BIOPOLYMERS
VOL. 15, 2155-2166 (1976)
Raman Spectroscopic Study of the Proteins of Egg White P. C. PAINTER and J. L. KOENIG, Department of Macromolecular Science, Case Western Reserve University, Cleveland, Ohio 44106
Synopsis The Raman spectra of ovalbumin, ovomucoid, and conalbumin are reported. Spectral shifts in the conformationallysensitive amide I and amide I11 lines as a result of thermal denaturation indicate the formation of intermolecular 0-sheets. A medium intensity line a t 1260 cm-' in the spectra of ovomucoid and ribonuclease is demonstrated to contain a substantial contribution from tyrosine residues.
INTRODUCTION Egg white is a key ingredient in many food products principally because of its ability to coagulate upon heating. I t is a predominantly protein system with only a trace of lipid and carbohydrate; the protein composition is listed in Table I.' Only two of these proteins, ovalbumin and conalbumin, are heat coagulable but together they constitute almost 70% of the protein content (by weight).' The major protein of egg white is ovalbumin. This was one of the first proteins to be obtained in pure form and as such has been extensively studied.2 However, most of these studies have utilized ovalbumin as a model protein in, for instance, denaturation experiments and, considering the vast literature concerning this protein, relatively little is known about its structure and function. Even less is known about the conformation of most of the other proteins of egg white, the exception being lysozyme where the full three-dimensional structure has been determined by X-ray crystallography. In this paper we will present the results of our application of Raman spectroscopy to a study of the conformation of the three principal proteins of egg white, ovalbumin, conalbumin, and ovomucoid. In view of the importance of these proteins to the food industry, we also studied the conformational changes that accompany the thermal denaturation and aggregation process (coagulation). In this respect, Raman spectroscopy has a unique advantage over other spectroscopic techniques in t h a t both solution and solid samples can be studied directly. We will not discuss in detail the literature concerning these proteins, but refer to recent review p a p e r ~ . 2 - ~However, we will briefly describe some aspects of their structure t h a t are relevant to this investigation. 2155 (c:
1976 by John Wiley & Sons, Inc.
2156
PAINTER A N D KOENIG TABLE I Protein Composition of Egg White % (approx) of Egg
White Solids Ovalbumin Conalbumin Ovomucoid Lysozyme Ovomucin Flavoprotein Ovoinhibitor Avidin Unidentified proteins
54 13 11 3.5
1.5 0.8
0.1 0.05 8
Ovalbumin is a glycoprotein, carbohydrate being present to the extent of 3.2%. Its molecular weight is approximately 45,000 and it is a compact, roughly spherical molecule. There are four thiol groups and one disulphide bridge.5 ORD studies have indicated that there may be 25% a-helix and 25% &sheet present.6 One of the more interesting properties of this protein is its ability to be irreversibly converted to a more stable form, known as S-ovalbumin, in solutions of pH 9-10.7 S-ovalbumin is highly resistant to the thermal aggregation process that gives ovalbumin its intrinsic value as a food ingredient.8 This conversion to a more stable form also occurs in uiuo during the storage of eggs and is therefore also significant from a commercial point of view. It was originally thought that a shift in the position of the disulphide bond might be responsible for the difference in properties, but a comparison of the soluble peptides obtained from tryptic digests did not reveal any significant changes in the peptides linked by this bond.g Conalbumin is an iron binding protein of approximately 87,000 molecular weight. ORD studies have indicated a helical content ranging from 5 to 31%.1° Results obtained for ovomucoid were more consistent indicating the presence of approximately 22% a-helix.'o Ovomucoid is a glycoprotein that inhibits the action of trypsin and does not coagulate upon heating; however, its anti-tryptic activity is destroyed.*
EXPERIMENTAL Samples of ovalbumin were obtained from Schwarz-Mann, Orangeburg, N.Y. Conalbumin (iron free) and ovombcoid were obtained from Sigma Chemical Company, St. Louis, Mo. The Raman instrumentation and sampling techniques have been described previous1y.l' All protein solutions were run at 10%w/w in 0.05 M NaCl solutions. S-ovalbumin was prepared from ovalbumin by the method of Smith and Back.7 Laser power was 350 mW for solutions, 200 mW for solid samples. The scan speed was 0.5 cm-l/sec a t a time constant of 4
sec.
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RESULTS AND DISCUSSION Ovalbumin and S-Ovalbumin The Raman spectrum of ovalbumin in aqueous solution is shown in Figure 1. Four lines are resolved in the conformationally sensitive amide I11 region of the spectrum. The strongest of these lines is observed at 1247 cm-I and the other three are resolved as shoulders at 1255; 1235, and 1276 cm-l. Weak lines near 1270 cm-' have been unambiguously assigned to a helical structures in the spectra of highly ordered po1ypeptides.l' However, in the spectra of the highly a-helical proteins tropomyosin,12bovine serum a l b ~ m i n , ' ~and . ' ~ a-keratin15 the amide I11 region is chacterized by weak broad scattering and no easily assigned lines are separately resolvable. Consequently, the assignment of the lines observed at 1270 and 1274 cm-l to the a-helical in the spectra of carboxypeptidase A16 and a-la~talbuminl~ regions of these proteins should not be regarded as certain, particularly as these proteins have less than 20% a-helical structure compared to 90% for tropomyosin. Furthermore, we will demonstrate below that a line near 1280 cm-' in the spectrum of S-ovalbumin remains unshifted upon full hydrogen-deuterium exchange of the protein; consequently this line is not due to an amide I11 vibrational mode. Polypeptides and proteins in the antiparallel @-sheetconformation are characterized by intense amide I11 lines between 1230 and 1240 cm-1.11J8J9 The weak line at 1235 cm-' in the spectrum of ovalbumin therefore indicates the presence of a small amount of @-sheetstructure. Medium-intensity amide I11 lines between 1243 and 1249 cm-l are characteristic of the spectra of most denatured and neutral solutions of the polyelectrolytes poly(L-lysine)and poly(L-glutamic acid).18
1500
1000
Fig. 1. Raman spectrum of 10% (w/w) aqueous solution of ovalbumin.
5 0 0 cm-1
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PAINTER A N D KOENIG
Such lines can therefore be assigned to an extended conformation. Even though this conformation is usually considered “disordered,” there is some evidence to support the hypothesis of regions of local order in the polyelectrolytes,21and until this controversy is resolved the terms “random coil” and “disordered” should be used with circumspection. Equal care should be used in applying these terms to the regions of proteins that have no regularly folded structure. The conformation of a protein is a product of millions of years of evolution that has resulted in a close structure/function relationship; the conformation may be irregular but it is certainly not “random.” The Raman spectra of those proteins considered to have no regular ordered stuctures such as a-helices and P-sheets, have been reported.12.22 In contrast to most extended, denatured proteins the amide I11 lines are all found at 1254 cm-I, although the relative intensities and broadness of these lines are comparable to the former. Consequently, the line observed at 1255 cm-l in the spectrum of ovalbumin can be assigned to the “irregular” regions of the polypeptide chain. However, it would be anomalous to assign the 1247-cm-l line to an extended, possibly disordered conformation. Studies using a range of technique^^-^ have established that ovalbumin is a spherical, compact molecule. We suggest that this line may indicate the presence of some parallel P-sheet structure. The 0-sheet regions of carboxypeptidase A are disordered to an extent that there is a substantial parallel arrangement of chains. This protein has an amide I11 line a t 1247 cm-’ that has been assigned to the parallel P-sheet regions,16 and it is therefore possible that such a parallel arrangement of chains may be present in the ovalbumin molecule. The amide I mode is not as useful as the amide I11 for the investigation of protein structure, principally because it is not resolved into lines that can be assigned to the various types of secondary structure that may be present. In the Raman spectrum of ovalbumin, the arnide I is observed at 1665 cm-1, and this would indicate that this protein has more irregularly folded structure than a-helices or P-sheets.12,22 As ovalbumin has only one cystine disulfide bridge, the characteristic line near 500 cm-l is too weak to observe. The spectrum of S-ovalbumin was found to be identical to that of ovalbumin, supporting the proposition that the differences in the properties of these two forms of the protein are not due to any major difference in secondary s t r u ~ t u r e . ~ - ~
Ovomucoid The glycoprotein ovomucoid has a carbohydrate content of 10-15%.4 The carbohydrate moiety is thought to be predominantly glucosamine. Figure 2 shows the Raman spectrum of this protein and the lines observed a t 1328,1111,945, and 513 cm-l are characteristic of the glucose ring.
RAMAN SPECTRA OF EGG-WHITE PROTEIN
2159
Two lines are observed in the amide I11 region of the spectrum: at 1266 and 1248 cm-l. Medium-intensity lines near 1260 cm-l in the spectra of ribonuclease16 and g l ~ c a g o have n ~ ~ been assigned to the a-helical regions of these proteins. However, as we pointed out in our discussion of the spectrum of ovalbumin, such assignments of weak- or medium-intensity lines to a-helical structures are dubious unless the protein is highly ahelical. This is certainly not the case for ribonuclease, which has only 15% a-helical structure. The superposition spectra of r i b o n ~ c l e a s indicate e~~ that lines near 1260 cm-’ could arise from tyrosine ring vibrations. One way to check this is to deuterate the protein fully whereupon the amide I11 lines shift to near 950 cm-l. Unfortunately, D20 solutions have a strong line near 1210 cm-l. This difficulty can be circumvented by exchanging the amide hydrogens of a solid sample in a saturated D20 atmosphere. A further difficulty is then to force complete hydrogen-deuterium exchange, as most proteins have “hard to exchange” amide hydr0gens.~5 In solution this can often be accomplished by heating. Our experimental approach was therefore to heat D20 solutions, freeze dry the product, and obtain the Raman spectrum with the sample exposed to a saturated D20 vapor in a sealed cell. Unfortunately, our samples of ovomucoid discolored slightly on heating. No such experimental difficulties were encountered with ribonuclease and we used this protein to establish the origin of the 1260-cm-I line. Two samples of ribonuclease in D20 solutions (5%w/w) were heated to 60°C for 1and 3 hr, respectively, lyophilized, and redissolved in D20 (5%w/w). The infrared spectra of these samples 1hr after dissolution are shown in Figure 3, compared to an untreated sample of ribonuclease 24 hr after dissolution. Only the sample heated for 3 hr had no residual amide I1 absorption near
J 1500
KKK)
Fig. 2. Raman spectrum of 1Wo (w/w)aqueous solution of ovomucoid.
UK)
cm-1
2160
PAINTER AND KOENIG
1550 cm-', demonstrating complete hydrogen-deuterium exchange. Freeze-dried portions of this sample were placed in a sealed optical cell containing a beaker of D20 to produce a saturated DzO atmosphere. The Raman spectrum was taken after 6 hr and is shown compared to that obtained from an unexchanged sample in Figure 4. The absence of any lines
v
1640
1800
1600
1400 CM'l
Fig. 3. Infrared spectrum of ribonuclease in DzO (5%w/w): curve A-after 24 hr; curve B-heated 1 hr 6OoC in D20, lyophilized, redissolved in DzO, spectrum obtained 1 hr after dissolution; curve C-heated 3 hr 6OoC in DzO, spectrum obtained 1 hr after dissolution.
''
1400
1200
Fig. 4. Raman spectrum of lyophilized ribonuclease: curve A-HzO B-fully exchanged sample, DzO sample.
atmosphere; curve
RAMAN SPECTRA OF EGG-WHITE PROTEIN
2161
in the fundamental N-H stretching region of the spectrum near 3300 cm-I is additional evidence for complete exchange. However, in the spectrum of this fully exchanged sample, the line a t 1260 cm-l is still evident. Consequently, this line is probably not an amide I11 vibrational mode, but more likely a tyrosine ring vibrational mode. We therefore infer that the line a t 1266 cm-I in the spectrum of ovomucoid can also be assigned t o tyrosine residues. This interpretation of the origin of lines near 1260 cm-I is not in agreement with the earlier interpretation of Yu and co-workers.16,23,26 T h e Raman spectrum of crystalline g l ~ c a g o nclearly ~ ~ demonstrates a weakto medium-intensity line a t 1266 cm-I, which was assigned t o the a-helical regions of this hormone. However, a shoulder a t this frequency is clearly discernible in the spectrum obtained from aqueous solutions, when the polypeptide chain is “disordered,” and also in the spectrum of the @sheet gel. Glucagon has two tyrosines out of a total of 29 amino-acid residues, a ratio comparable to ribonuclease. Further difficulties in interpretation arise when we consider the original amino-acid superposition spectrum of ribonuclease reported by Lord and Yu. The intensity of the tyrosine line near 1260 cm-’ is weaker than the line observed in our fully exchanged sample. However, it should be noted that the superposition spectrum was obtained a t p H 1.0. Yu and co-workers have demonstrated considerable changes in the relative intensities of lines due t o tyrosine between ribonuclease samples in the solid state and in solution a t various pH’s. Consequently, the interpretation of changes in this region of the spectrum pre-
B
i 1100
1000
Fig. 5. Raman spectrum of lyophilized conalbumin: curve A-native coagulated sample.
CM”
state; curve B
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PAINTER AND KOENIG
sents an acute problem for the spectroscopist. Most spectral charges observed upon dissolution of lyophilized samples of proteins have been interpreted in terms of conformational change. However, it is well known that the substitution of the stronger water-protein for interpeptide hydrogen bonds has a considerable effect on the electronic spectra of biopolymers27and such changes in electronic properties would also be expected to affect the Raman spectrum. In general, such factors have been neglected in interpreting spectral changes and these arguments indicate that more research is still required in order to decide these questions. This is demonstrated in the spectrum of fully exchanged ribonuclease where there is much larger contribution from tyrosine residues to the intensity of the line observed near 1260 cm-’ than previously considered. The amide I and I11 lines at 1665 and 1248 cm-l are characteristic of “irregular” structure12,22and these results indicate that this protein has little “ordered” secondary structure. This is in substantial agreement with the results of ORD studies, which indicated that this protein consists of “irregular” structure and a-helices only,l0with the proportion of the latter conformation being approximately 20%.
Conalbumin The Raman spectrum of conalbumin is shown in Figure 5 together with the spectrum of the thermally denatured protein. This latter spectrum will be discussed later. The amide I11 region of the native conalbumin
UOO
lo00
Fig. 6. Raman spectrum of whole egg white, lyophilized: curve A-native B-coagulated sample.
600 CM-’
state, curve
RAMAN SPECTRA OF EGG-WHITE PROTEIN
2163
spectrum is weak, broad, and structureless, making it very similar to the amide I11 of bovine serum albumin and a-keratin.’S--15 Both these proteins have 50% or more a-helical structure and we infer the presence of similar extensive a-helical regions in conalbumin, substantially more than the 5-31% range indicated by ORD studies.1° The amide I line a t 1667 cm-l is characteristic of the “irregular” regions of the protein polypeptide chain. Conalbumin is a poor Raman scatterer and difficulties were encountered in obtaining the spectrum. Consequently, the low signal-to-noise ratio renders a detailed discussion of the other lines observed in the spectrum of limited value.
The Thermal Denaturation of the Proteins of Egg White The Raman spectra of whole egg white (lyophilized) compared to the insoluble coagulum formed by heating a sample a t 70°C for 10 min is shown in Figure 6. For experimental purposes, this latter sample was lyophilized. The spectrum of whole egg white is almost identical to that of ovalbumin, which is not surprising in that this protein comprises 54%,by weight, of the system’ and that the other major protein (conalbumin) is a poor Raman scatterer. Upon thermal denaturation, an intense amide I11 line a t 1236 cm-’ becomes apparent and the amide I is shifted from 1667 to 1672 cm-’. These spectral changes demonstrate the formation of extensive regions of antiparallel 0-sheet between ovalbumin molecule^.^ I t has been proposed that interchain disulphide bonds play a n important role in the coagulation p r o ~ e s s . ~However, -~ aggregation still occurs in the presence of cysteine and p -chloromercuribenzoate28 and we observed no lines due 1~15,18319
--
3300
1200
900 cm”
,
Fig. 7. Raman spectrum: top-ovalbumin coagulated by heating D20 solution of 1 hr, aggregate lyophilized; bottom-S-ovalbumin, heating in D20 solution for 1 hr, clear solution lyophilized. Both spectra obtained in saturated D20 atmosphere.
PAINTER AND KOENIG
2164
to formation of new S-S bridges appearing near 500 cm-'. We conclude that the formation of stable intermolecular @-sheetstructures is of central importance in the thermal denaturation and aggregation of egg white. Ovalbumin demonstrates identical spectral changes to whole egg white upon coagulation. Similar changes are observed in the spectra of conalbumin upon thermal denaturation. The amide I line shifts to 1672 cm-1 and an intense amide I11 line at 1239 cm-l appears, demonstrating the formation of intermolecular @-sheetstructures. The ease with which ovalbumin aggregates upon heating has made it difficult to determine the extent of unfolding, it any, prior to c o a g ~ l a t i o n . ~ ~ The difficulties are experimental in that the presence of aggregated species in solutions renders the interpretation of ORD data,29or the obtaining of Raman spectra, difficult. However, it is possible to use Raman spectroscopy to gain some insight into this question by utilizing the capability of obtaining spectra in both solid and solution form and by comparing the extent of hydrogen-deuterium exchange of heated samples of ovalbumin and S-ovalbumin. Solutions of this latter form of the protein do not exhibit aggregation upon heating. A sample of S-ovalbumin was heated to 7OoC slowly over the course of 1hr. The clear solution was lyophilized and the Raman spectrum obtained after 3 hr exposure to a saturated D2O vapor in a sealed optical cell. A solution of ovalbumin heated in the same fashion
Fort
HEAT
PARTIAL UNFOLDING
OVALBUMIN; NATIVE STATE
INTERMOLECULAR p SHEETS
-
. HEAT
5- OVALBUMIN
RESTRICTED UNFOLDING
Fig. 8. Proposed thermal denaturation mechanism for ovalbumin.
RAMAN SPECTRA OF EGG-WHITE PROTEIN
2165
displayed considerable aggregation. T h e aggregate was separated by centrifugation and lyophilized and the Raman spectrum was obtained under identical experimental conditions to the sample of S-ovalbumin. The two spectra are compared in Figure 7. T h e absence of any scattering in the fundamental N-H stretching region of the spectrum of S-ovalbumin demonstrates that the protein had completely exchanged. However, the coagulated ovalbumin sample has weak residual scattering near 3300 cm-' and an unshifted amide I11 line a t 1254 cm-I is also observed. These results suggest that unfolding occurs in S-ovalbumin upon heating solutions of the protein. This unfolding is sufficient t o allow the exchange of amide hydrogens in the hydrophobic core but restricted in scope, probably by intramolecular crosslinks, so that aggregation does not occur. In contrast, the incomplete extent of hydrogen-deuterium exchange of ovalbumin indicates that only partial unfolding occurs before aggregation. However, the region or regions of the polypeptide chain that do unfold probably do so in an unrestricted fashion, so that intermolecular forces become important and the P-sheets are formed. This thermal denaturation mechanism is illustrated in Figure 8.
CONCLUSIONS There are three sets of conclusions resulting from this work, relating to the structure of the major proteins of egg white, the conformational changes that occur as a result of thermal denaturation and aggregation, and finally the origin of the medium-intensity line near 1260 cm-* in the Raman spectra of some proteins. The conformation of the ovalbumin polypeptide chain is predominantly irregular, though small amounts of P-sheet, some of which may be parallel rather than antiparallel, are also present. Ovomucoid has little ordered secondary structure but conalbumin has extensive amounts of a-helix. Upon thermal denaturation, both ovalbumin and conalbumin form intermolecular P-sheets. Ovalbumin only partially unfolds prior t o the formation of these intermolecular structures. Hydrogen-deuterium exchange studies demonstrated t h a t the line observed near 1260 cm-' in some proteins has a considerably larger contribution from a tyrosine ring vibration than previously considered.
References 1. Parkinson, T. L. (1966), J . Sci.Food Agric. 17,101-111. 2. Taborsky, T. (1974) Aduan. Protein Chem. 28,l-210. 3. Marshall, R. D. (1972) Ann. Reu. Biochem. 41,673-702. 4. Fevold, H. L. (1951) Aduan. Protein Chem. 6,188-252. 5. Fothergill, (1970) Riochem. J . 116,555-561. 6. Cho, K. H., Ph.D. dissertation Univ. Microfilms, Ann Arbor, Mich., Order 7019,772. 7. Smith, M. B. & Back, J. F. (1965), Aust. J . Biol. Sci. 18,365-377.
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8. Smith, M. B. & Back, J. F. (1968) Aust. J.Biol. Sci. 21,539-548. 9. Smith, M. B. & Back, J. F. (1968) Aust. J.Biol. Sci. 21,549-558. 10. Tomimatsu, Y. & Gaffield, W. (1965) Biopolymers 3,509-517. 11. Frushour, B. G. & Koenig, J. L. (1974) Biopolymers 13,455-474. 12. Frushour, B. G. & Koenig, J. L. (1974)Biopolymers 13,1809-1819. 13. Bellocq, A. M., Lord, R. C. & Mendelsohn, R. (1972) Biochem. Biophys. Acta 257, 280-287. 14. Lin. V. & Koenig, J. L. (1976) Biopolymers 15,203-218. 15. Lin, V. & Koenig, J. L. (1976) Textile Rs. J. (submitted). 16. Yu. N. T. & Jo, B. H. (1973) J . Amer. Chem. SOC.95,5033-5037. 17. Yu, N. T. (1974) J. Amer. Chem. Soc. 96,4664-4668. 18. Yu, T. J., Lippert, J. L. & Peticolas, W. L. (1973) Biopolymers 12,2161-2176. 19. Frushour, B. G. & Koenig, J. L. (1975) Biopolymers 14,649-662. 20. Chen, M. C., Lord, R. C. & Mendelsohn, R. (1974) J. Amer. Chem. SOC.9 6 , 3 0 3 s 3042. 21. Tiffany, M. L. & Krimm, S. (1969) Biopolymers 8,347-359. 22. Frushour, B. G. & Koenig, J. L. (1975) Biopolymers 14,379-391. 23. Yu. N. T. & Liu, C. S. (1972)J. Amer. Chem. SOC.94,5127-5128. 24. Lord, R. C. & Yu, N. T. (1970) J. Mol. Biol. 51,203-213. 25. Willunsen, L. (1971) Ct. R. Trau. Lab. Carlsberg 38,223-295. 26. Yu. N. T., Jo, B. H. & Liu, C. S. (1972) J. Amer. Chem. SOC.94,7572-7575. 27. Tinoco, I., Halpern, A. & Simpson, W. T. (1962) in Polyamino Acids Polypeptides and Proteins, Stahmann, M. A., Ed., pp. 147-160. 28. McKenzie, H. A., Smith, M. B. & Wake, R. G. (1963) Biochim. Biophys. Acta 69, 222-239. 29. Holme, J. (1963) J. Phys. Chern. 67,782-788.
Received August 14,1975 Accepted September 29,1975
VOL. 15, 2155-2166 (1976)
Raman Spectroscopic Study of the Proteins of Egg White P. C. PAINTER and J. L. KOENIG, Department of Macromolecular Science, Case Western Reserve University, Cleveland, Ohio 44106
Synopsis The Raman spectra of ovalbumin, ovomucoid, and conalbumin are reported. Spectral shifts in the conformationallysensitive amide I and amide I11 lines as a result of thermal denaturation indicate the formation of intermolecular 0-sheets. A medium intensity line a t 1260 cm-' in the spectra of ovomucoid and ribonuclease is demonstrated to contain a substantial contribution from tyrosine residues.
INTRODUCTION Egg white is a key ingredient in many food products principally because of its ability to coagulate upon heating. I t is a predominantly protein system with only a trace of lipid and carbohydrate; the protein composition is listed in Table I.' Only two of these proteins, ovalbumin and conalbumin, are heat coagulable but together they constitute almost 70% of the protein content (by weight).' The major protein of egg white is ovalbumin. This was one of the first proteins to be obtained in pure form and as such has been extensively studied.2 However, most of these studies have utilized ovalbumin as a model protein in, for instance, denaturation experiments and, considering the vast literature concerning this protein, relatively little is known about its structure and function. Even less is known about the conformation of most of the other proteins of egg white, the exception being lysozyme where the full three-dimensional structure has been determined by X-ray crystallography. In this paper we will present the results of our application of Raman spectroscopy to a study of the conformation of the three principal proteins of egg white, ovalbumin, conalbumin, and ovomucoid. In view of the importance of these proteins to the food industry, we also studied the conformational changes that accompany the thermal denaturation and aggregation process (coagulation). In this respect, Raman spectroscopy has a unique advantage over other spectroscopic techniques in t h a t both solution and solid samples can be studied directly. We will not discuss in detail the literature concerning these proteins, but refer to recent review p a p e r ~ . 2 - ~However, we will briefly describe some aspects of their structure t h a t are relevant to this investigation. 2155 (c:
1976 by John Wiley & Sons, Inc.
2156
PAINTER A N D KOENIG TABLE I Protein Composition of Egg White % (approx) of Egg
White Solids Ovalbumin Conalbumin Ovomucoid Lysozyme Ovomucin Flavoprotein Ovoinhibitor Avidin Unidentified proteins
54 13 11 3.5
1.5 0.8
0.1 0.05 8
Ovalbumin is a glycoprotein, carbohydrate being present to the extent of 3.2%. Its molecular weight is approximately 45,000 and it is a compact, roughly spherical molecule. There are four thiol groups and one disulphide bridge.5 ORD studies have indicated that there may be 25% a-helix and 25% &sheet present.6 One of the more interesting properties of this protein is its ability to be irreversibly converted to a more stable form, known as S-ovalbumin, in solutions of pH 9-10.7 S-ovalbumin is highly resistant to the thermal aggregation process that gives ovalbumin its intrinsic value as a food ingredient.8 This conversion to a more stable form also occurs in uiuo during the storage of eggs and is therefore also significant from a commercial point of view. It was originally thought that a shift in the position of the disulphide bond might be responsible for the difference in properties, but a comparison of the soluble peptides obtained from tryptic digests did not reveal any significant changes in the peptides linked by this bond.g Conalbumin is an iron binding protein of approximately 87,000 molecular weight. ORD studies have indicated a helical content ranging from 5 to 31%.1° Results obtained for ovomucoid were more consistent indicating the presence of approximately 22% a-helix.'o Ovomucoid is a glycoprotein that inhibits the action of trypsin and does not coagulate upon heating; however, its anti-tryptic activity is destroyed.*
EXPERIMENTAL Samples of ovalbumin were obtained from Schwarz-Mann, Orangeburg, N.Y. Conalbumin (iron free) and ovombcoid were obtained from Sigma Chemical Company, St. Louis, Mo. The Raman instrumentation and sampling techniques have been described previous1y.l' All protein solutions were run at 10%w/w in 0.05 M NaCl solutions. S-ovalbumin was prepared from ovalbumin by the method of Smith and Back.7 Laser power was 350 mW for solutions, 200 mW for solid samples. The scan speed was 0.5 cm-l/sec a t a time constant of 4
sec.
RAMAN SPECTRA OF EGG-WHITE P R O T E I N
2157
RESULTS AND DISCUSSION Ovalbumin and S-Ovalbumin The Raman spectrum of ovalbumin in aqueous solution is shown in Figure 1. Four lines are resolved in the conformationally sensitive amide I11 region of the spectrum. The strongest of these lines is observed at 1247 cm-I and the other three are resolved as shoulders at 1255; 1235, and 1276 cm-l. Weak lines near 1270 cm-' have been unambiguously assigned to a helical structures in the spectra of highly ordered po1ypeptides.l' However, in the spectra of the highly a-helical proteins tropomyosin,12bovine serum a l b ~ m i n , ' ~and . ' ~ a-keratin15 the amide I11 region is chacterized by weak broad scattering and no easily assigned lines are separately resolvable. Consequently, the assignment of the lines observed at 1270 and 1274 cm-l to the a-helical in the spectra of carboxypeptidase A16 and a-la~talbuminl~ regions of these proteins should not be regarded as certain, particularly as these proteins have less than 20% a-helical structure compared to 90% for tropomyosin. Furthermore, we will demonstrate below that a line near 1280 cm-' in the spectrum of S-ovalbumin remains unshifted upon full hydrogen-deuterium exchange of the protein; consequently this line is not due to an amide I11 vibrational mode. Polypeptides and proteins in the antiparallel @-sheetconformation are characterized by intense amide I11 lines between 1230 and 1240 cm-1.11J8J9 The weak line at 1235 cm-' in the spectrum of ovalbumin therefore indicates the presence of a small amount of @-sheetstructure. Medium-intensity amide I11 lines between 1243 and 1249 cm-l are characteristic of the spectra of most denatured and neutral solutions of the polyelectrolytes poly(L-lysine)and poly(L-glutamic acid).18
1500
1000
Fig. 1. Raman spectrum of 10% (w/w) aqueous solution of ovalbumin.
5 0 0 cm-1
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PAINTER A N D KOENIG
Such lines can therefore be assigned to an extended conformation. Even though this conformation is usually considered “disordered,” there is some evidence to support the hypothesis of regions of local order in the polyelectrolytes,21and until this controversy is resolved the terms “random coil” and “disordered” should be used with circumspection. Equal care should be used in applying these terms to the regions of proteins that have no regularly folded structure. The conformation of a protein is a product of millions of years of evolution that has resulted in a close structure/function relationship; the conformation may be irregular but it is certainly not “random.” The Raman spectra of those proteins considered to have no regular ordered stuctures such as a-helices and P-sheets, have been reported.12.22 In contrast to most extended, denatured proteins the amide I11 lines are all found at 1254 cm-I, although the relative intensities and broadness of these lines are comparable to the former. Consequently, the line observed at 1255 cm-l in the spectrum of ovalbumin can be assigned to the “irregular” regions of the polypeptide chain. However, it would be anomalous to assign the 1247-cm-l line to an extended, possibly disordered conformation. Studies using a range of technique^^-^ have established that ovalbumin is a spherical, compact molecule. We suggest that this line may indicate the presence of some parallel P-sheet structure. The 0-sheet regions of carboxypeptidase A are disordered to an extent that there is a substantial parallel arrangement of chains. This protein has an amide I11 line a t 1247 cm-’ that has been assigned to the parallel P-sheet regions,16 and it is therefore possible that such a parallel arrangement of chains may be present in the ovalbumin molecule. The amide I mode is not as useful as the amide I11 for the investigation of protein structure, principally because it is not resolved into lines that can be assigned to the various types of secondary structure that may be present. In the Raman spectrum of ovalbumin, the arnide I is observed at 1665 cm-1, and this would indicate that this protein has more irregularly folded structure than a-helices or P-sheets.12,22 As ovalbumin has only one cystine disulfide bridge, the characteristic line near 500 cm-l is too weak to observe. The spectrum of S-ovalbumin was found to be identical to that of ovalbumin, supporting the proposition that the differences in the properties of these two forms of the protein are not due to any major difference in secondary s t r u ~ t u r e . ~ - ~
Ovomucoid The glycoprotein ovomucoid has a carbohydrate content of 10-15%.4 The carbohydrate moiety is thought to be predominantly glucosamine. Figure 2 shows the Raman spectrum of this protein and the lines observed a t 1328,1111,945, and 513 cm-l are characteristic of the glucose ring.
RAMAN SPECTRA OF EGG-WHITE PROTEIN
2159
Two lines are observed in the amide I11 region of the spectrum: at 1266 and 1248 cm-l. Medium-intensity lines near 1260 cm-l in the spectra of ribonuclease16 and g l ~ c a g o have n ~ ~ been assigned to the a-helical regions of these proteins. However, as we pointed out in our discussion of the spectrum of ovalbumin, such assignments of weak- or medium-intensity lines to a-helical structures are dubious unless the protein is highly ahelical. This is certainly not the case for ribonuclease, which has only 15% a-helical structure. The superposition spectra of r i b o n ~ c l e a s indicate e~~ that lines near 1260 cm-’ could arise from tyrosine ring vibrations. One way to check this is to deuterate the protein fully whereupon the amide I11 lines shift to near 950 cm-l. Unfortunately, D20 solutions have a strong line near 1210 cm-l. This difficulty can be circumvented by exchanging the amide hydrogens of a solid sample in a saturated D20 atmosphere. A further difficulty is then to force complete hydrogen-deuterium exchange, as most proteins have “hard to exchange” amide hydr0gens.~5 In solution this can often be accomplished by heating. Our experimental approach was therefore to heat D20 solutions, freeze dry the product, and obtain the Raman spectrum with the sample exposed to a saturated D20 vapor in a sealed cell. Unfortunately, our samples of ovomucoid discolored slightly on heating. No such experimental difficulties were encountered with ribonuclease and we used this protein to establish the origin of the 1260-cm-I line. Two samples of ribonuclease in D20 solutions (5%w/w) were heated to 60°C for 1and 3 hr, respectively, lyophilized, and redissolved in D20 (5%w/w). The infrared spectra of these samples 1hr after dissolution are shown in Figure 3, compared to an untreated sample of ribonuclease 24 hr after dissolution. Only the sample heated for 3 hr had no residual amide I1 absorption near
J 1500
KKK)
Fig. 2. Raman spectrum of 1Wo (w/w)aqueous solution of ovomucoid.
UK)
cm-1
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PAINTER AND KOENIG
1550 cm-', demonstrating complete hydrogen-deuterium exchange. Freeze-dried portions of this sample were placed in a sealed optical cell containing a beaker of D20 to produce a saturated DzO atmosphere. The Raman spectrum was taken after 6 hr and is shown compared to that obtained from an unexchanged sample in Figure 4. The absence of any lines
v
1640
1800
1600
1400 CM'l
Fig. 3. Infrared spectrum of ribonuclease in DzO (5%w/w): curve A-after 24 hr; curve B-heated 1 hr 6OoC in D20, lyophilized, redissolved in DzO, spectrum obtained 1 hr after dissolution; curve C-heated 3 hr 6OoC in DzO, spectrum obtained 1 hr after dissolution.
''
1400
1200
Fig. 4. Raman spectrum of lyophilized ribonuclease: curve A-HzO B-fully exchanged sample, DzO sample.
atmosphere; curve
RAMAN SPECTRA OF EGG-WHITE PROTEIN
2161
in the fundamental N-H stretching region of the spectrum near 3300 cm-I is additional evidence for complete exchange. However, in the spectrum of this fully exchanged sample, the line a t 1260 cm-l is still evident. Consequently, this line is probably not an amide I11 vibrational mode, but more likely a tyrosine ring vibrational mode. We therefore infer that the line a t 1266 cm-I in the spectrum of ovomucoid can also be assigned t o tyrosine residues. This interpretation of the origin of lines near 1260 cm-I is not in agreement with the earlier interpretation of Yu and co-workers.16,23,26 T h e Raman spectrum of crystalline g l ~ c a g o nclearly ~ ~ demonstrates a weakto medium-intensity line a t 1266 cm-I, which was assigned t o the a-helical regions of this hormone. However, a shoulder a t this frequency is clearly discernible in the spectrum obtained from aqueous solutions, when the polypeptide chain is “disordered,” and also in the spectrum of the @sheet gel. Glucagon has two tyrosines out of a total of 29 amino-acid residues, a ratio comparable to ribonuclease. Further difficulties in interpretation arise when we consider the original amino-acid superposition spectrum of ribonuclease reported by Lord and Yu. The intensity of the tyrosine line near 1260 cm-’ is weaker than the line observed in our fully exchanged sample. However, it should be noted that the superposition spectrum was obtained a t p H 1.0. Yu and co-workers have demonstrated considerable changes in the relative intensities of lines due t o tyrosine between ribonuclease samples in the solid state and in solution a t various pH’s. Consequently, the interpretation of changes in this region of the spectrum pre-
B
i 1100
1000
Fig. 5. Raman spectrum of lyophilized conalbumin: curve A-native coagulated sample.
CM”
state; curve B
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PAINTER AND KOENIG
sents an acute problem for the spectroscopist. Most spectral charges observed upon dissolution of lyophilized samples of proteins have been interpreted in terms of conformational change. However, it is well known that the substitution of the stronger water-protein for interpeptide hydrogen bonds has a considerable effect on the electronic spectra of biopolymers27and such changes in electronic properties would also be expected to affect the Raman spectrum. In general, such factors have been neglected in interpreting spectral changes and these arguments indicate that more research is still required in order to decide these questions. This is demonstrated in the spectrum of fully exchanged ribonuclease where there is much larger contribution from tyrosine residues to the intensity of the line observed near 1260 cm-’ than previously considered. The amide I and I11 lines at 1665 and 1248 cm-l are characteristic of “irregular” structure12,22and these results indicate that this protein has little “ordered” secondary structure. This is in substantial agreement with the results of ORD studies, which indicated that this protein consists of “irregular” structure and a-helices only,l0with the proportion of the latter conformation being approximately 20%.
Conalbumin The Raman spectrum of conalbumin is shown in Figure 5 together with the spectrum of the thermally denatured protein. This latter spectrum will be discussed later. The amide I11 region of the native conalbumin
UOO
lo00
Fig. 6. Raman spectrum of whole egg white, lyophilized: curve A-native B-coagulated sample.
600 CM-’
state, curve
RAMAN SPECTRA OF EGG-WHITE PROTEIN
2163
spectrum is weak, broad, and structureless, making it very similar to the amide I11 of bovine serum albumin and a-keratin.’S--15 Both these proteins have 50% or more a-helical structure and we infer the presence of similar extensive a-helical regions in conalbumin, substantially more than the 5-31% range indicated by ORD studies.1° The amide I line a t 1667 cm-l is characteristic of the “irregular” regions of the protein polypeptide chain. Conalbumin is a poor Raman scatterer and difficulties were encountered in obtaining the spectrum. Consequently, the low signal-to-noise ratio renders a detailed discussion of the other lines observed in the spectrum of limited value.
The Thermal Denaturation of the Proteins of Egg White The Raman spectra of whole egg white (lyophilized) compared to the insoluble coagulum formed by heating a sample a t 70°C for 10 min is shown in Figure 6. For experimental purposes, this latter sample was lyophilized. The spectrum of whole egg white is almost identical to that of ovalbumin, which is not surprising in that this protein comprises 54%,by weight, of the system’ and that the other major protein (conalbumin) is a poor Raman scatterer. Upon thermal denaturation, an intense amide I11 line a t 1236 cm-’ becomes apparent and the amide I is shifted from 1667 to 1672 cm-’. These spectral changes demonstrate the formation of extensive regions of antiparallel 0-sheet between ovalbumin molecule^.^ I t has been proposed that interchain disulphide bonds play a n important role in the coagulation p r o ~ e s s . ~However, -~ aggregation still occurs in the presence of cysteine and p -chloromercuribenzoate28 and we observed no lines due 1~15,18319
--
3300
1200
900 cm”
,
Fig. 7. Raman spectrum: top-ovalbumin coagulated by heating D20 solution of 1 hr, aggregate lyophilized; bottom-S-ovalbumin, heating in D20 solution for 1 hr, clear solution lyophilized. Both spectra obtained in saturated D20 atmosphere.
PAINTER AND KOENIG
2164
to formation of new S-S bridges appearing near 500 cm-'. We conclude that the formation of stable intermolecular @-sheetstructures is of central importance in the thermal denaturation and aggregation of egg white. Ovalbumin demonstrates identical spectral changes to whole egg white upon coagulation. Similar changes are observed in the spectra of conalbumin upon thermal denaturation. The amide I line shifts to 1672 cm-1 and an intense amide I11 line at 1239 cm-l appears, demonstrating the formation of intermolecular @-sheetstructures. The ease with which ovalbumin aggregates upon heating has made it difficult to determine the extent of unfolding, it any, prior to c o a g ~ l a t i o n . ~ ~ The difficulties are experimental in that the presence of aggregated species in solutions renders the interpretation of ORD data,29or the obtaining of Raman spectra, difficult. However, it is possible to use Raman spectroscopy to gain some insight into this question by utilizing the capability of obtaining spectra in both solid and solution form and by comparing the extent of hydrogen-deuterium exchange of heated samples of ovalbumin and S-ovalbumin. Solutions of this latter form of the protein do not exhibit aggregation upon heating. A sample of S-ovalbumin was heated to 7OoC slowly over the course of 1hr. The clear solution was lyophilized and the Raman spectrum obtained after 3 hr exposure to a saturated D2O vapor in a sealed optical cell. A solution of ovalbumin heated in the same fashion
Fort
HEAT
PARTIAL UNFOLDING
OVALBUMIN; NATIVE STATE
INTERMOLECULAR p SHEETS
-
. HEAT
5- OVALBUMIN
RESTRICTED UNFOLDING
Fig. 8. Proposed thermal denaturation mechanism for ovalbumin.
RAMAN SPECTRA OF EGG-WHITE PROTEIN
2165
displayed considerable aggregation. T h e aggregate was separated by centrifugation and lyophilized and the Raman spectrum was obtained under identical experimental conditions to the sample of S-ovalbumin. The two spectra are compared in Figure 7. T h e absence of any scattering in the fundamental N-H stretching region of the spectrum of S-ovalbumin demonstrates that the protein had completely exchanged. However, the coagulated ovalbumin sample has weak residual scattering near 3300 cm-' and an unshifted amide I11 line a t 1254 cm-I is also observed. These results suggest that unfolding occurs in S-ovalbumin upon heating solutions of the protein. This unfolding is sufficient t o allow the exchange of amide hydrogens in the hydrophobic core but restricted in scope, probably by intramolecular crosslinks, so that aggregation does not occur. In contrast, the incomplete extent of hydrogen-deuterium exchange of ovalbumin indicates that only partial unfolding occurs before aggregation. However, the region or regions of the polypeptide chain that do unfold probably do so in an unrestricted fashion, so that intermolecular forces become important and the P-sheets are formed. This thermal denaturation mechanism is illustrated in Figure 8.
CONCLUSIONS There are three sets of conclusions resulting from this work, relating to the structure of the major proteins of egg white, the conformational changes that occur as a result of thermal denaturation and aggregation, and finally the origin of the medium-intensity line near 1260 cm-* in the Raman spectra of some proteins. The conformation of the ovalbumin polypeptide chain is predominantly irregular, though small amounts of P-sheet, some of which may be parallel rather than antiparallel, are also present. Ovomucoid has little ordered secondary structure but conalbumin has extensive amounts of a-helix. Upon thermal denaturation, both ovalbumin and conalbumin form intermolecular P-sheets. Ovalbumin only partially unfolds prior t o the formation of these intermolecular structures. Hydrogen-deuterium exchange studies demonstrated t h a t the line observed near 1260 cm-' in some proteins has a considerably larger contribution from a tyrosine ring vibration than previously considered.
References 1. Parkinson, T. L. (1966), J . Sci.Food Agric. 17,101-111. 2. Taborsky, T. (1974) Aduan. Protein Chem. 28,l-210. 3. Marshall, R. D. (1972) Ann. Reu. Biochem. 41,673-702. 4. Fevold, H. L. (1951) Aduan. Protein Chem. 6,188-252. 5. Fothergill, (1970) Riochem. J . 116,555-561. 6. Cho, K. H., Ph.D. dissertation Univ. Microfilms, Ann Arbor, Mich., Order 7019,772. 7. Smith, M. B. & Back, J. F. (1965), Aust. J . Biol. Sci. 18,365-377.
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8. Smith, M. B. & Back, J. F. (1968) Aust. J.Biol. Sci. 21,539-548. 9. Smith, M. B. & Back, J. F. (1968) Aust. J.Biol. Sci. 21,549-558. 10. Tomimatsu, Y. & Gaffield, W. (1965) Biopolymers 3,509-517. 11. Frushour, B. G. & Koenig, J. L. (1974) Biopolymers 13,455-474. 12. Frushour, B. G. & Koenig, J. L. (1974)Biopolymers 13,1809-1819. 13. Bellocq, A. M., Lord, R. C. & Mendelsohn, R. (1972) Biochem. Biophys. Acta 257, 280-287. 14. Lin. V. & Koenig, J. L. (1976) Biopolymers 15,203-218. 15. Lin, V. & Koenig, J. L. (1976) Textile Rs. J. (submitted). 16. Yu. N. T. & Jo, B. H. (1973) J . Amer. Chem. SOC.95,5033-5037. 17. Yu, N. T. (1974) J. Amer. Chem. Soc. 96,4664-4668. 18. Yu, T. J., Lippert, J. L. & Peticolas, W. L. (1973) Biopolymers 12,2161-2176. 19. Frushour, B. G. & Koenig, J. L. (1975) Biopolymers 14,649-662. 20. Chen, M. C., Lord, R. C. & Mendelsohn, R. (1974) J. Amer. Chem. SOC.9 6 , 3 0 3 s 3042. 21. Tiffany, M. L. & Krimm, S. (1969) Biopolymers 8,347-359. 22. Frushour, B. G. & Koenig, J. L. (1975) Biopolymers 14,379-391. 23. Yu. N. T. & Liu, C. S. (1972)J. Amer. Chem. SOC.94,5127-5128. 24. Lord, R. C. & Yu, N. T. (1970) J. Mol. Biol. 51,203-213. 25. Willunsen, L. (1971) Ct. R. Trau. Lab. Carlsberg 38,223-295. 26. Yu. N. T., Jo, B. H. & Liu, C. S. (1972) J. Amer. Chem. SOC.94,7572-7575. 27. Tinoco, I., Halpern, A. & Simpson, W. T. (1962) in Polyamino Acids Polypeptides and Proteins, Stahmann, M. A., Ed., pp. 147-160. 28. McKenzie, H. A., Smith, M. B. & Wake, R. G. (1963) Biochim. Biophys. Acta 69, 222-239. 29. Holme, J. (1963) J. Phys. Chern. 67,782-788.
Received August 14,1975 Accepted September 29,1975
