charged and cross-links through sulfhdryl-disulfied exchange, contributing to the 46 structural integrity of the gel (Shimada and Matsushita, 1980; 1981) Ferry (1948) explained the protein gelation theory as a two step process: Native ---->denatured protein ----- > aggregated protein (long chains) (associated network) Comparison of the rate of the denaturation step verus that of the aggregation step helps determine gel characteristics (Gossett et al., 1984). Hermansson (1979) suggested a gel network with a certain degree of order can be attained if aggregation occurs slower than denaturation; giving denatured protein molecules time to orient themselves before aggregation. Ferry (1948) suggested that unfolding of a single domain protein in solution contain two species of molecules; those that are folded and those that are unfolded. There are no intermediate species found in a pure co-operative process. Therefore, disruption of part of the molecule leads to unfolding of the entire molecule (Creighton, 1984). Koseki et al. (1989) developed a model showing high molecular weight ovalbumin aggregates as a "string of beads". Ovalbumin linear aggregates were formed by hydrophobic interaction forming a gel (Tung, 1974; Kato et al. 1983). Salts in the lyotropic series were used to probe the importance of hydrophobic interactions to protein network 47 formation (von Hippel and Schleich, 1969; Melander and Horvath, 1977). The driving force for such interactions arise from the specific effect each salt exerts on structure of the water molecules around them (Damodaran and Kinsella, 1982). By manipulating solvent conditions the structure of the protein will change due to decreasing hydrophobic interaction (Tanford, 1979). Measurement of the rate of heat flow by differential scanning calorimetry (DSC) can determine effects of storage, pH or stabilizing treatments on protein thermolability (Donovan et al., 1975). Ovalbumin displayed optimum thermal stability in the pH range 6 to 10 with a denaturation temperature of 79°C and denaturation enthalpy of 3.64 cal/g at pH 7.0 (Donovan et a1., 1975). Addition of 170 mM NaCl had little effect on denaturation temperature, but 17 mM CaC12 decreased denaturation temperature 2-3°C at pH values above the pI. Nondestructive dynamic rheological testing is used to measure viscous and elastic properties of a protein solution during heating and gives information about structure development in gel-forming proteins (Tung, 1978). Dynamic rheological analysis was used to observe structure development measured by an increase in storage modulus (G') in ovalbumin during heating from 30°C to 95°C at 2°C/min (Arntfield et al., 1990 c). The researchers reported the G' of a 10% ovalbumin solution decreased with increased salt 48 concentration (NaCl and Na2804 at 1.0M and 0.5M). Temperature of structure development as determined by changes in G' decreased with lower pH. The decrease in G' was attributed to increased intramolecular hydrophobic interaction. The objectives of this project were to influence the heat-induced gelation mechanism of hen egg ovalbumin by changing the pH or salt type then (1) to investigate the relationship between denaturation and structure formation with DSC and dynamic rheological testing and (2) determine thermodynamic and rheological properties of the gelation process. Materials and Methods Material Ovalbumin (Grade V, lot 19F 8105) was purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. Moisture content was determined using AOAC (1984) 17.006-17.007. Solutions of 80 mg\ml ovalbumin were prepared in (1) 0.5M NaCl at pH 3.0, 7.0 and 9.0 or (2) 0.5M NaI, 0.5M NaC1 and 0.5M Na2804 at pH 7.0. Samples were equilibrated for 30 min before testing. All solutions were prepared and tested in duplicate. Electrophoresis Ovalbumin was checked for purity by sodium dodecyl 49 sulfate polyarcylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970) using a Mini-Protean II Dual slab cell (BIO-RAD, Richmond, CA) and power supply (BIO-RAD model 1000/500, Richmond, CA). A running gel of 12% acrylamide and a stacking gel 4% acrylamide were used. The protein samples were run at a constant voltage of 200. Proteins were stained with Coomassie Brilliant Blue R-250 (Bio-Rad) in 40% ethanol and 7% acetic acid and then destained in 7% acetic acid. Differential Scanning Calorimetry A Dupont 990 Thermoanalyzer equipped with a Dupont 910 Cell Base and standard DSC cell were used to determine onset denaturation temperature (To), thermal denaturation temperature ”'6’ and enthalpy (Heal) of ovalbumin. Approximately 15 mg of ovalbumin solution was sealed in a Dupont aluminium hermetic pan (part 900793-901) and lid (part 900794-903). Samples were heated from 30°C to 110°C at 10°C/min. Temperature calibration and calibration coefficient E for the DSC cell were determined using weighed samples of indium over a scanning range of 25-200°C. The reference pan contained an identical ovalbumin solution that was previously heat-denatured in the same temperature range as suggested by de Witt (1981 a). Rescanning the cooled ovalbumin (Patel et al., 1990) sample showed no denaturation peak, suggesting irreversible denaturation. The cell was 50 flushed with nitrogen at 50 mL min'1 to maintain an inert environment for all experiments. All scans showed an endothermic heat flow. Onset denaturation temperature (T0) was the temperature at which a change in slope of the curve occurred as determined by DuPont 9900 General V 2.2A software program. Ovalbumin Td was defined as the endothermic peak temperature and corresponded to 50% unfolding of ovalbumin as determined by the DuPont 9900 General V 2.2A software program. The apparent enthalpy of calorimetry (Heal) was calculated using DuPont 9900 General V 2.2A software as follows: (1) Heal = A/M (60BEqs) where A is the area (cmz), M is the mass of the sample (mg), B is the time base (min cmfl), E is the cell calibration coefficient, and qs is the Y axis range (mW/cm). Peak area baselines were constructed as a single straight line from the begining to the end of the endotherm. The beginning of the endotherm was determined as the initial increase in the integrating curve and endotherm termination was the integrating curve plateau. Activation energy of ovalbumin denaturation was calculated by a method developed by Patel et al. (1990) for whey protein using DSC. The equation, reduced to linear form, was: (2) ln (da/dt) = ln KO - Ea/RT + n [ln (1-a)] 51 The reaction rate (da/dt), at any temperature, T, is calculated as the ratio of peak height to total area. The fraction of denatured protein (a) was calculated as the ratio of partial area to total peak area. Values of the preexponential factor (KO), activation energy (Ea), and reaction order (n) are obtained from multilinear regression (Eisensmith, 1989) performed using 1n (da/dt) as dependent variable and 1/T and 1n (1-a) as two independent variables. The gas constant is R. Thermodynamic Calculations The following two thermodynamic terms were calculated by the methods defined by Bertazzon and Tsong (1990 a). The van't Hoff enthalpy is defined as follows: (3) Hm = 4RT2CP(max) / Hem1 where R is the gas constant, T is the temperature (K), Cp(max) is the maximum heat capacity of the DSC excess heat capacity curve. The cooperativity ratio is defined by: (4) I'IVH / Hcal Dynamic Rheological Testing Storage moduli of ovalbumin solutions during heating from 60°C to 100°C at 2°C/min and 15 min isothermal experiments were monitored using a Rheometrics Fluids Spectrometer Model 8400 (Piscataway, NJ), equipped 52 with a 1-100 g-cm torque transducer and a silicon oil circulation system controlled by a Nelsprit Temperature Programmer. One and a half milliliters of ovalbumin solution was placed between the cone and plate geometry (radius 12.5 mm, 0.02 cone angle and 50 um gap) and equilibrated for 5 min before heating. The G' was recorded continuously at a fixed frequency of 1 rad/sec and strain of 1.0%. Limits of constant viscoelasticity were determined by conducting frequency (0.1 to 100 rad/sec) and strain sweeps (0.1 to 100 %) in preliminary experiments. Significant rheological structure was designated when 6' reached 10 Pa since it was a common point of comparison for all treatments in preliminary studies. Temperatures for isothermal heating experiments were selected based on temperatures at which G' reached at least 10 Pa between 0.1 and 15 min heating period. Thermal scanning experiments (heating from 60°C to 100°C at 2°C/min) were used to determine the temperature at which G' equalled 10 Pa. This temperature was defined as the onset temperature for structure development (Ta). Activation energy of structure formation (Eaa) was calculated from isothermal heating experiments at different temperatures using time-temperature superpositioning (Ferry, 1980). Values for time (t) were taken directly from the curve of G' versus time in which the time needed to reach a G' of 10 Pa was used. The Arrenhius equation was used to 53 Icalculate the activation energy of structure formation: (5) ln t = lnA -Ea/RT where t is the time needed to reach 10 Pa, A is the preexponential factor (s'l), E8 is the activation energy (J mol'l), R is the gas constant and T is the temperature (K). Activation energy was calculated from the slope of the plot ln t versus 1/T. Statistics Analysis MSTATC software (version c, East Lansing, MI) was used for basic statistics and two way analysis of variance (replication x treatment) on a complete randomized design experiment. Tukey's honestly significant difference test (P < 0.05) and standard error of the means were used to evaluate the significant differences between means. Multilinear regression analysis for determining Ead and reaction order was conducted using Plotit (Eisensmith, 1985). All tests were performed in duplicate. RESULTS AND DISCUSSION Proximate Analysis Grade V ovalbumin had a moisture content of 0.35% and the electrophoregram exhibited one band at 45,000 (Powrie and Nakai, 1986) which is the MW of ovalbumin. Donovan and Mapes (1976) observed different Td for ovalbumin and S-ovalbumin at pH 9.0 of 84.5°C and 92.5°C, respectively. In 54 this research the Td at pH 9.0 was 90.6°C. Based on this information, S-ovalbumin was used in this experiment. Effect of pH on Thermal Denaturation The T0 and Ta of S-ovalbumin denaturation was lower at pH 3.0 compared to pH 7.0 or 9.0 (Table 5). The decrease in Td at pH 3.0 may be explained by an increase in number of positive charges that increases repulsive forces within the molecules causing S-ovalbumin to unfold. Luescher et al. (1974) showed that T6 of ovalbumin determined by DSC was lower at pH 3.0 (63°C) than at pH 7.0 (78°C). Donovan et al. (1975) observed that ovalbumin Td Table 5 - Influence of pH on thermal denaturation of 8% (w/v) S-ovalbumin in 0.5M NaC1 using differential scanning calorimetry pH TO TC Edd n *Ead (°C) (°C) (kJ/mOI) (kJ/mOI) 3 68.0° 80.5° 97.1° 1.2° 16.7° 7 83.6” 90.6” 304.0” 1.3° 61.1” 9 82.4” 90.6” 278.5” 1.9° 51.3” T onset temperature T: thermal denaturation temperature Ead activation energy of denaturation n reaction order *Ead activation energy of denaturation calculated when n=1 Column values with different superscripts are significantly different (P < 0.05). 55 was the same at pH 7.0 and 9.0. Arntfield et al. (1981) observed lowering pH from 7.0 to 3.0 decreased the Td from 84°C to 64°C. S-ovalbumin Ead*was lowest at pH 3.0 and not different (P > 0.05) between pH 7.0 and 9.0 (Table 5). The lower S- ovalbumin Ead at pH 3.0 indicated the energy needed to drive the reaction from native to denatured state and temperature dependence of the reaction were decreased. Donovan and Mapes (1976) assumed denaturation of ovalbumin was irreversible and reported an activation energy of 73.3 kJ/mol at pH 9.0 and 37°C. The apparent reason for the discrepancy between calculated Ea was due to the method of calculation. While Donovan and Mapes (1976) used a reaction order of one; this research used a reaction order depending on the fit of the data to the computer analysis program. Therefore, Ea was dependent on reaction order. S-ovalbumin reaction order for denaturation was not different (P > 0.05) (Table 5). Cheftel et al. (1985) reported denaturation of proteins was a first order reaction. Dwek and Navon (1972) estimated the Ea for denaturation of egg albumen based on thermal data to be 103 kJ/mol. Patel et al. (1990) reported reaction order of denatured whey protein (10%) at pH 6.34 to 6.38 ranged from 1.35 to 1.5. calculated using a multilinear regression equation. Dannenberg and Kessler (1988) calculated the reaction order for denaturation of a-lactalbumen and B-lactoglobulin at pH 6.6 to be 1.1 and 56 1.5, respectively. A reaction order greater than suggested more than one molecular event occurring such as unfolding of a-helix and B-sheet. Kokini (1991) suggested that gelation might be considered to have two reaction orders, one for denaturation and another for aggregation. The *Ead decreased with decreased pH (Table 5) suggesting that pH 3.0 required less thermal energy to drive the native protein to denaturation compared to pH 7.0 or pH 9.0. The *Ead and Bad were 5 fold different from each other indicating the concentration of S-ovalbumin may contributed to the calculation. Using a reaction order of one for the Ea calculation decreased the error due to concentration that might include aggregation. The presence of high S-ovalbumin concentration during heat-induced gelation may cause aggregation without denaturation causing inaccurate Ea calculation. S-ovalbumin enthalpy decreased with decreased pH (Table 6). At pH 3.0 ovalbumin enthalpy was lower compared to pH 7.0 or pH 9.0. The decrease in enthalpy might be due to partially unfolded and aggregated molecules compared to completely unfolded molecules at pH 7.0 or 9.0. Because aggregation occurred without denaturation S-ovalbumin did not totally unfolded therefore, decreasing the potential heat energy absorption. Privalov and Khechinashvilli (1974) reported that aggregation is a exothermic process that will 57 Table 6- Effect of pH on the enthalpic contribution to the stability of 8% (w/v) S-ovalbumin in 0.5M NaCl calorimetric van't Hoff pH enthalpy enthalpy CR (kJ/mol) (kJ/mol) 3 41.2° 16.6° 0.40a 7 62.2” 52.1” 0.83” 9 77.6c 50.4” 0.65” CR-cooperative ratio Column values with different superscripts are significantly different (P < 0.05). decrease enthalpic values. Ma et a1. (1988) and Patel et al. (1990) reported a decrease in enthalpy in whey protein due to thermal denaturation at acidic pH as measured by DSC. Ovalbumin denaturation enthalpies have been reported ranging from 706.5 kJ/mol (Donovan and Mapes, 1976) to 392.9 kJ/mol (Fujita and Noda, 1981). Some variation in the calculation and instrument sensitivity might account for difference in reported values. The unfolding step of ovalbumin is usually highly cooperative, and requires heat, which is seen as an endothermic peak in DSC thermograms (Kitabatake et al., 1990). No endothermic peak suggests a fully denatured molecule (de Wit, 1980). A cooperativity ratio (CR) below one suggests the presence of domains or intermediate steps in the melting process. A value higher than one indicates 58 an increased cooperativity of structure and aggregation (Bertazzon and Tsong, 1990 b). The CR for S-ovalbumin was lowest at pH 3.0 and differred (P< 0.05) compared to pH 7.0 or 9.0 (Fig. 6). The sharpest endothermic peaks was exhibited at pH 7.0. Ovalbumin has not been identified as containing domains, therefore an intermediate form might be present. Effect of pH on Structure Development The G' in Tables 7—9 were determined after 15 min isothermal heating. The highest isothermal temperature in which G' was measured was larger and differred (P < 0.05) from the lowest isothermal temperature. The results suggested higher temperature enhanced S-ovalbumin unfolding causing cross-linking and more structure development. The time taken to reach 10 Pa decreased with increased temperature, suggesting higher temperatures increased the rate of S-ovalbumin unfolding. At pH 3.0 the maximum temperature used based on the time necessary to reach 10 Pa was lower than at pH 7.0 or 9.0. Suggesting pH 7.0 and 9.0 were more thermally stable than at pH 3.0. S-ovalbumin was positive charged at pH 3.0 causing electrostatic repulsion between molecules facilitating unfolding. S-ovalbumin Ta increased with pH (Table 10). At pH 3.0 59 Temperature (C) Fig. G-Effect of pH on the differential scanning calorimetry thermogram of 8% 10°C/min. (w/v) S-ovalbumin in 0.5M naci heating at 60 S-ovalbumin was negatively charged and partially unfolded because of electrostatic repulsion accelerating gel structure formation. S-ovalbumin has a balance of attractive (hydrophobic interaction, S-S and hydrogen bonding) and repulsive (electrostatic) forces at pH 7.0 and 9.0, therefore higher temperatures were needed to form a G' at 10 Pa. Arntfield et al. (1989) reported the T8 of ovalbumin at pH 3.0 was 72°C while at pH 7.0 and 9.0 T8 was 95°C. At each pH where thermal onset temperatures (To) from the DSC were compared to T8, T0 preceded TB indicating that partial unfolding of S-ovalbumin occurred before structure development. This observation was also noted by Hegg et a1. (1979). Beveridge (1985 a) using small amplitude oscillatory and Arntfield et al. (1989) using dynamic rheological testing observed that development of rheological structure of ovalbumin did not begin until most of the ovalbumin had been denatured. The use of parallel plates compared to cone and plate geometry, different ovalbumin concentration and salt concentration might be responsible for the different results. S—ovalbumin Eaa at pH 7.0 was lower than at pH 3.0 or pH 9.0 (Table 10). Higher Eaa indicated more energy was required to form cross-linked aggregates which was associated with a slower reaction. Harwalkar (1980) using 61 Table 7-Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 3.0 in 0.5M NaC1 during isothermal heating for 15 min Temperature 6' Time (°C) (Pa) (min) 65 14.73 10.4‘1 70 32.2” 5.8” 75 125.6” 3.3” 80 253.7” 1.4d G' storage modulus determined from isothermal heating for 15 min Time period to reach 10 Pa during isothermal heating Column values with different superscripts are significantly different (P < 0.05). Table 8-Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaC1 during isothermal heating for 15 min Temperature 6' Time (°C) (Pa) (min) 80 105.2° 6.91‘ 85 364.5” 4.4” 90 680.7°” 3.4” 95 1378.1°” 2.3”” 100 2001.3” 0.8” G' storage modulus determined from isothermal heating for 15 min Time period taken to reach 10 Pa during isothermal heating Column values with different superscripts are significantly (P < 0.05). Table 9-Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 9.0 in 0.5M NaCl during isothermal heating for 15 min Temperature G' Time (°C) (Pa) (min) 80 25.3° 12.723 85 192.5” 8.9” 90 368.5” 6.4c 95 2218.7” 1.8” 100 1926.1” 0.8” G' storage modulus determined from isothermal heating for 15 min Time period of time taken to reach 10 Pa during isothermal heating Column values with different superscripts are significantly different (P < 0.05). Table 10- Influence of pH on structure development of 8% (w/v) S-ovalbumin in 0.5M NaCl during heating pH T. Eaa (°C) (kJ/mol) 3 73.7a 596.3” 7 83.9” 315.7” 9 89.9c 676.2a Ts temperature of structure formation Ea. activation energy for structure development Column values with different superscripts are significantly different (P < 0.05). 63 the Arrhenius plot calculated the E8 of thermal denaturation of B-lactoglobulin at pH 6.7 to be 71.6 kJ/mol. The researcher suggested that variation in Eaa due to pH or salt type, may be because of different mechanisms of thermal denaturation. Activation energies calculated from dynamic testing may have had mechanical energy transferred into the system (oscillatory motion of the geometry) that may influence the calculations. Decreasing S-ovalbumin concentration to avoid torque overload and increasing the strain might decrease the variation in Bag. Goldsmith and Toledo (1985) used NMR data to calculate activation energies of gel strenght (structure) in the range of 183.68 to 188.44 kJ/mol. Beveridge et al. (1984) reported an Ea of 138.15 kJ/mol for freeze-dried egg albumen using rate constants obtained at 85°C and 90°C. Effect of Salt Type on Thermal Denaturation Thermal denaturation temperature of ovalbumin at pH 7.0 decreased as a function of salt type in the order of the Hoffmeister series Nast4 > NaC1 > NaI (Table 11). The low Td for NaI may have resulted from the disruption of the hydrophobic interaction within the S-ovalbumin molecule producing instability of the protein structure. In contrast Nazso4 increased intramolecular hydrophobic interaction, therefore stabilizing the native S-ovalbumin structure. 64 Damodaran and Kinsella (1982) suggested that hydrophobic interaction between apolar residues is the major stabilizing force of the native conformation of protein. The ions which salt-out hydrocarbons should enhance hydrophobic interaction in the protein and provide more stabilizing energy. Arntfield et al. (1989) suggested that salts have an impact on the attractive-repulsive balance associated with network formation. At salt concentrations of 0.5M and higher the stabilizing influence was attributed to strengthened hydrophobic interactions. The reseachers reported a decrease in Td and enthalpy using 10% ovalbumin in 0.5M NaSCN as compared to 0.5M NaCl and 0.5M Na2804. Arntfield et al. (1989) observed a significant increase between ovalbumin Td using 0.5M Na2804 compared to 0.1M NaCl and 0.5M C2H3Na02. Damodaran (1988) observed an increase in soy isolate T3 at pH 8.0 in 0.5M Na2804 compared to 0.5M NaC104. Arntfield et al. (1986) and Ismond et al. (1986) used DSC to rank salts in terms of their ability to stabilize faba bean proteins by monitoring changes in Td. The Td values using DSC coincided with their position in the lyotropic series and reflected the importance of hydrophobic interactions to the stability of soy proteins. Activation energies of S-ovalbumin thermal denaturation as a function of salt type did not differ (P > 0.05) 65 (Table 11). The reaction orders for S-ovalbumin in Na2804, NaCl and NaI were 1.1, 1.3 and 2.4, respectively. The reaction order might suggest the occurrance of molecular events within the ovalbumin molecules, such as unfolding of a a-helix or B-sheet. Therefore, it was hypothesized that NaI decreases hydrophobic interaction causing uncoiling of S-ovalbumin molecules increasing the reaction order. When reaction order was one the activation energy of S-ovalbumin in 0.5M NaI differed (P < 0.05) compared to 0.5M NaCl or 0.5M Na2804. The NaI was less temperature dependent than the other two neutral salts suggesting that the energy needed to unfold the molecule might be influenced by ionic interaction that salt imparts on water structure. Table 11- Influence of salt type on thermal denaturation of 8% (w/v) S-ovalbumin at pH 7.0 using differential scanning calorimetry Salt T T o d Ead n *Ead (0.5M) (”C) (”C) (kJ/mol) (kJ/mol) NaI 79.3” i 86.7“, 395.3” 2.4” 38.08” NaC1 83.6” 90.6” 304.0” 1.3” 61.05” Na2804 86.4” 94.5” 238.7” 1.1” 60.69” To onset temperature Td thermal denaturation temperature Ead activation energy of denaturation n reaction order *Ead activation energy of denaturation calculated when n=1 Column values with different superscripts are significantly different (P < 0.05). 66 Enthalpy values for S-ovalbumin in NaZSO4, NaCl and NaI did not differ (P < 0.05) (Table 12). The addition of neutral salts to ovalbumin did not result in any denaturation as evidenced by the similarity in enthaply. This was supported by the CR that did not differ (P < 0.05) between salt types. Artifield et al. (1990a) observed no significant difference in enthalpy between neutral salts at 0.5M concentrations. Effect of Salt Type on Structure Development The G' in Tables 13-15 were determined after 15 min of isothermal heating. The highest isothermal temperature in which G' was measured was larger and differred (P < 0.05) from the lowest isothermal temperature. The results Table 12-Effect of salt type on the enthalpic contributions to the stability of 8% (w/v) S-ovalbumin at pH 7.0 calorimetric van't Hoff Salt enthalpy enthalpy CR (0.5M) (kJ/mol) (kJ/mol) NaI 62.2” 45.6” 0.73” NaC1 62.4” 52.1” 0.83” NaZSO4 65.5a 56.3a 0.89a CR-cooperativity ratio Column values with different superscripts are significantly different (P < 0.05) 67 suggested higher temperatures facilitated the unfolding of S-ovalbumin causing cross-linking and structure development. There were large errors between each treatment temperature that might have been caused by some sample dehydration. The time taken to reach 10 Pa had less error between treatments. Because the time take to reach 10 Pa was less than 15 min which was the time when G' was determined. All cases showeed that as isothermal temperatures increased the time decreased suggesting hihger temperatures increase the unfolding rate of S-ovalbumin. The time for NaI were higher for most time-temperature comparisons suggesting tht disrupting of the water structure and hydrophobic interaction was caused by the chaotropic activity of NaI compared to NaCl or Na2S04. Arntfield et al. (1990 a) reported that G' increased with the inclusion of salts at the destabilizing end of the lyotropic series. The authors concluded that the involvement of hydrophobic interactions were factors in determining the strength of heat-induced ovalbumin networks. Both NaCl and Na2504 stabilize protein structure by promoting hydrophobic interactions within the native structure, whereas, NaI destabilizes protein structure by weakening intramolecularhydrophobic interactions (von Hippel and Schleich, 1969; Catsimpoosas and Meyer, 1970; Babajimopoulos et al., 1983). Arntfield et al. (1989) 68 observed 0.5M NaC1 and 0.5M Na2804 followed the Hofmeister series in their research with 10% ovalbumin. S-ovalbumin TB did not differ (P > 0.05) (Table 15) suggesting salt type does not influence the temperature at which rheological structure was formed. Arntfield et al. (1989) noted T8 for 10% ovalbumin solutions was unaffected by various salt environments. The researchers suggested it was possible that salts have an impact on attractive-repulsive balance associated with network formation. The Eaa of S-ovalbumin in NaC1, NaI and Nazso4 were not different (P > 0.05) (Table 15) suggesting salt type did not change temperature dependency for cross-linking and aggregate formation. Harte (1989) reported an average Ea for denaturation / gelation was 183.47 kJ/mol although a value of 169.32 kJ/mol was reported using the Arrhenius kinetic theory and the temperature-time history. Effect of Concentration on Thermal Denaturation and Structure Development The T0 for 4.0% S-ovalbumin solution occurred 2.3°C before the T0 for 8.0% S-ovalbumin solution. Td did not change with increased S-ovalbumin concentration suggesting that different concentrations of S-ovalbumin did not influence thermal denaturation (Table 16). Changes in Td due to S-ovalbumin concentration have not been discussed in 69 literature, but Kitabatake et al. (1989; 1990) observed that increase in soy bean protein concentration increased Td. The Ead and reaction order for 4.0% S-ovalbumin was the same as 8.0% S-ovalbumin solution. The 4.0% S-ovalbumin enthalpy was less than 8.0% S-ovalbumin indicating less total heat energy was absorbed during denaturation due to the higher protein concentration (Table 17). There was no difference (P > 0.05) in CR between 4.0% and 8.0% S-ovalbumin solution indicating that two state unfolding theory (Ferry, 1948) was not a function of concentration. The G' of 4% S-ovalbumin solution was not detected after 15 min isothermal heating until 90°C suggesting inadequate protein concentration for crosslinking at temperatures below 90°C (Table 18). The T8 for 4.0% S- ovalbumin solution was higher compared to 8.0% S-ovalbumin solution suggesting less protein was present to form a measurable cross-linked structure. The higher TB of 4% S- ovalbumin solution was needed to detected structure formation suggesting more molecular unfolding to furhter extend the molecule for cross-linking had to occur compared to the 8.0% ovalbumin solution. The Eaa did not differ (P > 0.05) between 4.0% and 8.0% ovalbumin solution indicating no additional energy was needed to form a cross-linked structure due to concentration. The reaction order did not differ (P > 0.05) between S-ovalbumin concentrations suggesting the number of molecular events 70 Table 13-Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaI during isothermal heating for 15 min Temperature 6' Time (°C) (Pa) (min) 75 62.6” 9.7” 80 441.7”” 3.7” 85 910.1”” 5.2” 90 1418.8”” 2.1” 95 1134.7””” 1.4”” 100 1795.2” 0.4” G' storage modulus determined from isothermal heating for 15 min Time period taken to reach 10 Pa during isothermal heating for 15 min Column values with different superscripts are significantly different (P < 0.05). Table 14-Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M Nazsoyduring isothermal heating for 15 min Temperature 6' Time (°C) (Pa) (min) 80 43.11‘ 7.4a 85 86.0” 4.9”” 90 1251.4” 3.3”” 95 541.2” 2.0”” G' storage modulus determined from isothermal heating for 15 min Time period of time taken to reach 10 Pa during isothermal heating for 15 min Column values with different superscripts are significantly different (P < 0.05). 71 Table 15- Influence of salt type on structure development of 8% (w/v) S-ovalbumin at pH 7.0 during heating Salt Ta Eaa (0.5M) (°C) (kJ/mol) NaI 83.9” 350.4” NaC1 83.9” 311.5” Na2804 85.6” 384.1” Ta temperature of structure formation Eaa activation energy of structure formation Column values with different superscripts are significantly different (P < 0.05). Table 16- Influence of S-ovalbumin concentration at pH 7.0 in 0.5M NaC1 on thermal denaturation using differential scanning calorimetry concen- To Td Ead n *Ead tration (%) (°C) (°C) (kJ/m01) (kJ/mol) 8 83.6” 90.6” 304.0” 1.3” 66.24” 4 81.3” 90.6” 239.6” 1.0” 61.05” To onset temperature Ta thermal denaturation temperature Ead activation energy of thermal denaturation n reaction order *Ead activation energy of thermal denaturation when n=1 Column values with different superscripts are significantly different (P < 0.05) 72 Table 17- Effect of S-ovalbumin concentration at pH 7.0 in 0.5M NaC1 on thermodynamic properties concen- calormetric van't Hoff CR tration enthalpy enthalpy (%) (kJ/mol) (kJ/mol) 8 62.4” 52.1” 0.83” 4 45.1” 28.6” 0.65 CR-cooperativity ratio Column values with different superscripts are significantly different (P < 0.05). Table lS-Influence of temperature on structure development of 4% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaC1 Temperature 6' Time (°C) (Pa) (min) 90 46.1a 10.0a 95 131.5”” 6.2” 100 262.3” 3.6”” 105 286.8” 1.7” 6' storage modulus determined from isothermal heating for 15 min Time period taken to reach 10 Pa during isothermal heating for 15 min Column values with different superscripts are significantly different (P < 0.05). 73 Table 19- Effect of S-ovalbumin concentration at pH 7.0 in 0.5M NaCl on structural properties during heating concentration TB Ea (%) (°C) (kJ7mol) 8 83.9a 311.58 4 91.4” 624.2” TB temperature of structure development Eaa activation energy of structure development Column values with different superscripts are significantly different (P < 0.05). that occur with respect to 4.0% and 8.0% S-ovalbumin were the same. There was no difference (P > 0.05) between 4.0% and 8.0% S-ovalbumin *Ead indicating that no additional energy was needed to drive the denaturation reaction due to concentration. The Bad of 4.0% and 8.0% S-ovalbumin was approximately 4 and 5x larger, respectively than *Ead. Conclusion The present study suggested that DSC and dynamic rheological testing are complementary methods in the study of ovalbumin gelation when influenced by pH and salt type. At pH 3.0 or 0.5M NaI the thermal stability of S-ovalbumin 74 decreased due to increased electrostatic repulsion or decreased hydrophobic interaction, respectively. At pH 9.0 or 0.5M Na2804 S-ovalbumin had a higher *Ead suggesting increased thermal energy was needed to unfold the protein compared to pH 3.0 or 0.5M NaI. Variation in Bad was dependent on the order of the reaction. The variation might be attributed to the high concentration of ovalbumin used in the experiment or the regression routine used. IV. STUDY 2 Denaturation and Structure Development of S-Ovalbumin as Influenced by Guanidine Hydrochloride and B-Mercaptoethancl ABSTRACT Effect of guanidine hydrochloride (GuHCl) and B- mercaptoethanol (B-ME) on the thermal denaturation and structure development of S-ovalbumin in 0.5M NaCl, pH 7.0 were studied. Increasing GuHCl and B-ME decreased the denaturation temperature 10°C and 9°C, respectively. Enthalpy did not differ (P > 0.05) at any of the GuHCl concentrations but decreased with increased B-ME (62.4-44.6 kJ/mole). Onset temperature for S-ovalbumin structure formation decreased and storage moduli increased with increased concentrations of GuHCl and B-ME. 75 76 INTRODUCTION The ability to manufacture desirable egg containing products comes from understanding the behavior of proteins in different environments. Heat-induced gelation of egg proteins, particularly ovalbumin, is an important functional property in food systems. Protein gelation is a two step process involving denaturation and aggregation as defined by Ferry (1948): Xpn < ----- > XPd ----- > (Pd)x where x is the number of native (n) or denatured (d) protein molecules (P). Several researchers have studied egg protein heat denaturation, aggregation, coagulation and gelation (Donovan and Mapes, 1976; Gossett et al., 1984; Beveridge and Arntfield, 1985; Arntfield et al., 1990 a; b; c), however more information is needed to understand the gelation mechanism. Guanidine hydrochloride (GuHCl) is a denaturant that is used for the disrupting and unfolding proteins by destabilizing hydrogen bonding and hydrophobic interactions (Shimada and Matsushita, 1981; Xiong and Kinsella, 1990) . The exact nature of the interactions of guanidine with protein groups or solvent molecules is not well understood. Lee and Timasheff (1974) reported that GuHCl binds preferentially to the peptide backbone and aromatic amino acid side chains. The interaction between GuHCl and protein weakens hydrogen bonds that stabilize the globular conformation. Gordon (1972) suggested that 77 induced changes exclusively at the surface of the protein molecule by increasing the solubility of nonpolar side chains. The increased solubility of nonpolar amino acid side chains diminish the magnitude of the hydrophobic effect by up to one third, producing unfolding of the protein (Creighton, 1984). Strambini and Gonnelli (1986) observed that GuHCl penetrates into the interior of liver alcohol dehydrogenase decreasing intramolecular interactions resulting in an increased fluidity. Bismuto and Irace (1988) observed a decrease in structural organization and reported a decline in cooperativity of a two-state gelation process using fluorescence. The unfolding of component polypeptides and the transition from an ordered to random coiled structure .allowed buried functional groups, e.g. SH of cysteine, to Ibecome exposed. Flexibility of protein molecules after ‘unfolding enhanced the availability of some functional igroups and facilitated their reactivity (Xiong and Kinsella, 1990). Addition of B-mercaptoethanol (B-ME) cleaved disulfide bonds disrupting the secondary and tertiary structure of proteins (Chang et al., 1978). Hirose et al. (1986) reported that the addition of 70 mM B-ME induced egg white gelation at room temperature. The researchers concluded conalbumin (which contains 30 half-cystine residue/molecule) ‘was involved in intramolecular disulfide linkages caused by 78 reduction of cystine. Harwalkar and Ma (1987) and Zarins and Marshall (1990) observed the Td of oat globulin and glycinin decreased with addition of 5% B-ME. Proteins that contain disulfide bonds between polypeptide chains will be retained after denaturation with GuHCl if reagents to break disulfide bonds are not added to the protein solution (Tanford, 1968). Denaturation of ovalbumin in 6.0M GuHCl and 0.1M B-ME yielded completely unfolded molecules (Ansari et al., 1972; Ahmad and Salahuddin, 1974). The GuHCl disrupted hydrogen bonds while B-ME reduced disulfied linkages, causing unfolding of ovalbumin. Differential scanning calorimetry (DSC) measures the heat capacity (Cp) of a protein as a function of temperature and can be used to determine protein denaturation temperature (Td) and denaturation enthalpy (de Wit and Swinkels, 1980). Donovon and Mapes (1976) used DSC to distinguish between ovalbumin and S-ovalbumin. The researchers determined the T3 of ovalbumin and S-ovalbumin was 84.5°C and 92°C, respectively at pH 9.0. Dynamic rheological testing is a nondestructive method used to monitor changes in viscous and elastic properties of a protein solution and thus structure formation during heating (Tung, 1978). Arntfield et al. (1989) reported the storage modulus (G') of 10% ovalbumin decreased with salt type (NaC1 > Na2804) and salt concentration (0.1M > 0.5M). 79 Arntfield et al. (1990 a) observed the temperature of structure development as determined by changes in G' decreased with lower pH and was attributed to intramolecular hydrophobic interaction. The objectives of this project were to influence the heat-induced gelation mechanism of hen egg ovalbumin by the addition of GuHCl or B-ME then (1) to investigate the relationship between denaturation and structure formation with DSC and dynamic rheological testing, respectively and (2) calculate transition temperatures and thermodynamic properties of the gelation process. Materials and Methods Material S-ovalbumin purity, moisture determination and electrophoresis methods are described in study 1. S-ovalbumim was prepared at a concentration of 80 mg/ml at pH 7.0 in 0.5M NaCl and (1) 0.5M, 1.0M, or 2.0M GuHCl (2) 0.5%, 1.0%, 2.0% or 3.0% B-ME. All samples were allowed to equilibrate for 30 min prior to testing. All samples were prepared and tested in duplicate. 80 Differential Scanning Calorimetry. Dupont 990 Thermoanalyzer equipped with a Dupont 910 Cell Base and standard DSC cell were used to determine thermal properties of ovalbumin. Approximately 15 mg of ovalbumin solution was sealed in Dupont aluminium hermetic pan (part 900793-901) and lid (part 900794-903). Samples were heated from 30°C to 110°C at 10°C/min. Temperature calibration and calibration coefficient E for the DSC cell were determined using weighed samples of indium over a scanning range of 25-200°C. The reference pan contained an identical ovalbumin solution that had been previously heat-denatured in the same temperatue range (de Witt, 1981). Rescanning of the cooled ovalbumin (Patel et al., 1990) sample showed no denaturation peak, indicating irreversible denaturation. The cell was flushed with nitrogen at 50 mL min"1 for all runs. All scans indicated an endothermic heat flow. Onset denaturation temperature (T0) was determined by drawing a tangent line from the Td to the base line of the endotherm performed by the DuPont General V 2.2A software program. The Td was defined as the peak temperature in the endotherm determined by the DuPont General V 2.2A software program. The apparent enthalpy of calorimetry (Heal): (1) Heal = A / M (GOBEqs) where A is the area (cmz), M is the mass of the sample (mg), B is the time base (min cm‘l), E is the cell calibration 81 coefficient, and qs is the Y axis range (mW cm'l). Peak areas baselines were constructed as a single straight line from the beginning to the end of the endotherm and the area of the endotherm was integrated. The beginning of the endotherm was determined as the initial increase in the integrating curve and endotherm termination was when the integrating curve became constant. Activation energy (Ead) was calculated by a method developed by Patel et a1. (1990) for whey protein using DSC. The equation, reduced to linear form was: (2) ln (do / dt) = 1n KO - Ea / RT + n [1n (1-a)] The reaction rate (da/dt), at any temperature, T, was calculated as the ratio of peak height to total area. The fraction of denatured protein (a) is calculated as the ratio of partial area to total peak area. Values of preexponential factor (KO), activation energy (Ea), and reaction order (n) are obtained from multilinear regression (Eisensmith, 1989) performed using ln (do / dt) as dependent variable and l/T and ln (l-a) as two independent variables. The gas constant is R. Thermodynamic Calculations The following two thermodynamic terms were calculated by the methods defined by Bertazzon and Tsong (1990). The van't Hoff enthalpy is defined as follows: (3) Hm = 4RT2 Cp(max) / Hcan 82 where R is the gas constant, T is the temperature (K), Cp(max) is the maximum heat capacity of the DSC excess heat capacity curve. The cooperativity ratio, is defined by (4) five / Hcal Dynamic Rheologcial Testing The (G') of ovalbumin solutions during heating from 60°C to 100°C at 2°C/min and 15 min isothermal experiments were monitored using a Rheometrics Fluids Spectrometer Model 8400 (Piscataway, NJ), equipped with a 1-100 g-cm torque transducer and water circulation system controlled by a Nelsprit Temperature Programmer. One and one-half milliliters of ovalbumin solution was placed between the cone and plate geometry (radius 12.5 mm, 0.02 cone angle and 50 um gap) and equilibrated for 5 min prior to heating. The G' was recorded continuously at a fixed frequency of 1 rad/sec and strain of 1.0%. Limits of constant viscoelasticity were determined by conducting frequency (0.1 to 100 cmfl) and strain sweeps (0.1 to 100 %) in preliminary studies. Significant rheological structure was designated when G' reached 10 Pa since it was a common point of comparison for all treatments in preliminary studies. Temperatures for isothermal heating experiments were selected based on 83 temperatures at which G' reached at least 10 Pa between 0.1 and 15 min heating period. Thermal scanning experiments (heating from 60°C to 100°C at 2°C/min) were used to determine the temperature at which an initial increase in G' was observed. This temperature was defined as the onset temperature for structure development (T3) . Activation energy of structure formation (Bag) of ovalbumin was calculated using time-temperature superpositioning (Ferry, 1980) at temperatures between 65°C and 100°C for 15 min. Values for time (t) were taken directly from the curve of 6' versus time in which time need to reach 10 Pa was used. The Arrenhius equation was used to calculate activation energy, (5) ln t = lnA -Ea/RT where t is the time needed to reach 10 Pa, A is the preexponential factor (8'1), Ea is the activation energy (J/mol'l), R is the gas constant and T is the temperature (K). Activation energy was calculated from the slope of the plot ln t versus 1 / T. Statistics MSTATC software (version C, East Lansing, MI) was used for basic statistics and two way analysis of variance (replication x treatments) on a complete randomized design experiment. Tukey's honestly significant difference test (P 84 < 0.05) and standard error of the means were used to evaluate the significant differences between means. Multilinear regression analysis for determining Edd and reaction order was conducted using Plotit (Eisensmith, 1985). All treatments were tested in duplicate. RESULTS AND DISCUSSION Effect of Guanidine Hydrochloride on Thermal Denaturation S-ovalbumin To decreased with increased GuHCl concentration (Table 20) suggesting that denaturation occurred at a lower temperature with higher concentrations of GuHCl. A decrease in S-ovalbumin Td at all GuHCl concentrations suggest destabilized hydrogen and hydrophobic bonds compared to the absence of the denaturant. S- ovalbumin T0 was 8°C to 10°C lower than Td (Table 20) suggesting structure development occurred before Td. Privalov (1979) reported lysozyme Td decreased with increased GuHCl concentrations at pH 2.3 and 4.5. S-ovalbumin Ead at 0.5M GuHCl was different (P < 0.05) from the 2.0M GuHCl treatment (Table 20). The decrease in Bad might be due to a decrease in water structure and a decrease in hydrophobic interactions that lead to protein 85 Table 20- Influence of guanidine hydrochloride (GuHCl) on thermal denaturation of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaCl using differential scanning calorimetry GuHCl To Td Edd n *Ead (M) (°C) (°C) (kJ/moll (kJ/mol) 0.0 83.6” 90.6” 304.0”” 1.3” 61.15” 0.5 78.6” 86.2” 437.4” .2.4” 45.2”” 1.0 77.2” 85.2” 257.3”” 1.5” 40.4” 2.0 66.3” 76.2” 123.4” 1.6” 14.5” To onset temperature Td denaturation temperature Ead activation energy of denaturation n reaction order *Ead activation energy of denaturation when n=1 Column values with different superscripts are significantly different (P < 0.05). .unfolding at higher GuHCl concentration, therefore the energy needed to drive the reaction from native to denatured state was reduced. Kauzmann.(1959) reported the heat capacity was strictly dependent on the ordering of water molecules around exposed hydrophobic groups and a change of heat capacity at denaturation should be connected with disruption of hydrophobic bonds. The 0.5M GuHCl reaction order was different (P < 0.05) compared to the other GuHCl concentrations (Table 20). At 0.5M GuHCl the solubility of ovalbumin might have been enhanced through a salting in effect. The reaction order may give information on the number of molecular events occurring in denaturation. As the number of events 86 (uncoiling of the molecule) increased so did the reaction order suggesting that 0.5M GuHCl had more molecular events occurring compared to 2.0M GuHCl due to temperature dependency. In addition the S-ovalbumin concentration might be too high to detect denaturation specifically and the reaction order and Bad may included aggregation. Kokini (1991) suggested that gelation might be considered to have two reaction orders one for denaturation and another for aggregation. Further research is necessary to investigate the reaction order during gelation. The S-ovalbumin *Ead for the control was different (P <0.05) compared to 1.0M or 2.0M GuHCl (Table 20) suggesting that the control was more temperature dependent. The *Ead seems to be more consistent compare to Ead with what would be expected. Since GuHCl disrupts and unfolds protein molecules one would expect a significant difference between the control and 2.0M GuHCl which is seen in *Ead but not in Bad. S-ovalbumin enthalpy did not differ (P > 0.05) (Table 21) with the addition of GuHCl suggesting that no substantial unfolding occurred because of the denaturant. Privalov (1979) suggested that enthalpies associated with protein unfolding due to temperature, pH and GuHCl were the same. von Hippel and Schleich (1969) reported that guanidine salts followed the same Hofmeister 87 Table 21 - Effect of guanidine hydrochloride (GuHCl) on the enthalpic contributions to stability of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaC1 GuHCl calorimetric van't Hoff CR enthalpy enthalpy (kJ/mol) (kJ/mol) 0.0 62.4” 52.1” 0.83” 0.5 56.4” 45.1” 0.80” 1.0 55.3” 38.2”” 0.69” 2.0 56.5” 22.4” 0.40” CR-cooperativity ratio Column values with different superscripts are significantly different (P < 0.05). series as all neutral salts. Arntfield et al. (1989) observed addition of stabilizing salts to ovalbumin did not result in any substantial protein unfolding as indicated by the similarity in the enthalpy values. Pfeil and Privalvo (1976) reported lysozyme enthalpy at pH 2.0 decreased with increased concentrations of GuHCl. A The researchers observed no difference in enthalpy between GuHCl denatured and heat denatured lysozyme. A cooperativity ratio (CR) equal of one is a true two step gelation process, whereas a CR < 1 suggest intermediate steps in thermal denaturation (Bertazzon and Tsong, 1990). Complete protein unfolding by GuHCl is known, although there is some doubt whether complete unfolding can be achieved by thermal denaturation (Pfeil and Privalov, 1976). Ovalbumin CR decreased with increased GuHCl concentrations (Table 21) 88 indicating the two state unfolding theory (Ferry, 1948) may not hold true under these conditions. Privalov (1979) observed 6.0M GuHCl unfolded lysozyme from its native structure and Tanford (1966) concluded from NMR (sharpness of transition) results indicated denaturation was highly cooperative. Pfeil (1981) suggested the cooperativity of protein folding seemed independent on the nature of the denaturing action. Cooperativity ratio was 0.7 for G-actin, suggesting that denaturation is not a simple two-state process, and therefore can be resolved into multiple steps. The higher the CR and Td the greater stability (Bertazzon et al. 1990 c). Bismuto and Irace (1988) used fluorescence intensity to monitor the effect of GuHCl on the structure of apomyoglobin. 'The researchers reported that apomyoglobin exhibited cooperativity with increasing GuHCl concentrations (0-4M, pH 7.0). They further suggested that GuHCl acted on specific structural regions of apomyoglobin instead of a general loosening of structure as reported with liver alcohol dehydrogenase. Doi et al. (1987) reported that ovalbumin contained residual secondary structure after heating but was mainly random coil after treatment with 5.0M GuHCl as monitored with circular dichroism (CD). For B-lactoglobulin significant differences between GuHCl and heat denaturation were found, as judged from optical rotation (Harwalkar, 1979). Suresh Chandra et a1. (1984) used CD to determine the effect of GuHCl on the 89 secondary structure of glycinin. A disordered structure was observed at 15°C that appeared to become more ordered at higher temperatures. Effect of Guanidine Hydrochloride on Structure Development S-ovalbumin G' in Tables 22-24 were determined after 15 min of isothermal heating. The highest isothermal temperature in which 6' was measured was larger and differred (P < 0.05) from the lowest isothermal temperature at all GuHCl concentrations. The results suggested higher temperatures enhanced the unfolding of S-ovalbumin casuing cross-linking and more structure development. The presence GuHCl disrupted hydrogen bonding allowing S-ovalbumin to unfold at a lower temperature compared to the control. Unfolding at a lower temperature enabled formation of higher molecular weight molecules increasing G' at higher GuHCl concentrations. S-ovalbumin was more stable at 0.5M than at the control as addition of low GuHCl concentrations might have enhanced protein solubility through a salting-in effect. The time taken to reach 10 Pa decreased with increased temperatures which suggested higher temperatures increased the rate of unfolding. Katsusta and Kinsella (1990) observed an increase in G' of 10% B-lactoglobulin in 6.0M GuHCl at 25°C during a 12 hr incubation period. The 90 Table 22-Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M guanidine hydrochloride (GuHCl) and 0.5M NaC1 during isothermal heating for 15 min Temperature G' Time (°C) (Pa) (min) 80 66.1” 9.3” 85 285.0”” 4.2” 90 390.8”” 2.0”” 95 638.4” 0.8” 6' storage modulus determined from isothermal heating for 15 min Time period of time taken to reach 10 Pa during isothermal heating for 15 min Column values with different superscripts are significantly different (P < 0.05). Table 23-Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 1.0M guanidine hydrochloride (GuHCl) and 0.5M NaC1 during isothermal heating for 15 min Temperature G' Time (°C) (Pa) (min) 75 12.2” 14.9” 80 129.6” 6.5” 85 230.3” 4.9” 90 299.3” 1.4” 95 650.9” 0.3” G' storage modulus determined from isothermal heating for 15 min Time period of time taken to reach 10 Pa during isothermally heating for 15 min Column values with different superscripts are significantly different (P < 0.05). . 91 researchers suggested that 6.0M GuHCl decreased the fludity of B-lactOglobulin solution and the gel became more rigid as network-cross links were formed. S-ovalbumin TB control differred (P < 0.05) from 2.0M GuHCl (Table 25). Lower salt concentrations enhanced solubility, and increased S-ovalbuminheat stability compared to the control. At 2.0M GuHCl disruption of hydrogen bonds facilitated unfolding of S-ovalbumin and decreasing T8. S-Ovalbumin Ea8 calculated from a series of different isothermal temperatures increased with increased GuHCl concentrations (Table 25). The increase in Ea8 indicates more energy is needed at 2.0M than at the control to form cross-linked aggregates. The Ea8 calculated from rheological data had an inverse relationship as compared to Ead this may be due to the rate of thermal treatment (rate) and the addition of mechanical energy input into the gel system. The data supports the idea that the control is less temperature dependent compared to 2.0M GuHCl to drive the native S-ovalbumin molecule to denaturation. Effects of B-Mercaptoethanol on Thermal Denaturation The onset temperature (To) for S-ovalbumin decreased with increased fl-ME concentrations (Table 26). The Td of S- ovalbumin decreased with increasing B-ME concentrations (Table 26) suggesting that disulfied bonds were not reduced. The Td should remain constant when the S-S bond is reduced. Table 24-Influence of temperature on structure develompent of 8% (w/v) S-ovalbumin at pH 7.0 in 2.0M guanidine hydrochloride (GuHCl) and 0.5M NaC1 during isothermal heating for 15 min Temperature G' Time (°C) (Pa) (min) 65 16.5” 13.0” 70 68.5” 7.7” 75 212.4” 3.3” 80 538.2” 0.4” G' storage modulus determined from isothermal heating for 15 min Time period of time taken to reach 10 Pa during isothermal heating for 15 min Column values with different superscripts are significantly different (P < 0.05). Table 25- Influence of guanidine hydrochloride (GuHCl) on structural properties of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaC1 during heating GuHCl' T Ea S S (K) (°C) (kJ/mol) 0.0 83.9” 311.5” 0.5 88.8” 598.9” 1.0 85.7”” 731.1” 2.0 75.9” 837.5d Ta temperature of structure formation Eaa activation energy of structure formation Column values with different superscripts are significantly different (P < 0.05). 93 Ma et al. (1987) observed the addition of 5.0% B-ME lowered Td of oat globulin suspension from 108°C to 96.7°C and caused a reduction in enthalpy. They attributed these effects to B-ME acting as a monohydric alcohol that destabilized proteins by weakening hydrophobic interactions. Zarins and Marshall (1990) concluded that the reduction in Td of soy glycinin as a consequence of increased B-ME was due to the destabilization of hydrophobic bonds in the protein interior. de Wit and Klarenbeek (1981) observed a decrease in T3 of B-lactoglobulin in the presence of 1.5 x 10'6 mol B-mercaptoethanol. S-ovalbumin Ead (Table 26) did not differ (P > 0.05) in the presence of B-ME suggesting that disulfide bonds do not have a large influence on the temperature dependence of ovalbumin unfolding. S-Ovalbumin Ead was inconsistent suggesting the disulfide bonds was not completely reduced at any B-ME concentrations making it difficult to calculate an accurate Ead. As discussed in the GuHCl section there seems to be more consistency in the *Ead data than Ead. Since B-ME cleaves disulfied bonds causing disruption and unfolding of the molecules one would expect a significant difference between the control and samples containing higher concentrations of the denaturant. There was a difference (P < 0.05) between the control and 3.0% B-ME in *Ead. 94 Table 26-Influence of B-mercaptoethanol (B-ME) on thermal denaturation of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaC1 using differential scanning calorimetry ME To Td Ead n *Ead (%) (°C) (°C) (kJ/m01) (kJ/m01) 0.0 83.6” 90.6” 304.0” 1.3””” 61.15” 0.5 75.5” 86.2” 228.3” 1.6””” 32.8”” 1.0 75.7” 85.6” 310.9” 2.0” 49.1”” 2.0 71.3” 81.2” 146.6” 1.2” 28.8” 3.0 69.0” 77.9” 210.7” 1.5””” 41.9”” To onset temperature Td denaturation temperature Ead activation energy of denaturation n reaction order *Ead activation energy of denaturation when n=1 Column values with different superscripts are significantly different (P < 0.05). S-ovalbumin enthalpy at 3.0% B-ME was different (P < 0.05) compared to all other B-ME concentrations (Table 27). This implied that 3.0% B-ME treated S-ovalbumin was not in its native state before enthalpy measurement, therefore partial unfolding of ovalbumin due to reduction of disulfide bond occurred. Zarins and Marshall (1990) observed that increasing B-ME concentration had little effect on the enthalpy of denaturation. S-ovalbumin CR decreased with increased B-ME concentrations (Table 27) suggesting transient states of partially unfolded S-ovalbumin. de Wit and Klarenbeek (1981) reported a broadening of the B-lactoglobulin endotherm in the presence of B-ME. 95 Table 27- Effect of B-mercaptoethanol (B-ME) on the enthalpic contributions to stability of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaC1 ME calormetric van't Hoff CR enthalpy enthalpy (%) (kJ/mol) (kJ/mol) 0.0 62.4” 52.1” 0.83” 0.5 68.0” 46.7” 0.69”” 1.0 60.1” 39.8” 0.67”” 2.0 66.1” 43.9” 0.66”” 3.0 44.6” 26.4” 0.59” CR-cooperativity ratio Column values with different superscripts are significantly different (P < 0.05). Effect of S-Mercaptoethanol on Structure Development S-ovalbumin G'in Tables 28-31 were determined after 15 min of isothermal heating. The highest isothermal temperature in which G' was measured was larger and differred (P < 0.05) from the lowest isothermal temperature at all B-ME concentrations. As the B-ME concentration increased the S-S bond became partially reduced in ovalbumin due further unfolding of the molecules enhancing cross-linking. Exposed hydrophobic groups interacted intermolecularly increased gel structure. The time taken to reach 10 Pa decreased at higher temperatures suggesting Table 28-Influence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5% B-mercaptoethanol (B-ME) and 0.5M NaCl during isothermal heating for 15 min Temperature G' Time (°C) (Pa) (min) 75 23.0” 12.1” 80 247.2” 6.9” 85 926.5”” 3.9” 90 1664.4”” 2.5”” 95 2132.1”” 1.5”” 100 4962.3” 0.4” G' storage modulus determined from isothermal heating at for 15 min Time period taken to reach 10 Pa during isothermal heating for 15 min Column values with different superscripts are significanly different (P < 0.05). Table 29-Inf1uence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 1.0% B-mercaptoethanol (B-ME) and 0.5M NaC1 during isothermal heating for 15 min Temperature 6' Time (°C) (Pa) (min) 80 454.1” 5.9” 85 1323.3” 3.9” 90 1791.7”” 1.9”” 95 2023.6” 1.1” G' storage modulus determined form isothermal heating for 15 min Time period taken to reach 10 Pa during isothermal heating for 15 min Column values with different superscripts are significantly different (P < 0.05). 97 Table 30-Inf1uence of temperature on structure development of 8% (w/v) S-ovalbumin at pH 7.0 in 2.0% B-mercaptoethanol (B-ME) and 0.5M NaC1 during isothermal heating for 15 min Temperature G' Time (°C) (Pa) (min) 70 15.0” 13.1” 75 676.5”” 7.3” 80 620.5”” 2.2” 85 889.5”” 1.6” 90 1434.8” 0.6” G' storage modulus determined from isothermal heating for 15 min Time period taken to reach 10 Pa during ‘ isothermal heating for 15 min Column values with different superscripts are significantly different (P < 0.05). Table 31—Influence of temperature on structure development of 8%(w/v) S-ovalbumin at pH 7.0 in 3.0% B-mercaptoethanol (B-ME) and 0.5M NaC1 during isothermal heating for 15 min Temperature G' Time (°C) (Pa) (min) 65 13.4” 13.7” 70 133.8” 7.1” 75 693.5”” 2.1” 80 . 1077.2”” 1.4”” 85 2201.7” 0.7” G' storage modulus determined from isothermal heating for 15 min Time period taken to reach 10 Pa during isothermal heating for 15 min Column values with different superscripts are significantly different (P < 0.05). 98 an increased rate of unfolding. Hirose et al. (1986) reported gel hardness of egg white increased with increased thiol concentration (0 to 0.2M) at 35°C over a period of 24 hr. Shimada and Cheftel (1988) suggested that partial reduction of disulfide bonds in the whey proteins enhanced interations between exposed hydrophobic regions. Ovalbumin T8 decreased with increased B-ME concentrations (Table 32). The T0 occurred before Ta indicating partial unfolding was needed before any structure formation in all ovalbumin treatments. Ovalbumin Eaa increased with increased B-ME concentrations (Table 32). The increase in EaB with B-ME concentrations, suggested that the aggregation reaction was temperature dependent. As discussed for Bad, B-ME ovalbumin system may be in a transition from oxidized to reduced state, therefore affecting the accuracy of Eaa calculations. Comparing DSC to Dynamic Rheological Testing Comparing DSC to rheological data a relationship between unfolding To and Ts'was exhibited. In both GuHCl and B-ME treatments To occurred before Ta indicating that partial unfolding of ovalbumin was necessary for structure development. Work by other authors investigating DSC and rheological changes induced by chemical perturbation of ovalbumin was not found. Arntfield et al. (1989) reported that T6 of ovalbumin occurred before Ta as with salts and pH Table 40-Influence of B-mercaptoethanol (B-ME) on structural properties of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaC1 during heating B-ME T,3 Eaa (%) (°C) (kJ/mol) 0.0 83.9” 311.5” 0.5 82.5”” 477.9” 1.0 83.5” 330.2” 2.0 79.7” 639.7” 3.0 72.9” 641.8” G' storage modulus determined from isothermal heating at 80°C for 15 min Ta temperature of structure development Eaa activation energy of structure development Column values with different superscripts are significantly different (P < 0.05). although Herald (1991) observed the opposite trend. This could have been do to the different geometries used (parallel plates versus cone and plate, respectively). Conclusion . Increased concentrations of GuHCl or B-ME decreased the thermal stability of S-ovalbumin. Suggesting that GuHCl and B-ME disrupted the bonding of S-ovalbumin causing unfolding of at lower temperatures. The time taken to reach 10 Pa decreased with increased denaturant concentration at all temperatures indicating S-ovalbumin unfolded due to the presence of GuHCl and B-ME. Differential scanning calorimetry and dynamic rheological testing was complementary in quantifying changes in thermal properties of ovalbumin. V. STUDY 3 neat-Induced Changes in the Secondary Structure of Sen Egg S-Ovalbumin ABSTRACT Solutions of hen egg S-ovalbumin at pD 3.0, 7.0 and 9.0 were heated at temperatures between 30°C to 90°C to study changes in secondary structure by Fourier transform infrared spectroscopy (FT-IR). Second derivative infrared spectra of native S-ovalbumin at pD 7.0 and 9.0 revealed protein absorption bands for B-sheet at 1626 cm'l, BIO-helix at 1638 cm“, a-helix at 1656 curl, and turns at 1682 curl. The B-sheet absorption band was not observed for ovalbumin at pD 3.0. The quantity of a-helix and B-sheet structure decreased as heating temperature was inCreased. 100 101 INTRODUCTION Ovalbumin is the major hen egg white protein that contributes gel properties to egg containing food systems. Conformational changes occur in ovalbumin during heating and perturbation (induced environmental change) as indicated by changes in thermal denaturation temperatures and viscoelastic properties (Arntfield et al., 1989; Herald, 1991). Monitoring heat-induced changes in the secondary structure of ovalbumin will help to understand the mechanism involved in gelation and texture formation of egg proteins. This information may be used to manipulate protein conformation to optimize textural properties for specific food systems. The secondary structure of ovalbumin and S-ovalbumin has been determined using Raman spectroscopy. Painter and Koenig (1976) were unable to detect any conformational difference between 10% ovalbumin and S-ovalbumin in 0.05M NaCl. Both proteins contain 50% random coil, 25% a-helix or 25% B-sheet. Kint and Tomimatsu (1979) reported 7% ovalbumin solutions in 0.05M KCl, pH 8.0 contained a higher a-helix content than B-sheet. S-ovalbumin had a 3-4% higher B-sheet content than ovalbumin. Using circular dichrosim (CD), Egelandsdal (1986) reported that a 10% ovalbumin solution at pH 9.5 contained 30% a—helix and 40% B-sheet. The a-helixical content 102 increased when ovalbumin was solubilized in 0.04M NaCl as compared to water. Kato and Takagi (1988) observed an increase in B-sheet and a loss of a-helix when 0.28% ovalbumin solution at pH 7.0 was heated to 80°C and measured using CD. Doi et al. (1987) using CD observed more a-helix and B-sheet than unordered structure in ovalbumin. Prestrelski et al. (1991 a) and Susi and Byler (1988) reported that CD is unable to detect turns or distinguish between 310-helix and a-helix. The secondary structure of proteins can be examined using Fourier transform infrared spectroscopy (FT-IR) with second derivative analysis. The FT-IR is a non-dispersive technique that provides better wavelength accuracy and higher signal to noise ratio than disperive techniques (Susi and Byler, 1986). Good correlation between FT-IR and x-ray crystallography has been reported in measuring protein secondary structure of a-lactalbumin (Prestrelski, 1991 a). The FT-IR technique exhibited a higher percentage of a-helix and B-sheet than was reported by researchers using Raman or CD (Painter and Koenig, 1976; Kint and Tomimatsu, 1979; Egelansdal 1986). The Amide I band (1620-1700 cmfl), caused by carbonyl stretching vibration of the peptide backbone, is detected using FT-IR and used to monitor secondary structure of proteins (Byler and Susi, 1986; Surewicz and Mantsch, 1988; Havel, 1989). Vibrational transitions associated with 103 a-helix, BIO-helix, B-sheet, turn and unordered structure give rise to bands at specific frequencies in the Amide I region (Prestrelski et al., 1991 b). Second derivative spectra analysis allows for resolution enhancement of overlapping bands in FT-IR spectra providing a qualitative means for following subtle change in protein conformation (Byler and Susi, 1988; Byler and Purcell, 1989). Using FT-IR spectroscopy in aqueous solution is difficult because water absorbs strongly throughout much of the mid-IR.region (4000-400 cmfl). Particular problems occur in the Amide I region because of the strong HOH bending mode that absorbs around 1644 cmfl. Susi and Byler (1986) reported that deuterium oxide (D20) does not absorb in the Amide I region and is a good solvent to use when studying protein structure by FT-IR. The FT-IR has been used to investigate secondary structure of whey proteins (Byler and Purcell, 1989; Prestreiski et al., 1991 b), bovine serum albumin, carbonic anhydrase, lysozyme (Byler and Susi, 1986), hemoglobin, and ribonuclease A (Susi and Byler, 1983). During heating, a-lactalbumin retained more of its native conformation compared to B-lactoglobulin or bovine serum albumin (Byler and Purcell, 1989). No research has been reported that used FT-IR with second dervative analysis to resolve the secondary structure of native ovalbumin and subsequent conformational changes due to heating and pH. The 104 objectives of the present research were to monitor changes in secondary structure of S-ovalbumin as a function of temperature at pD 3.0, 7.0, and 9.0. Materials and Methods S-ovalbumin and all reagents were stored in a vacuum dessicator with phosphorous pentoxide to absorb water vapor. Ovalbumin (Grade V, lot 19F 8105) was purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. S-ovalbumin was prepared as 1.75% (w/v) solutions in 25 mM KD2P04 made up in D20. Samples were prepared in a glove box under an atmosphere of dry nitrogen. The 99.9% nitrogen was passed through a container of CaSO4 dessicant prior to entering the glove box. The pD of the S- ovalbumin solution was adjusted with 30% DCl or 40% NaOD to pD 3.0, 7.0 or 9.0 by adding 0.4 to the pH reading (Covington et al., 1968) measured using a Corning pH meter (Model 107, Corning, NY). S-ovalbumin solutions (200 ul) were placed in capped glass vials (2 mL capacity) which were purged with dry nitrogen for 2 min before heating at a constant temperature in a Fisher Programable Isotemp Oven for 30 min (Model 230F, Pittsburg, PA). After heating samples were placed inside the glove box and allowed to stand approximately 5 min until cool. S-ovalbumin heat treatments at pD 7.0 and 9.0 were control, 80°C, 85°C, and 90°C. Heat treatments at pH 3.0 105 were control, 60°C, 65°C, 70°C and 80°C. Heat treatments at pD 7.0 in 2.0M GuHCl or 3.0% B-ME were 30°C and 80°C. Treatments were selected based upon S-ovalbumin thermal denaturation temperatures as determined by differential scanning calorimetry (Herald, 1991). The cooled sample was again purged with dry nitrogen gas for 2 min and 100 ul was transferred to a Circular Demountable Cell (Model P-E N930-1117, Perkin-Elmer, Norwalk, CT) with CaFZ windows and 0teflon spacers (75 um pathlength), which had been purged with dry nitrogen gas for 10 min before sample loading. Infrared (IR) spectra were collected at ambient temperature using a model 1800 FTIR spectrometer (Perkin-Elmer) equipped with a incandescent wire source, a potassium bromide coated beam splitter and a broad range” mercury/cadmium/telluride (MCT) detector. Before the IR spectrum was recorded, the instrument sample chamber and cell were purged with dry nitrogen gas for 15 min. All spectra were scanned 1000 times and recorded at a resolution of 2 cm'l. The sample chamber was continuously purged with dry nitrogen. Resolution enhancement by second-derivative analysis (CDS-3 Applications software, Perkin-Elmer) was performed using the Savitzky-Golay derivative routine (Savitzky and Golay, 1964) and a 13-data point (13 cm'l) window. Spectral contributions from a D20 blank and residual water vapor were subtracted. Each treatment was tested in triplicate. 106 The FT-IR spectra of 19 proteins with known secondary structure (Clark et al., 1981; Byler and Susi, 1986; Susi, and Byler, 1987; Halloway and Mantsch, 1989) were used as a means to identify the ovalbumin secondary structure. For example the crystalline structure of hemoglobin contains 86% a-helix and the FT-IR spectrum of hemoglobin has it's largest peak at 1655 cmfl. Therefore, the FT-IR band assignment for a-helix is 1655 cmfl. Band assigments for 8- sheet are at 1624 and 1627 cm"1 based on concanavalin A and immunoglobulin G which both contain 86% B-sheet (Levitt and Greer, 1977; Dong et al. 1990). a-lactalbumin contains 30% 310ehelix with a band assignment at 1639 cm'1 (Acharya et al., 1989; Halloway and Mantch, 1989). B-lactoglobulin which contains 46% turn has FT-IR bands at 1670 and 1680 cm.’1 (Papiz et al., 1986). Since B-helix, unordered, and B-sheet can be assigned to other bands, the bands located at 1666, 1672, 1680 and 1688 cmfliwere assigned to turn structures (Dong et al., 1990). RESULTS AND DISCUSSION Secondary Structure of Native S-Ovalbumin The crystalline structure of ovalbumin is known (Stein et al., 1990; Wright, 1990). However, the FT-IR spectral bands have not been assigned. The second derivative FTIR spectra of native ovalbumin were identified at pD 3.0, 7.0, and 9.0 at 30°C. 107 Five Amide I component bands were observed at pD 3.0 (Fig. 7) with peaks at 1638.2 cm”1 (310-helix), 1656.3 cm'1 (a-helix) , 1682.5 cm"1 turn and two shoulders at 1625 cm"1 (B-sheet) and 1669.6 cm"1 (turn). There is strong evidence suggesting the band at 1639 cm"1 is 310-helix for globular proteins (Acharya et al., 1989; Byler and Purcell, 1989; Prestrelski et al. 1991 a). However, X-ray crystallograpy has not identified a 310-helix in the secondary structure of ovalbumin (Stein et al. 1990; Wright et al. 1990). In addition the band at 1639 cmflihas also been assigned to B-sheet (Dong et al. 1990). Therefore, the band at 1639 cm'1 is still under investigation. In the presence research the band at 1639 cmfl'was considered.310-helix due to the strong evidence found in other globular proteins. Second derivative FT-IR spectrum of native S-ovalbumin at pD 7.0 exhibited 4 Amide I peaks and a shoulder (Fig. 8). The spectrum was similar to that of S-ovalbumin at pD 3.0. A distinguishable peak rather than a shoulder was observed at 1627.5 cm'1 (B-sheet). Peak intensities were observed at pD 7.0 were larger than pD 3.0 at 1638 cm.“1 (310 helix). However, pD 3.0 exhibited greater peak intensities at 1656 cm“1 (a-helix) and 1681 cm'1 (turn). Second derivative FT-IR spectrum of native S-ovalbumin at pD 9.0 exhibited 4 Amide I peaks and a shoulder similar to the spectrum at pD 7.0 (Fig. 9). The band representing 108 310-helix was larger at pD 9.0 than at pD 3.0 and the peak intensity representing a-helical was smaller at pD 9.0 than at pD 3.0. Effect of pD and Heating on Secondary Structure In all cases increasing the temperature shifted bands frequencies of ovalbumin by 2-3 wavenumbers. S-ovalbumin, pD 3.0, heated to 60°C (Fig. 8), exhibited an amplified band at 1614 cm‘1 (intermolecular hydrogen bonding) compared to ovalbumin at 30°C. The band intensity at 1682.5 cm'1 (turn) increased and was broader at 60°C compared to 30°C. Intermolecular hydrogen bonding increased with higher treatment temperatures suggesting that hydrogen bonds were formed due to gel formation upon cooling. The increased hydrogen bonds might be responsible for gel rigidity. Byler and Purcell (1989) observed the appearance of sharp peaks at 1614 cm"1 and 1684 cm"1 prior to gelation of B-lactoglobulin and bovine serum albumin at 80°C and 75°C, respecitively. The researchers assigned the bands to intermolecular hydrogen bonds. 109 a Pig. 7-Subtracted second dervative ”-3! absorption spectra of 1.75% S-ovalbumin in 0,0 (pD 3.0) and 25 mM EbzPO‘ after different heat treatments for 30 min. ”SO .Wavenumber (cm. ') 1680 . u m WW «39.5553 \ ocean-53¢ 1750 110 a rig. 8-Subtracted second dervative ”-1! absorption spectra of 1.75% S-ovalbumin in D20 (pD 7.0) and 25 mM EnzPO‘ after different heat treatments for 30 min. 111 a Pig. 9-Subtracted second dervative PT-IR absorption spectra of 1.75% S-ovalbumin in D20 (pD 9.0) and 25 mM kbzPO‘ after different heat treatments for 30 min. 112 As the denaturation temperature of S-ovalbumin was approached the development of structure as determined by dynamic rheological testing was reported (Herald, 1991) and an increased intensity of the peaks at 1614 cm“1 (intermolecular hydrogen bonding) and 1684 cm'1 (turn) suggest intermolecular cross-linking between the intrmolecular hydrogen bond and turn. The turn at 1684 cm’1 might be a type III which is a portion of a 310-helix (Creighton, 1984). Therefore, the turn extentsion might be the result of the the 310-helix uncoiling. S-ovalbumin, pD 3.0, heated to 65°C exhibited an increase in band intensities at 1614.4 cm"1 (intermolecular hydrogen bonding) and 1684.5 cm"1 (type III turn) compared to pD 3.0 heated to 60°C. Peaks at 1637.2 cm '1 (310- helix) and 1654.8 cm '1 (a-helix ) decreased in intensity. No B-sheet structure was observed. The spectrum of S-ovalbumin, pD 3.0, heated to 70°C exhibited an increased band intensity at 1617.3 cm'1 as compared to the 65°C treatment indicating additional intermolecular hydrogen bonding. The quantity of 1640.2 cm‘1 (310-helix) and 1658.2 cm"1 (oz-helix) decreased. The 1658.2 cm'1 peak decreased to a shoulder indicating uncoiling of helical structure. The absorption band 1684.4 cm"1 (type III turn) increased in intensity compared to 65°C. 113 The pD 3.0 S-ovalbumin spectrum at 80°C was similar to the 70°C spectrum except a more intense bonds at 1615 cm'1 (intermolecular hydrogen bonding) and 1685.5 cm.”1 (type III ‘turn) was observed. The band at 1641.7 cm'1 (310-helix) ‘was broader and the band at 1657.8 cm'1 (aéhelix) was not observed. Because of the broadness of the peak at 1641.7 cm'1 smaller peaks representing disorder structure (1645 cm’l) and a-helix (1657.8 cm’l) may be masked. The thermal denaturation temperature of S-ovalbumin as determined by DSC at pH 3.0 was 80°C (Herald, 1991). Subsequently a decline in secondary structure due to uncoiling of 310-helix and a-helix at 80°C were observed. Herald (1991) reported a storage modulus of 10 Pa (defined as rheologically significant) when S-ovalbumin at pH 3.0 was heated isothermally at 65°C for 15 min using dynamic rheological testing. Below 65°C no structure was evident. An increase in intermolecular hydrogen bonding and a decrease in a-helix at 65°C correlate with dynamic rheological testing in measuring structure formation. S-ovalbumin at pD 7.0 and heated to 80°C (Fig. 8) exhibited an increase in band intensity at 1613.4 cm'1 (intermolecular hydrogen bonding) and 1681.6 cm“1 (type III turn) at the expense of the bands at 1638.7 cm’1 (310- helix), 1655.8 cm"1 (oz-helix) and 1627.1 cm'1 (B-sheet). The decrease in band intensity indicated unfolding of the BIO-helix, c-helix and B-sheet for S-ovalbumin. A new band 114 at 1670.9 cm"1 was observed and was identified as a turn (Byler and Susi, 1988). S-ovalbumin at pD 7.0 and heated to 85°C exhibited an increase in band intensity at 1613.9 cm'1 (intermolecular hydrogen bonding) and 1682.5 cm'1 (type III turn). Compared to the 80°C S-ovalbumin treatment the band intensities at 1627.1 cml' (B-sheet), 1637.8 cm"1 (310 -helix) and 1653.8 cm'1 (a-helix) remained unchanged. S-ovalbumin at pD 7.0 and heated to 90°C showed an increased band intensity at 1614.9 cm'1 (intermolecular hydrogen bond) and 1682.5 cm'1 (type III turn) and the band at 1627.1 cm“1 (B-sheet) was not observed. A decrease in band intensity at 1639.7 cm'1 (310-helix) and 1657.7 cm'1 (a-helix) was observed compared to the 85°C treatment. A small shoulder at 1647.5 cm":l was observed and was assigned to unordered structure (Byler and Susi, 1988). Increased intermolecular hydrogen bonding and turn at 85°C correlated with rheological structure development as detected by isothermal and temperature ramping methods (Herald, 1991). Thermal denaturation temperature of S-ovalbumin as determined by DSC at pH 7.0 was 90.6°C. Uncoiling of 310-helix, c-helix, B-sheet and formation of unordered structure occurred during thermal denaturation. Intermolecular hydrogen bond formation occurred before thermal denaturation of ovalbumin at pH 7.0 as determined by DSC (Herald, 1991) suggesting structure formation occurred 115 before thermal denaturation. Herald (1991) reported that temperature in which S-ovalbumin structure was formed as measured by dynamic rheological testing occurred before thermal denaturation temperature. This is supported by FT-IR in which changes in secondary structure occurred before Td. S-ovalbumin, pD 9.0, heated to 80°C exhibited a new band at 1614.4 cm’1 associated with intermolecular hydrogen bonding (Byler and Purcell, 1989). A slight decrease was observed in the band at 1653.8 cm"1 (a-helix) and no changes in band intensities were exhibited at 1627.1 cm'1 (B-sheet), 1636.8 cm'1 (310-helix), 1670.9 cm"1 (turn) and 1680.6 cm"1 (type III turn). S-ovalbumin, pD 9.0, heated to 85°C an increase in band intensity at 1614.9 cm"1 (intermolecular hydrogen bonding increased). No other changes were exhibited between the 80°C and 85°C treatments. . S-ovalbumin at pD 9.0 and heated to 90°C exhibited conformational changes compared to the 85°C treatment. Increase in band intensity 1615.4 cm"1 (intermolecular hydrogen bonding) and 1683.5 cm'1 (type III turn) were observed. The band intensity at 1636.3 cm'1 (BIO-helix) and 1653.8 cm"1 (a-helix) decreased. The bands at 1624.6 cm"1 (B-sheet) and 1670.9 cm'1 (turn) were not present. Herald (1991) reported 8% S-ovalbumin heated at 80°C for 15 min had a G' of 25 Pa at pH 9.0 using dynamic 116 rheological testing. Both dynamic rheological testing and FT-IR support the theory that conformational changes occur before structure development takes place as indicated by increase in intermolecular hydrogen bonding and an increase in G'. Thermal denaturation temperature of S-ovalbumin as determined by DSC at pH 9.0 was 90.6°C (Herald, 1991). Therefore, uncoiling of 310-helix, a-helix and loss of B-sheet were necessary before thermal denaturation occurred. The most stable of all pD treatments observed was pD 9.0 as indicated by the amount of original structure left after heating at 90°C. At 30°C native S-ovalbumin's secondary structure did not change at pD 3.0, 7.0 or 9.0 as measured by FT-IR. Conformational changes were observed after heating suggesting pD alone does not influence secondary structure of S-ovalbumin. At pH 9.0 more 310-helix and a-helix were retained than either at pD 3.0 or pD 7.0 suggesting a more stable environment. S-ovalbumin activation energy (Ea) calculated from DSC data was less at pH 3.0 than at pH 7.0 or pH 9.0 (Herald, 1991), suggesting that energy needed to drive the reaction from the native to denatured state and temperature dependence of the reaction was over lower at pH 3.0. S-ovalbumin at pD 3.0 did not contain B-sheet at 30°C and 117 there was a decrease in intensity of 310-helix at 80°C compared at pD 7.0 or 9.0. Therefore, Ea can be related to conformational changes of S-ovalbumin due to pD. Effect of Denaturants on Secondary Structure Second derivative FT-IR spectrum of S-ovalbumin at 30°C in 2.0M GuHCl (Fig. 10) exhibited one band at 1624 cm'1 inthe Amide I region. This band was attributed to B-sheet based on model systems (Susi and Byler, 1983). One other band at 1582 cm"1 outside the Amide I region was observed. The 310- helix, a-helix and turn structure were not observed in the 2.0M GuHCl ovalbumin sample as compared to the native ovalbumin, suggesting that GuHCl disrupted most of the secondary structure of native S-ovalbumin. There was a decrease in intensity in the second derivative FT-IR spectrum of ovalbumin heated to 80°C in 2.0M GuHCl. A peak at 1628 cm'1 was exhibited in the Amide I region. Temperature influenced the S-ovalbumin FT-IR spectrum in the presence of 2.0M GuHCl. The few small peaks that are observable are believe to be incomplete H-D exchange between D20 and GuHCl. Conclusion The present study suggested that FT-IR using second derivative analysis can be used to follow changes in secondary structure of ovalbumin. Changes in ovalbumin 118 a rig. 1o-Subtracted second dervative rT-IR absorption spectra of 1.75% S-cvalbumin in D O (pD 7.0), 25 mM EDZPO‘ and 2.0M guanidine hydrochloride after di ferent heat treatments for 30 min. 118 ATEOV LonEoco>o>> camp 08' 0.5.0. 4 8: out. ,JeqwnuerM/eouquosqv 119 conformation using FT-IR occurred prior to structural changes reported using DSC and dynamic rheological testing. Unfolding of S-ovalbumin was related to thermal denaturation .temperatures and an increase in storage modulus at different isothermal temperatures. VI. STUDY 4 Effects of pH, Salt Type and Denaturants on the Microstructure of Hen Egg S-Ovalbumin as Determined by Scanning Electron Microscopy ABSTRACT Changes in microstructure of heat-induced hen egg S-ovalbumin gels influenced by pH (3.0, 7.0, 9.0), salt type (NaC1, NaI, Na2804) and denaturants (guanidine hydrochloride and B-mercaptoethanol) were studied using low temperature scanning electron microscopy (LTSEM) and chemical fixation procedures with scanning electron microscopy (SEM). The LTSEM of 8% S-ovalbumin gels were honey comb in appearance, while ovalbumin gels prepared by chemical fixation exhibited a microstructure of grape-like clusters. S-ovalbumin gels prepared at pH 3.0 or in 0.5M Na2804 exhibited the smallest pore size as viewed using LTSEM. Using SEM the largest void volumes exhibited by the S-ovalbumin gels were prepared at pH 3.0 or 0.5M NaI. 120 121 INTRODUCTION Protein-protein interactions are responsible for providing structural integrity to gel systems, while entrapping other food ingredients such as water and flavors (Arntfield et al., 1990 a). Ovalbumin is the major egg white protein and is responsible for structure development in egg containing products. Herald (1991) characterized ovalbumin properties due to heating and perturbation with pH, salt type and denaturants. The researcher reported that structure development, unfolding and secondary structure were all influenced by perturabation of environmental conditions. Scanning electron microscopy (SEM) has provided information on microstructure of heat-induced egg white gels that assist in interpreting textural characteristics of food systems (Stanely and Tung, 1976; Heertje and van Kleef, 1986). Microstructure can illustrate the effect of attractive and repulsive molecular forces on the formation of the protein gel network (Heertje and Van Kleef, 1986) by visual analysis of pore size, strand thickness and shape (Clark et al., 1981). Ferry (1948) suggested that an optimal pH balance between attractive and repulsive forces would provide a uniform gel matrix possessing high gel strength and optimum water-binding properties. Beyond optimal pH repulsive forces may be too strong resulting in 122 1980). Harte (1989) reported the balancing of attractive and repulsive forces were responsible for an optimal ovalbumin network at pH 6.0 and 7.0. Although SEM micrographs of egg white gels have been published using chemical fixation and critical point drying (CPD) little information is available on the low temperature scanning electron microscopy (LTSEM) method. The LTSEM procedure is less time-consuming, decrease induced artefacts associated with chemicals and reduces specimen shrinkage (Sargent, 1988). Freeman and Shelton (1991) and Sargent (1988) have reviewed the advantages of LTSEM for determining ultrastructure of food products. The objectives of this investigation were (1) to compare chemical fixation and low temperature methods for ovalbumin gels examined by SEM and (2) to evaluated network structure of heat-induced ovalbumin gels prepared under different conditions by varying pH, salt type, and denaturants. MATERIALS AND METHODS Materials Pure ovalbumin (grade V, lot 19F 8105) was purchased from Sigma Chemical Co.( St. Louis, MO) and used without further purification. Ovalbumin was prepared at a concentration of 80 mg/ml in the following aqueous environments (1) pH 3.0, 7.0 and 9.0 in 0.5M NaC1, (2) 0.5M 123 environments (1) pH 3.0, 7.0 and 9.0 in 0.5M NaCl, (2) 0.5M NaI, NaC1 and Na2$O4 at pH 7.0 (3) 0.5M, 1.0M and 2.0M guanidine hydrocholride (GuHCl) at pH 7.0 and 0.5M NaCl (4) 0.5%, 1.0%, 2.0% or 3.0% fl-mercaptoethanol (B-ME) at pH 7.0 and 0.5M NaCl. All samples were rechecked after 30 min to ensure pH stability. The ovalbumin solutions were deaerated prior to heating at 80°C for 30 min. The 80°C temperature was selected based on the other studies used in the dissertation. Two representative micrographs of each sample were obtained for comparison. Electron Microscopy Two scanning electron microscopy techniques were used to examine ovalbumin gel morphology. The first was LTSEM that involved examination of ovalbumin gels at low temperature and high vaccuum. The second technique involved chemical fixation in which four methods of ovalbumin gel preparation were compared (1) gluteraldehyde (2) osmuim tetroxide (3) osmium-thiocarohydrazide osmium (OTO) (4) osmium-tannic acid-uranyl acetate (OTU). LTSEM A portion of S-ovalbumin gel (less than 5 mmz) was mounted on a copper stub and submerged into a nitrogen slush maintained at -190°C to -160°C and held under vacuum of 10"2 - 10‘3 Torr (EMScope SP-ZOOO Sputter-Cryo, Ashford, Kent, 124 sample was then transferred to the preparation chamber ( < -160°C) and carefully fractured using a knife maintained at the same low temperature. The ovalbumin gel was transferred to the cold stage of the SEM (Model JSM-35C, Japan Electron Optics Limited, Tokyo, Japan) and etched for approximately 8 min at -65°C to remove the top layer of water which, may obscure surface detail. After the ovalbumin gel temperature was lowered to -165°C, it was transferred back to EMScope SP2000 Sputter-Cryo and gold sputter-coated at 40 mA for 6 min. The prepared ovalbumin gel was transferred to the cold stage of the microscope and viewed using a 15kV beam, working distance of 39 mm and condenser lens setting of 600 Chemical Fixation Methods (1) Glutaraldehyde fixation method. Portions of ovalbumin gel (5 mm x 2 mm) were cut with a razor blade and fixed for 6 hr in 2% glutaraldehyde in 0.1M Na2P04 buffer pH 6.0 (2) OSO‘jprocedure (Beveridge and Ko, 1984) ovalbumin gels were fixed in gluteraldehyde then postfixed in 1% 0804 for 2 hr at 23°C (3) 0T0 procedure (Woodward and Cotterill, 1985) Ovalbumin gels were fixed in gluteraldehyde and were post-fixed in 1% 0304 for 2 hr at 23°C. Samples were rinsed three times in deionized water (dH§O) and placed in 1% thiocarbohydrazide solution for 30 min. After rinsing six times in dHZO, samples were placed in 2% OsO4 for 1 hr 125 (4) OTU procedure (Woodward and Cotterill, 1985) S- ovalbumin gels fixed in glutaraldehyde and post-fixed in 0804 were washed in dHfiO and placed in 1% tannic acid for 1 hr at 23°C. After three rinses with dHfiO, S-ovalbumin gels were placed in 0.5% uranyl acetate for 1 hr at 23°C and then rinsed again. .Following fixation using one of the four above mentioned procedures, all samples were stored overnight in dHZO at 4°C and dehydrated sequentially in 25%, 50%, 75%, 95% and three changes of 100% ethanol (v/v) 15 min/step. Fixed S-ovalbumin gels were placed in critical point drying (CPD) baskets in 100% ethanol and then transferred immediately to the CPD chamber (Balazer FL 9496 Critical Point Dryer, Balzer's Union, Furstentum, Liechtenstein, West Germany) and dried using C02 as the transitional fluid. Following CPD with C02, the fixed and dried S-ovalbumin gels were mounted to aluminum mounts (Electron Microscopy Sciences, Ft. Washington, PA). A fine line of Conducting Graphite Paint (Ladd 60780, Burlington, VT) was applied from the edge of the sample over the edge of the metal stub to prevent charging. The mounted samples were sputter coated with gold (Emscope SC 500, Emscope, Ashord, Kent, England) under a vacuum of 0.006 Torr for 6 min at 30 mA. Dried and gold coated samples were stored in a vacuum desiccator jar. Gels were examined at 10,000 X magnification in a JEOL JSM-35 scanning electron microscope equippied with a 126 tungsten electron gun at 15 kV accelarating voltage. Working distance was 15 mm with a condenser lens of 600. Micrographs were made using Polaroid film (665P/N, Polariod Corp., Cambridge, MA). RESULTS AND DISCUSSION LTSEM At 2000 X magnification the S-ovalbumin gel network at pH 3.0, 7.0 and 9.0 exhibited a honey comb appearance (Fig. 11). The interspaces of ovalbumin gel at pH 9.0 (Fig. 11c) were 15 um compared to 10 um in diameter for pH 3.0. The large interspaces between cells might be explained by ice crystal formation (Harte, 1989). Davis and Gordon (1984) decreased the interspaces by adding 30% surcrose to a 10% collagen gel providing a cryoprotectant effect. The researchers noted that caution should be taken when evaluating gel microstructure in the presence of a cryoprotectant. Harte (1989) reported similar structure with 5% ovalbumin gels at pH 7.0. Herald (1991) reported the storage modulus (G') of S-ovalbumin decreased with increased pH suggesting that at higher pH S-ovalbumin gels were weak and that less structure development had occurred. Therefore, this accounts for a more open microstructure at pH 9.0 compare to pH 3.0. The S-ovalbumin gel prepared in 0.5M NaI (Fig. 12a) 127 a Pig. 11-The use of low temperature scanning electron microscopy in evaluating the influence of pH on the microstructure of 8% (w/v) S-ovalbumin gel in 0.5M MaCl heated at 80°C for 30 min A. pH 3.0 8. pH 7.0 C. pH 9.0. 128 a Fig. 12-The use of low temperature scanning electron microscopy in evaluating the influence of denaturants on the microstructure of 8% (w/v) S-ovalbumin gel at pH 7.0 heated at 80°C for 30 min A. 0.51! Ma! B. 0.5)! NaC1 C. 0.5! NazSO‘. 129 exhibited the same honey comb structure as 0.5M NaC1 (Fig. 11b), although the pore size was larger. The 0.5M Na2804 (Fig. 12c) S—ovalbumin gel structure was denser with smaller diameter strands than either gel prepared in 0.5M NaCl or 0.5M NaI. The microstructure suggested that the stabilizing salt (Na2804) produced a weaker protein network than the destabilizing salt types under the conditions measured. Ovalbumin gels compared at pH 7.0 in 2.0M GuHCl (Fig. 13a) or 3.0% B-ME (Fig. 14b) at 2000 X magnification exhibited similar network structure. Therefore, it was difficult to compare the effects of denaturants on the microstructure of S-ovalbumin at 2000 X magnification. Comparison of Chemical Fixation Techniques S-ovalbumin gels fixed in gluteraldehyde did not exhibit grape-like clusters as described by Clark et al. (1981) but is composed of small globular aggregates. S-ovalbumin gels prepared in glutaraldehyde appeared ruptured (Fig. 14a). This may have been due to high pressure imposed by CPD procedure that can shrink and collapse the spherical structure of the egg protein molecules (Woodward and Cotterill, 1986). The gluteraldehyde fixation procedure produced a S-ovalbumin gel which was fragile to handle therefore difficult to mount onto stubs. The 0504, OTO and OTU fixations provided good 130 a Pig. 13-The use of low temperature scanning electron microscopy in evaluating the influence of denaturants on the microstructure of 8% (w/v) S-ovalbumin gel at pH 7.0 in 0.5M NaC1 heated at 80°C for 30 min A. 2.0M guanidine hydrochloride 8. 3.0% S-mercaptoethanol. 151 a Pig. 14-Comparison of scanning electron microscopy chemical fixation techniques of 8% (w/v) S-ovalbumin gel at pH 7.0 in 0.5M NaC1 heated at 80°C for 30 min A. gluteraldehyde 8. osmium tetroxide C. osmium-thiocarbohydrasids-osmium D. osmium-tannic acid-uranyl acetate. 3.31 I... r 1. i” m 132 surface definition of the grape-like clusters (Fig. 14 b, d and e). The OTU prepared S-ovalbumin gel did not crumble as easily compared to other fixation methods (Fig. 14 e) therefore, it was the method of choice. Woodward and Cotterill (1985) reported 50% shrinkage of gluteraldehyde fixed egg white gels during CPD with pores size ranging from 0.1 to 0.2 um in diameter. Egg white gels fixed in OTU had pore size ranging from 0.1 to 0.8 um. Wollweber et al. (1981) reported treatment of peritoneal cells in OTU caused 5% shrinkage. Woodward and Cotterill (1985) attributed the enhanced structural stability of ovalbumin gel to the cross-linking of tannic acid with OsO4 and uranyl ions. Effect of pH on Microstructure Surface morphology of 8% ovalbumin in 0.5M NaCl at pH 3.0, 7.0 and 9.0 were compared (Fig. 15 a, b and c, respectively). Large irregular void areas, up to 6 um across were observed in ovalbumin gels at pH 3.0. The pI of ovalbumin is 4.5 therefore, at pH 3.0 electrostatic repulsion of the protein molecules resulted in large voids between clusters. Ovalbumin aggregates at pH 3.0 and 7.0 were approximately 0.5 um across while gel clusters at pH 9.0 were approximately 0.25 um indiameter. The large void areas exhibited in the S-ovalbumin gel at pH 3.0 were -not 133 a Pig. is-Microstructure of 8% (w/v) S-ovalbumin gel in 0.5M NaC1 heated at 80°C for 30 min prepared using the osmium- tannic acid-uranyl acetate fixation procedure A. pH 3.0 B. pH 7.0 C. pH 9.0. in in «III P. 3’0 134 found at pH 7.0 and 9.0. At pH 7.0 and 9.0 S-ovalbumin molecules were tightly clustered together leaving small irregular voids. The decrease in large void areas exhibited at pH 7.0 and 9.0 were due to the balance of attractive and repulsive forces. The storage modulus (G') at pH 3.0 was larger than at pH 7.0 or 9.0 (Herald, 1991). Indicating the area between aggregates at pH 3.0 allow for more elastic behavior than exhibited at pH 7.0 or 9.0. Woodward and Cotterill (1986) a fine structured gel at pH 9.0 in 1.0M NaC1 with numerous globules 0.1 to 1.2 um in size that were interconnected by fine protein strands and small and evenly distributed pores throughout the gel matrix. Harte (1989) described different sized grape-like clusters and void areas as non-homogeneous, in 5% ovalbumin gels at pH 6.0 and 7.0. Heertje and van Kleef (1986) suggested ovalbumin gelation at pH 5.0 occurred because of the presence of large aggregates that formed networks. Montejano et al. (1984) reported spherical particles in micrographs of low pH gels might be caused by random aggregation that resulted in non-homogenous structure. Arntfield et al. (1990b) used light microscopy to relate 10% ovalbumin gel microstructure to G'. The researcher observed that at pH 5.0 and 6.0 crosslinking was absent, therefore there was an increase in protein-solvent interaction. At pH 3.0 there was evidence of strand like structures that were not well cross-linked. Arntfield et 135 al. (1990 b) suggested that an increase in G' has been attributed to increased crosslinking with the network. Effect of Salt Type on Microstructure The ovalbumin gel prepared in NaI was more compact and had smaller grape-like clusters (0.5 um) compared to the gel prepared in NaQSO4 (1.0 um) (Fig. 16 a and b, respectively). The ovalbumin gel prepared with NaC1 contained different size clusters ranging from 0.5 to 1.0 um across (Fig. 14 c). The G'_at 80°C for 15 min NaI was larger than for NaCl and Nast4 suggesting the hydrophobic interaction was decreased causing an increased cross-linking. Woodward and Cotterill (1986) compared micrographs of egg white gels with and without salt at pH 9.0. Gels prepared in 1.0M NaC1 contained aggregated proteins, resulting in large particles that were clustered tightly together, leaving irregular voids. Gels prepared in a salt free environment were coarser and less aggregated than gels prepared in 1.0M NaCl. Hegg et al. (1979) reported thermal aggregation of 4.4% ovalbumin in a salt free environment only took place around the pI indicating the number of net charges on ovalbumin determines aggregation. Harte (1989) observed round spherical aggregates in ovalbumin gels containing 3.0% NaCl supporting the hypothesis of electrostatic interaction. Using a phosphate buffer gel system at pH 6.0, Harte (1989) 156 a Pig. 16-Microstructure of 8% (w/v) S-ovalbumin gel at pH 7.0 heated at 80°C for 30 min prepared using the osmium-tannic acid-uranyl acetate fixation procedure A. 0.5M III a. 0.511 mazso‘. 137 observered no difference between ultrastructure of 5.0% ovalbumin in 3.0% NaCl and without salt using OTO, CPD procedure. Hegg et al. (1979) and Hatt et al. (1986) reported the addition of 170 mM NaCl shielded repulsive charges, thus promoting protein aggregation. Arntfield et a1. (1990 c) reported that 10% ovablumin gel at pH 8.5 in 0.5M NaC1 formed well cross-linked strands whereas ovalbumin gels at pH 8.5 in 0.5M Nast4 exhibited little evidence of cross-linking when observed using a light microscopy. The authors reported the relationship between the storage modulus and the position of a salt in the lytropoic series at a concentration of 0.5M provided evidence for the involvement of hydrophobic interactions as factors in determining the strength of heat-induced ovalbumin networks. Effect of Guanidine Hydrochloride on Microstructure Ovalbumin gels prepared in 0.5M and 1.0M GuHCl (Fig. 17a) exhibited grape-like aggregates of approximately 1 um in diameter. The ovalbumin gel microstructure was composed of compact aggregates with irregular interstitial spaces. The 2.0M GuHCl ovalbumin gel was amorphous in structure (Fig. 17b) probably due to unfolding of ovalbumin molecules due to disruption of hydrogen bonds. The amorphous structure exhibited the ability of ovalbumin to 138 a Pig. 17-Microstructure of 8% (w/v) S-ovalbumin gel at pH 7.0 in 0.5M NaC1 heated at 80°C for 30 min prepared using the osmium-tannic acid-uranyl acetate fixation procedure A. 1.0M guanidine hydrochloride S. 2.0)! guanidine hydrochloride. 139 cross-link and increase the G' of the ovalbumin gel compared to lower GuHCl concentrations (Herald, 1991). Amphorous microstructure due to GuHCl has not been reported before in literature. Heertje and van Kleef (1986) investigated the effects of urea at pH 10 (that was attributed to disruption of non-covalent bonds) and found an amphorous, homogeneous and non-cellular structure. Effect of B-Mercaptoethanol on Microstructure S-ovalbumin gels prepared in 1.0% and 2.0% B-ME (Fig. 18a and b) were similar to each other in grape-like aggregate structure (approximately 1.2 um across) and irregular void area. The control had a denser microstructure than all B-ME containing treatments. The 3.0% ME (Fig. 18c) treatment was similar to 2.0M GuHCl in that an amorphous structure rather than the grape-like cluster structures were formed. Herald (1991) reported that as B-ME concentration increased G' for S-ovalbumin increased, however changes in surface morphology was exhibited only at 3.0% B-ME. Conclusion Changes in S-ovalbumin gel pores sizes and strand diameter were related to gel structure. A smaller pore size and larger diameter strands were associated with a higher storage modulus and a lower thermal denaturation temperature. 140 a Pig. 18-Microstructure of 8% (w/v) S-ovalbumin gel at pH 7.0 in 0.5M Neel heated at 80°C for 30 min prepared using the csmium-tannic acid-uranyl acetate fixation procedure A. 1.0% S-mercaptoethancl B. 2.0% B-mercaptoethanol C. 3.0% S- mercaptoethanol a. 0.514 Nazso‘. ;1 1.: lg tit . 1.0% at l- 141 VII. SUMMARY AND CONCLUSIONS Study 1. This study used DSC and dynamic rheological testing to characterize ovalbumin gelation as a function of pH and salt type. At pH 3.0, thermal denaturation and structure develoment temperatures were reduced compared to pH 7.0 or pH 9.0. Below the pI the thermal denaturation temperature declined due to electrostatic replusion of the ovalbumin molecules causing unfolding at a lower temperature. At pH 3.0 there might be some aggregation, therefore decreasing the temperature of structure development. Denaturation enthalpy determined by DSC may be influenced by the high concentration of ovalbumin (8.0%) used. As a decrease in enthalpy may be the result of S-ovalbumin aggregation. Inclusion of 0.5M NaI to 8% ovalbumin at pH 7.0 decreased thermal denaturation and structure development temperatures. The decrease in thermal stability suggest a reduction in water structure and an increase in exposed hydrophobic residues that were able to cross-link. The results suggest that pH and salt type are a significant factor in the thermal behavior of ovalbumin as measured by DSC and dynamic rheological testing. By manipulating pH and salt type a desired textural characteristics can be obtained under specific conditions of processing. r—‘1_ I 142 This study used DSC and dynamic rheological testing to characterize thermal behavior of ovalbumin as a function of GuHCl and B-ME concentration. Increasing concentrations of either denaturant decreased the thermal stability of 8% (w/v) S-ovalbumin at pH 7.0 in 0.5M NaC1 by either disrupting hydrogen bonds or reducing the disulfide bond. Using denaturants helped to provide information on 5 structural changes occurring during thermal processing and their influence on the storage modulus or denaturation temperature. Study III. This study used second derivative FTIR spectra to monitor changes in S-ovalbumin solution as a function of p0 and temperature. Three bands and 2 shoulders representing B-sheet, 310-helix, a-helix and turn were observed at 30°C. Type III turn and intermolecular hydrogen bonding increased at the expense of both helicies and B-sheet. Conformational changes in S-ovalbumin such as a decrease in 310-helix and an increase in type III turn related to denaturation temperatures and temperatures of structure formation as measured by DSC and dynamic rheological testing, respectively. 143 Using FT-IR to monitor secondary structure has provided an addition tool to the already established DSC and dynamic rheological testing methods. Research can now be done to relate specific conformations to functinal attributes that will aid food processor during manufacturing. Study IV. This study showed that LTSEM micrographs of S-ovalbumin gels exhibited honey comb structures compared to grape-like cluster shown for the chemical fixation procedures. LTSEM preparation and viewing of specimens was faster than the chemical fixation methods. A trend was observed when comparing the LTSEM method to the different S-ovalbumin gel treatments at 80°C. When the S-ovalbumin gel microstructure exhibited thick strands and small pore sizes the G' was greater as compared to thin strands and large pore sizes. The thin strands and large pore sizes were weaker due to the more heat stable environment provide by pH 9.0 or NaZSO4. The OTU method was not as conclusive in relating S-ovalbumin microstructure to G' due to similar grape like cluster sizes and void areas. Microscopy contributed physical meaning to ovalbumin gelation. However, more work must be done in order to understand the effects of fixation procedures on gel microstructure. 144 VIII. RECOMMENDATION FOR FUTURE RESEARCH Study the influence of prosthetic groups on ovalbumin secondary structure. Monitor interaction between the prosthetic group and polypeptide chain such as: isolate each ovalbumin variant (A1, A2 and A3) and compare secondary structure. remove mannose groups one at a time and compare secondary structure. Use modeling and FT-IR to determine if further differences between ovalbumin and S-ovalbumin can be discerned. Compare structure-function relationship between ovalbumin and other members of the serpin family. Use N-ethlylmaleimide (NEM) to block S-S formation during heating and monitor conformational change. Determine if partial or total loss of S-S formation has any significant influence on functionality (solubility or texture) and relate to changes in secondary structure. Study the effects of protein concentration on activation energy and reaction order and note if differences can be observed between aggregation versus denaturation. Study the effect of frozen storage of ovalbumin on structure function relationship. Does low temperature 145 change ovalbumin conformation that correlates to specific protein functions within food systems such as texture or solubility. Elucidate the presence of a BIO-helix or B-sheet at 1638 cm."1 by determining the number of psi, phi and omega angels of the 3-dimensional ovalbumin structure. LIST OF REFERENCES 146 I! REFERENCES Acharya, K.R., Stuart, D.I., Walker, N.P.C., Lewis, M. and Phillips, D.C. 1989. Refine structure of baboon a-lactablumin at 1.7 A resolution: Comparison with C- type lysozyme. J. Mol. Biol. 208: 99-127. Adler, A., Greenfield, N. and Fasman, G. 1973. Circular dichroism and optical rotary dispersion of proteins and polypeptides. Meth. Enzymol. 27: 675-735. Ansari, R., Aftab, B., Ahmad, R. and Salahuddin, A. 1972. The native and denatured states of ovalbumin. Biochem. J. 126: 447-448. Ahmad, F. and Salahuddin, A. 1974. Influence of temperature on the intrinsic viscosities of proteins in random coil conformation. Biochemistry 13 (2): 245-249. AOAC. 1980. "Official Methods of Analysis," 13‘”h ed. Association of Official Analytical Chemists, Washington, DC. Arntfield, S.D. and Murray, E.D. 1981. The influence of processing parameters on food protein functionality. I. Differential scanning calorimetry as an indicator of protein denaturation. Can Inst. Food Sci. Technol. J. 14: 289-294. Arntfield, S.D., Murray, E.D. and Ismond, M.A. 1986. Effect of salt on the thermal stability of storage proteins from fababean (Vicia faba). J. Food Sci. 51: 371-377. Arntfield, S.D., Murray, E.D., Ismond, M.A. and Bernatsky, A. 1989. Role of the thermal denaturation-aggregation relationship in determining the rheological properties of heat induced networks for ovalbumin and vicilin. J. Food Sci. 54: 1624-1631. Arntfield, S.D., Murray, E.D. and Ismond, M.A. 1990 a. Influence of salts on the microstructural and rheological properties of heat-induced protein networks from ovalbumin and vicilin. J. Agric. Food Chem. 38: 1335-1343. 147 Arntfield, S.D., Murray, E.D. and Ismond, M.A. 1990 b. Dependence of thermal properties as well as network microstructure and rheology on protein concentration for ovalbumin and vicilin. J. Texture Studies 21: 191-212. Arntfield, S.D., Murray, E.D. and Ismond, M.A. 1990 c. Influence of protein charge on the thermal properties as well as microstructure and rheology of heat induced networks for ovalbumin and vicilin. J. Texture Studies 21: 295-322. Babajimopoulos, M., Damodaran, S., Rizvi, S. and Kinsella, J. 1983. Effects of various anions on the rheological and gelling behavior of soy proteins: Thermodynamic observations. J. Agric. Food Chem. 31: 1270-1275. Bertazzon, A. and Tsong, T.Y. 1990 a. Effects of ions and pH on thermal stability of thin and thick filaments of skeletal muscle: High-sensitivity differential scanning calorimetric study. Biochemistry 29: 6447- 6452. Bertazzon, A. and Tsong, T.Y. 1990 b. Study of effects of pH on the stability of domains in myosin rod by high- resolution differential scanning calorimetry. Biochemistry 29: 6453-6459. Bertazzon, A., Tian, G., Lamblin, A. and Tsong, T. 1990 c. Enthalpic and entropic contribution to actin stability: Calorimetry, circular dichroism, and fluorescence study and effects of calcium. Biochemistry 29: 291-298. Beveridge, T., Arntfield, 8. K0, S. and Chung, J. 1980. Firmness of heat induced albumen coagulum. Poult. Sci. 59: 1229-1236. Beveridge, T., Jones, L. and Tung, M. 1984 a. Progel and gel formation and reversibility of gelation of whey, soybean and albumen protein gels. J. Agric. Food Chem. 32: 307-313. Beveridge, T. and Ko, S. 1984 b. Firmness of heat-induced whole egg coagulum. Poultry Sci. 63: 1372-1377. Beveridge, T. and Timbers, G. 1985a. Small amplitude oscillatory testing (SOAT). Instrumentation development and application to coagulation of egg albumen, whey protein concentrate and beef wiener emulsion. J. Texture Studies. 16: 333-343. 148 Beveridge, T., Arntfield, S. and Murray, E. 1985b. Protein structure development in relation to denaturation temperatures. Can. Inst. Food Sci. Technol. J. 18: 189-191. Bismuto, E and Irace, G. 1988. Protein conformational changes induced by guanidine at predenaturational concentrations. Int. J. Peptide Protein Res. 32: 321- 325. Bull, H. B. and Breeze, K. 1970. Water and solute binding by proteins. I. Electrolytes. Arch. Biochem. Biophys. 137: 299-305. Byler, D. and Susi, H. 1986. Examination of the secondary structure of proteins by deconvolved FT-IR spectra. Biopolymers 25: 469-487. Byler, D.M. and Susi, H. 1988. Application of computerized infrared and Raman spectroscopy to conformation studies of casein and other food proteins. J. Industr. Microbiol. 3: 73-88. Byler, D.M. and Purcell, J.M. 1989. FT-IR examination of thermal denaturation and gel-formation in whey proteins. SPIE 1145: 415-417. Cann, J.R. 1949. Electrophoretic analysis of ovalbumin. J. Am. Chem Soc. 71: 907-909 Catsimpoolas, N. and Meyer, E.W. 1970. Gelation phenomena of soybean globulins. 1. Protein-protein interactions. Cereal Chem. 47: 559-570. Chan, C., Wh, C. and Yang, J. 1978. Circular dichroic analysis of protein conformation:Inclusion of the B- turns. Anal. Biochem. 91: 13-31. Cheftel, J., Cuq, J. and Lorient, D. 1985. Amino acids, proteins and polypeptides. In "Food Chemistry," 2nd Fennema, 0. (Ed.), Marcel Dekker Inc., NY. Chen, R., Ker, Y. and Wu, C. 1990. Temperature and shear rate affecting the viscosity and secondary structural changes of soy 11$ globulin measured by a cone-plate viscometer and Fourier transform infrared spectroscopy. Agric. Biol. Chem. 54: 1165-1176. 149 Chin, J., Edward, K,. Jung, V., and Chan, Y. 1987. Structural basis of human erythrocyte glucose transporter function in proteoliposome vesicles: Circular dichrosim measurements. Proc. Natl. Acad. Sci. 84: 4113-4116. Cho, K.H. 1970. The conformation and denaturation of ovalbumin. Ph.D. dissertation, Princeton Univ., Princeton, NJ. Clark, A.H., Judge, F.J., Richards, J.B., Stubbs J. and Suggett, A. 1981 a. Electron microscopy of network structures in thermally-induced globular protein gels. Int. J. Peptide Protein Res. 17: 380-392. Clark, A.H., Saunderson, D.H. and Suggett, A. 1981 b. Infrared and laser-Raman spectroscopic studies of thermally-induced globular protein gels. Int. J. Peptide Protein Res. 17: 353-364. Clark, A.H. and Lee-Tuffnell, C.D. 1986. Gelation of globular proteins. In "Functional Properties of Food Macromolecules, " Mitchell, J. and Ledward, D. (Eds.) Elsevier Applied Science Publishers, NY. Covington, A.K., Paabo, M., Robinson, R.A. and Bates, R.G. 1968. Use of the glass electrode in deuterium oxide and the relation between the standardized pD (pad) scale and the operational pH in heavy water. Anal. Chem. 40: 700-706. Creighton, T. 1984. Proteins:Structures and molecular Properties. Freeman and Company, NY. Damodaran, S. and Kinsella, J. 1982. Effects of ions on protein conformation and functionality. In "Food Proteins Deterioration. Mechanisms and Functionality," ACS Symposium Series No. 206, ACS, Washington, DC. Damodaran, S. 1988. Refolding of thermally unfolded soy proteins during the cooling regime of the gelation process: Effect on gelation. J. Agric. Food Chem. 36: 262-269. Dannenberg, F. and Kessler, H. 1988. Reaction kinetics of the denaturation of whey proteins in milk. J. Food Sci. 53: 258-263. Davis, E.A. and Gordon, J. 1984. Microstructural analysis of gelling systems. Food Technol. 37 (5): 99-106, 109. 150 de Wit, J.N. and Swinkels, G. 1980. A differential scanning calorimetric study of the thermal denaturation of bovine B-lactoglobulin: Thermal behaviour at temperatures up to 100°C. Biochim. Biophy. Acta 624: 40-50. de Wit, J.N. 1981 a. Structure and functional behaviour of whey proteins. Neth. Milk Dairy J. 35: 47-64. de Wit, J.N. 1981 b. A differential scanning calorimetric study of the thermal behaviour of bovine B- lactoglobulin at temperatures up to 160°C. J. Dairy Res. 48: 293-302. de Wit, J.N. and Klarenbeek, G. 1981. A differential scanning calorimetric study of the thermal behaviour of bovine B-lactoglobulin at temperatures up to 160°C. J. Dairy Res. 48: 293-302. Delben, F. and Crescenzi, V. 1969. Thermal denaturation of lysozyme. A differential scanning calorimetry investigation. Biochim. Biophys. Acta 194: 615-618. Delben, F., Crescenzi, V. and Quadrifoglio, F. 1969. A study of the thermal denaturation of ribonuclease by differential scanning calorimetry. Intern. J. Protein Res. 1: 145-149. Doi, E., Koseki, T. and Kitabatake, N. 1987. Effects of limited proteolysis on functional properties of ovalbumin. JAOCS 64:1697-1703. Dong, A., Huang, P. and Caughey, W.S. 1990. Protein secondary structures in water from second-derivative Amide I infrared spectra. Biochem. 29: 3303-3308. Donovan, J.W., Mapes, C.J., Davis, J.G. and Garibaldi, J. A. 1975. A differential scanning calorimetric study of the stability of egg white to heat denaturation. J. Sci. Food Agric. 26: 73-83. Donovan, J.W. and Mapes, C.J. 1976. A differential scanning calorimetric study of conversion of ovalbumin to S-ovalbumin in eggs. J. Sci. Food Agric. 27: 197-204. Donovan, J.W. 1984. Emerging techniques: Scanning calorimetry of complex biological structures. T188 9: 340-344. 151 Dwek, R.A. and Navon, G. 1972. On boiling an egg. Nature 240: 491. Eisensmith, S. 1989. Plotit and integrated graph-statistical package. Scientific Programming Enterpresis. Haslett, MI. Egelandsdal, B. 1980. Heat-induced gelling in solutions of ovalbumin. J. Food Sci. 45: 570-575. Egelandsdal, B. 1984. A comparison between ovalbumin gels formed by heat and by guanidinium hydrochloride denaturation. J. Food Sci. 49: 1099-1102. Egelandsdal, B. 1986. Conformation and structure of mildly heat-treated ovalbumin in dilute solutions and gel formation at higher protein concentrations. Int. J. Peptide Protein Res. 28: 560-568. Feeney, R. 1964. Egg proteins. In "Symposium of Foods: Proteins and Their Reactions,” Schultz, H. and Anglemier, A. (Eds.), AVI Publishing Co., Westport, CT. Freeman, T. and Shelton, D. 1991. Microstructure of wheat starch: Form Kernel to bread. Food Technol. 3: 164-168. Fernandez-Diez, J., Osuga, D. and Feeney, R. 1964. The sulfhydryls of avian ovalbumins, bovine B-lactoglobulin and bovine serum albumin. Arch. Biochem. Biophys. 107: 448-458. Ferry, J. D. 1948. Protein gels. Adv. Protein Chem. 4: 1-79. Ferry, J. D. 1970. Viscoelastic properties of polymers 2nd ed. Academic press; New York. Ferry, J. D. 1980. Viscoelastic properties of polymers 3rd ed. Academic press; NY. Froning, G. Nutritional and functional properties of egg proteins. In "Development in Food Proteins-6," Hudson, B. (Ed.), Applied Science Publishers, NJ. Fujita, Y. and Noda, Y. 1981. The effect of hydration on the thermal stability of ovalbumin as measured by means of differential scanning calorimetry. Bull. Chem. Soc. Jpn. 54: 3233-3234. Goldsmith, S.M. and Toledo, R.T. 1985. Studies on egg albumin gelation using nuclear magnetic resonance. J. Food Sci. 50: 59-62. 152 Gordon, J. 1972. Denaturation of globular proteins. Interaction of guanidinium salts with three proteins. Biochemistry 11: 1862-1870. Gossett, P.W. Rizvi, S.S.H. and Baker, R.C. 1984. Quantitative analysis of gelation in egg protein systems. Food Technol. 5: 67-74, 96. Hagerdal, B. and Martens, H. 1976. Influence of water content on the stability of myoglobin to heat treatment. J. Food Sci. 41: 933-937. Halloway, P. and Mantsch, H.H. 1989. Structure of cytochrome b5 in solution by Fourier-transform infrared spectroscopy. Biochem. 28: 931-935. Harte, J. 1989. Ovalbumin thermal gelation: Prediction of gel strength as influenced by selected factors. Ph. D. dissertation, Michigan State Univ., East Lansing, MI. Harwalkar, V.R. 1979. Kinetics of thermal denaturation of B- lactoglobulin at pH 2.5. J. Dairy Sci. 63: 1052-1057. Harwalkar, V.R. 1980. Measurement of thermal denaturation of B-lactoglobulin at pH 2.5. J. Dairy Sci. 63:1043-1051. Hatta, H., Kitabatake, N. and Doi, E. 1986. Turbidity and hardness of a heat-induced gel of hen egg ovalbumin. Agric. Biol. Chem. 50: 2083-2089. Havel, H., Chao, R., Haskell, R. and Thamann, T. 1989. Investigations of protein structure with optical spectroscopy:Bovine growth hormone, Anal. Chem. 61: 642-650. Hayakawa, S. and Nakai, S. 1985. Contribution of hydrophobicity, net charge and sulfhydryl groups to thermal properties of ovalbumin. Can. Inst. Food Sci. Technol. J. 18(4): 290-295. Hayakawa, S. and Nakamura, R. 1986. Optimization approaches to thermally induced egg white lysozyme gel. Agric. 8161. Chem. 50: 2039-2046. Heertje, I. and van Kleef, F. 1986. Observations on the microstructure and rheology of ovalbumin gels. Food Microstructure 5: 91-98. 153 Hegg, P. O., Martens, H. and Lofqvist, B. 1979. Effects of pH and neutral salts on the formation and quality of ovalbumin. A study on thermal aggregation and denaturation. J. Sci. Food Agric. 30: 981-993. Herald, T.J. 1987. Rheological and functional properties of pasteurized liquid whole egg during frozen storage. M. S. thesis, Michigan State Univ., East Lansing, MI. Herald, T.J. 1991. Effect of pH, salt type and denaturants on the denaturation properties, storage modulus, secondary structure and microstructure of hen egg 8- ovalbumin heat-induced gels. Ph.D. dissertation, Michigan State Univ., East Lansing, MI. Hermansson, A. M. 1979. Aggregation and denaturation involved in gel formation. In "Functionality and Protein Structure,: A. Pour-El, ed,. ACS Symp. Series 92, Am. Chem. Soc., Washington, DC. Hermansson, A.M. and Lucisano, M. 1982. Gel characteristics-waterbinding properties blood plasma gels and methodological aspects on the waterbinding of gel systems. J. Food Sci. 47: 1955-1960. Hillier, R., Lyster, R. and Cheeseman, G. 1980. Gelation of reconstituted whey powders by heat. J. Sci. Food Agric. 31: 1152-1157. Hirose, M., Oe, H. and Doi, E. 1986. Thiol-dependent gelation of egg white. Agric. Biol. Chem. 50(1): 59-64. Holt, D.L., Watson, M.A., Dill, C.W, Alford, E.S, Edwards, R.L, Diehl, K.C and Garnder, F.A. 1984. Correlation of the rheological behavior of egg albumen to temperature, pH, and NaCl concentration. J. Food Sci. 49: 137-141. Ismond, M.A., Murray, E.D. and Arntfield, S.D. 1986. The role of non-covalent forces in micelle formation by vicilin from Vicia faba. II. The effect of stabilizing and destabilizing anions on protein interactions. Food Chem. 21: 27-46. Johnson, C. 1988. Secondary structure of proteins through circular dichrosim spectroscopy. Am. Rev. Biophys. Chem. 17: 145-166. Kakalis, L. and Regenstein, J. 1986. Effect of pH and salts on the solubility of egg white protein. J. Food Sci. 51: 1445-1447, 1455. 154 Kato, A. Tsutsui, N., Matsudomi, N., Kobayashi, K. and Kakai, S. 1981. Effects of partial denaturation on surface properties of ovalbumin and lysozyme. J. Biol. Chem. 45: 2755-2760. Kato, A., Nagase, Y., Matsudomi, N. and Kobayaski, K. 1983. Determination of molecular weight of soluble ovalbumin aggregates during heat denaturation using low angle laser light scattering technique. Agric. Biol. Chem. 47: 1829-1834. Kato, A. and Takagi, T. 1988. Formation of intermolecular B-sheet structure during heat denaturation of ovalbumin. J. Agric. Food Chem. 36: 1156-1159. Kato, A., Tanaka, A., Matsudomi, N. and Kobayashi, K. 1986. Deamidation of ovalbumin during S-ovalbumin conversion. Agric. Biol. Chem. 50: 2375-2382. Katsuta, K. and Kinsella, J.E. 1990. Spontaneous gelation of whey proteins in urea and guanidine hydrochloride. Agric. Biol. Chem. 54 (9): 2423-2424. Kauzmann, W. 1959. Some factors in the interpretation of protein denaturation. Adv. Protein Chem. 14: 1-63. Kauzmann, W. 1987. Thermodynamics of unfolding. Nature. 325: 763-765. Kilara, A. and Sharkasi, T. 1986. Effects of temperature on food proteins and its implications on functional properties. CRC Crit. Rev. Food Sci. Nutri. 23: 323-395. Kint, S. and Tomimatsu, Y. 1979. A Raman difference spectroscopic investigation of ovalbumin and S- ovalbumin. Biopolymers 18: 1073-1079. Kitabatake, N., Tahara, M. and Doi, E. 1989. Denaturation temperature of soy protein under low moisture conditions. Agric. Biol. Chem. 53: 1201-1202. Kitabatake, N., Tahara, M. and Doi, E. 1990. Thermal denaturation of soybean protein at low water contents. Agric. Biol. Chem. 54: 2205-2212. Kohnhorst, A.L. and Mangion, M.E. 1985. Prediction of the strength of whey protein gels based on composition. J. Food Sci. 50: 1403-1405. 155 Kokini, J.L. 1991. Starch structure and rheology. Paper 19, presented at 54th Annual Meeting of Inst. of Food Technologists, Dallas, TX, June 1-5. Koseki, T, Kitabatake, N. and Doi, E. 1989. Irreversible thermal denaturation and formation of linear aggregates of ovalbumin. Food Hydro. 3: 135-148. Koseki, T., Kitabatake, N. and Doi, E. 1990. Freezing denaturation of ovalbumin at acid pH. J. Biochem. 107: 389-394. Krimm, S. and Bandekar, J. 1986. Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. Adv. Protein Chem. 38: 183-364. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227:680-685. Lee, Y. and Montogomery, R. 1962. Glycoproteins from ovalbumin: The structure of the peptide chains. Arch. Biochem. Biophys. 97: 9-13. Lee, Y., Wu, Y. and Montgomery, R. 1964. Modification of emulsion action on asparaginyl-carbohydrate from ovablumin by dinitrophenylation. Biochem. J. 91: 9-15. Levitt, M and Greer, J. 1977. Automatic identification of secondary structure of globular proteins. J. Mol. Biol. 114: 181-239. Lewis, D. F. 1979. Meat products. In "Food Microscopy". J. G. Vaughan (Ed.), Academic Press, London. Li-Chan, E. and Nakai, S. 1989. Biochemical basis for the properties of egg white. Crit. Rev. Poultry Biol. 2 (1): 21-58. Longsworth, L.G. Cannon, R.K. and MacInnes, D.A. 1940. An electrohoretic study of the proteins of egg white. J. Luescher, M., Ruegg, M. and Schindler, P. 1974. Effect of hydration upon the thermal stability of tropocollagen and its dependence on the presence of neutral salts. Biopoly. 13: 2489-2503. Ma, C. and Holmes, J. 1982. Effect of chemical modification of some physiochemical properties and heat coagulation of egg albumen. J. Food Sci. 47: 1454-1458. 156 Ma, C., Khanzada, G. and Harwalkar, V. 1988. Thermal gelation of oat globulin. J. Agric. Food Chem. 36: 275-280. Matsudomi, N., Rector, D. and Kinsella, J. 1991. Gelation of bovine serum albumin and B-lactoglobulin;Effects of pH, salts and thiol reagents. Food Chem. 40: 55-69. Melander, W. and Horvath, C. 1977. Salt effects on hydrophobic interactions in precipitation and chromatography of proteins:An interpretation of the , lyotropic series. Arch. Biochem. Biophys. 183: 200-215. Montejano, J., Hamann, 0., Ball, H. and Lanier, T. 1984. Thermally induced gelation of native and modified egg white-rheological changes during processing; final ( strengths and microstructures. J. Food Sci. 49: 1249- 1257. Montogomery, R. Lee, Y. and wu, Y. 1965. Glycopeptides from ovalbumin. Biochem. 4: 1454-1459. MSTAT. 1985. Statistical package for agronomic research. Michigan State University, East Lansing, MI. Mulvihill, D. and Kinsella, J. 1988. Gelation of B- lactoglobulin:Effects of sodium chloride and calcium chloride on the rheological and structural properties of gels. J. Food Sci. 53: 231-236. Nakamura, R., Sugiyama, H. and Sato, Y. 1978. Factors contributing to heat-induced aggregation of ovalbumin. Agric. Biol. Chem. 42: 819-824. Nakamura, R., Harai, M. and Takemori, Y. 1980. Some differences noted between the properties of ovalbumin and S-ovalbumin in native state. Agric. Biol. Chem. 44: 149-153. Nandi, P. and Robinson, D. 1972. THe effects of salts on free energies of nonpolar groups in model polypeptides. J. Am. Chem. Soc. 94: 1308-1315. Neucere, J.N. and Cherry, J.P. 1982. Structural changes and metabolism of proteins following heat-denaturation. In "Food Protein Deterioration. Mechanisms and Functionality" Comstock, J. (Ed.) ACS Symposium Series. Am. Chem. Soc. New York, NY. Ngo, W. and Taranto, M. 1986. Effect of surcrose level on the rheological properties of cake batters. Cereal Foods World 31: 317-322. 157 Nisbet, A., Saundry, R., Moir, A., Fothergill, L. and Fothergill, J. 1981. The complete amino acid sequence of hen ovalbumin. Eur. J. Biochem. 115: 335-345. Oakenfull, D. 1987. Gelling Agents. CRC Crit. Rev. Food Sci. and Nutri. 26: 1-25. Olinger, J.M., Hill, D.M., Jakobsen, R.J. and Brody, R.S. 1986. Fourier transform infrared studies of ribonuclease in HfiO and 020. solutions. Biochim. Biophys. Acta 869: 89-98. Pace, C. N. 1975. The stability of globular proteins. CRC Crit. Rev. Biochem. 1-43. Painter, P.C. and Koenig, J.L. 1976. Raman Spectroscopic study of the proteins of egg white. Biopoly. 15: 2155-2166. Papiz, M.z., Sawyer, L., Eliopoulos, E.E., North, A.C.T., Findlay, J.B.C., Sivaprasadarao, R., Jones, T.A., Newcomer, M.E. and Kraulis, P.J. 1986. The structure of B-lactoglobulin and its similarity to plasma retinal-binding protein. Nature 324: 383-385. Park, H.K. and Lund, D.B. 1984. Calorimetric study of thermal denaturation of B-lactoglobulin. J. Dariy Sci. 67:1699-1706. Patel, M.T., Kilara, A. Huffman, L.M., Hewitt, S.A. and Houlihan A.V. 1990. Studies on whey protein concentrates. 1. Compositional and thermal properties. J. Dairy Sci. 73: 1439-1449. Perlmann, G.E. 1952. Enzymatic dephosphorylation of ovalbumin and plakalbumin. J. Gen. Physiol. 25: 711- 726. Pfeil, W. and Privalov, P.L. 1976. Thermodynamic investigations of proteins. II. Biophys. Chem. 4: 33 Powrie, W. and Nakai, S. 1986. The chemistry of eggs and egg products. In "Egg Science and Technology," 3 ed. Stadelman, W.J. and Cotterill, O.J. (Eds.). AVI Publishing Co., Inc., Westport, CT. Preston, K. 1989. Effects of neutral salts of the lyotropic series on the physical dough properties of a Canadian red spring wheat flour. Cereal Chem. 66: 144-148. 158 Prestrelski, S.J., Byler, D.M. and Liebman M.N. 1991 a. Comparison of various molecular forms of bovine trypsin:Correlation of infrared spectra with X-ray crystal structures. Biochem. In press. Prestrelski, S. J., Byler, D. M and Thompson, M. P. 1991 b. Spectroscopic discrimination of alpha- and 31 helices in globular proteins: Assignment of Amide I infrared bands to secondary structures in a-lactalbumin. Int. J. of Peptide and Protein Res. 30: 0000. Privalov, P.L. and Khechinashvili, N.N. 1974. A thermodynamic approach to the problem of stabilization of globular protein structure:A calorimetric study. J. Mol. Biol. 86: 665-684. Privalov, P.L. 1979. Stability of proteins:small globular proteins. Adv. Protein Chem. 33: 67-236. Richardson, J. 1981. The anatomy and taxonomy of protein structure. Adv. Protein Chem. 34: 167-363. Ruegg, M., Moor, U. and Blanc, B. 1977. A calorimetric study of the thermal denaturation of whey proteins in simulated milk ultrafiltrate. J. Dairy Res. 44: 509-520. Sargent, J. 1988. The application of cold stage scanning electron microscopy to food research. Food Microstruct. 7: 123-135. Savitzky, A. and Golay, J.E. 1964. Smoothing and differentiation of data by simplified least square procedure. Anal. Chem. 36: 1628-1639. Seideman, W., Cotterill, O. and Funk, E. 1963. Factors affecting heat coagulation of egg white. Poultry Sci. 42: 406-417. Shimada, L. and Matsushita, S. 1980a. Thermal coagulation of egg albumen. J. Agric. Food Chem. 28:409-412. Shimada, L. and Matsushita, S. 1980b. Relationship between thermocoagulation of proteins and amino acid composition. J. Agric. Food Chem. 28: 413-417. Shimada, L. and Matsushita, S. 1981. Effects of salts and denaturants on thermocoagulation of proteins. J. Agric. Food Chem. 29: 15-20. 159 Shimada, K. and Cheftel, J.C. 1988. Texture characteristics, protein solubility, and sulfydryl group/disulfide bond content of heat-induced gels of whey protein isolate. J. Agric. Food Chem. 36: 1018-1025. Shitamori, S., Kojima, E. and Nakamura, R. 1984. Changes in the heat-induced gelling properties of ovalbumin during its conversion to S-ovalbumin. Agric. Biol. Chem. 48: 1539-1543. Sichina, W. 1988. Application of the time-temperature superposition principle In "Guide to the use of the 983 DMA" DuPont Instrument Division, Wilmington, DE. Siemon, J., Meier, J., and Rudd, G. 1986. Development of creep resistant elastomeric mounts, Elastomerics 26: 23-27. Skoog, D. 1985. Thermal Methods. In "Principles of Instrumental Analysis 3rd ed.," D. Skoog (Ed.), Saunders College Publishing, NY. Slosberg, H., Hannson, H., Stewart, G. and LOwe, B. 1948. Factors influencing the effects of heat treatment on the leavening power of egg white. Poultry Sci. 27: 294-301. Smith, M. 1964. Studies on ovalbumin I. Denaturation byheat and the heterogeneity of ovalbumin. Aust. J. Biol. Sci. 17: 261-266. Smith, M. and Back, J. 1965. Studies on ovalbumin. II. The formation and properties of S-ovalbumin, a more stable form of ovalbumin. Aust. J. Biol. Sci. 18: 365-377. Smith, M. and Back, J. 1968. Studies on ovalbumin. IV. Trypsin digestion and the cystine peptides of ovalbumin and S-ovalbumin. Aust. J. Biol. Sci. 21: 549-556. Sone, T., Dosako, S. and Kimura, T. 1983. Microstructure of protein gels in relation to their rheological properties. In "Instrumental Analysis of Foods. Vol. 2." Charalambous, G. and Inglett, G. (Eds.) Academic Press, Inc., New York. Stanley, D. and Tung, M. 1976. Microstructure of food and its relation to texture. In "Rheology and Texture in Food Quality," de Mann, J. Voisey, P., Rasper, V. and Stanley, 0., (Eds.). Avi Publ. Co., Inc., Westport, CT. 160 Stein, E.P., Leslie A.G., Finch, J.T. Turnell W.G., McLaughlin, P.J. and Carrell, R.W. 1990. Crystal structure of ovalbumin as a model for the reactive centre of serpins. Nature 347: 99-102. Strambini, G. and Gonnelli, M. 1986. Effects of urea and guanidine hydrochloride on the activity and dynamical structure of equine liver alcohol dehydrogenase. Biochem. 25: 2471-2476. Suresh Chandra, B.R., Appu Rao, A.G., Narasinga Rao, M.S. 1984. Effect of temperature on the conformation of soybean glycinin in BM urea or 6M guanidine hydrochloride solution. J. Agric. Food Chem. 32: 1402- 1405. Surewicz, W.K. and Mantsch, H.H. 1988. New insight into protein secondary structure from resolution-enhanced infrared spectra. Biochim. Biophys. Acta 952: 115-130. Susi, H., Timasheff, S. and Stevens, L. 1967. Infrared spectra and protein conformations in aqueous solutions. I. The amide I band in H20 and D20 solutions. J. Biol. Chem. 242: 5460-5466. Susi, H. 1969. Infrared spectra of biological macromolecules In "Structure and Stability of Biological Macromolecules." Marcel Dekker, NY. Susi, H. and Byler, D. 1983. Protein structure by Fourier transform infrared spectroscopy:Second derivative spectra. Biochem. Biophys. Res.Comm. 115: 391-397. Susi, H. and Byler, D. 1986. Resolution-enhanced Fourier transform infrared spectroscopy of enzymes. Meth. Enzymol. 130: 290-309. Susi, H. and Byler, M.D. 1988. Fourier transform infrared spectroscopy in protein conformation studies. In ”Methods for Protein Analysis", Cherry, J.P. and Barford, R. A. (Eds.) American oil Chemists' Society, Champaign, IL. Tanford, C., Kawahora, K. and Lapanje, S. 1966. Proteins in 6M guanidine hydrochloride. Demonstration of random coil behavior. J. Biol. Chem. 241: 1921-1923. Tanford, C. 1968. Protein denaturation. Adv. Prot. Chem. 23: 121-282. 161 Tanford, C. 1979. Interfacial free energy and the hydrophobic effect. Proc. Natl. Acad. Sci. 76: 4175- 4176. Timasheff, S. Susi, H, and Stevens, L. 1967. Infrared spectra and protein conformations in aqueous solutions, II. Survey of globular proteins. J. Biol. Chem. 242: 5467-5473. Tolstoguzov, V., Braudo, E. and Gurov, A. 1981. On protein functional properties and the methods of their control. Part I. On the concept of protein functional properties. Nahrung 25: 232-239. Tombs, M.P. 1974. Gelation of globular proteins. Disc. Faraday Chem. Soc. 57: 158-164. Tung, M. 1978. Rheology of protein dispersions. J. Texture studies 9: 3-31. van Kleef, F.S.M., Boskamp, J.V. and Van Den Tempel, M. 1978. Determination of the number of cross-links in a protein gel from its mechanical and swelling properties. Biopolymers 17: 225-235. van Kleef, F.S.M. 1986. Thermally induced protein gelation. A study on the gelation and rheological characterization of highly concentrated ovablumin and soybean protein gels. Biopolymers 25: 31-59. von Hippel, P. and Schleich, T. 1969. The effects of neutral salts on the structure and conformational stability of macromolecules in solution. In "Structure and Stability of Biological Macromolecules," Timasheff, S., Fasman, G., (Eds.), Marcel Dekker, NY. Wollweber, L., Stracke, R. and Gothe, U. 1981. The use of a simple method to avoid cell shrinkage during SEM preparation. J. Microscopy 121: 185-189. Woodward, S.A. and Cotterill, O.J. 1985. Preparation of cooked egg white, egg yolk, and whole egg gels for scanning electron microscopy. J. Food Sci. 50: 1624-1628. Woodward, S.A. and Cotterill, O.J. 1986. Texture and microstructure of heat-formed egg white gels. J. Food Sci. 52: 333-339. 162 Wright, D. 1982. Application of scanning calorimetry to the study of protein behaviour in foods. In "Developments in Food Proteins-1," Hudson, B. J. (Ed.), Applied Science Publishers, NJ. Wright, H.T., Qian, H.K. and Huber, R. 1990. Crystal structure of plakalbumin, a proteolytically nicked form of ovalbumin: Its relationship to the structure of cleaved a-l-proteinase inhibitor. J. Mol. Biol. 213: 513-528. Xiong, Y. and Kinsella, J.E. 1990. Evidence of urea-induced sulfhydryl oxidation reaction in proteins. Agric. Biol. Chem. 54 (8): 2157-2159. Yang, J., Wu, C. and Martinez, H. 1986. Calculations of protein conformation from circular dichoism. Meth. Enzymol. 130: 208-269. Zarins, Z. and Marshall, W. 1990. Thermal denaturation of soy glycinin in the presence of 2-mercaptoethanol studied by differential scanning calorimetry. Cereal Chem. 67(1): 35-38. Zirbel, F. and Kinsella, J. 1989. Effects of thiol reagents and ethanol whey protein gels. Food Hydrocolloids. 2: 467-475. TATE UNIV. LIBRARIES MICHIGAN s IIHIHNIHHIIW I 3129 30090 1 | WI 2255 11111