ARCHIVES
OF BIOCHEMISTRY
Stimulation
AND
of Reduced WILLIAM Department
182, 705-711 (1977)
BIOPHYSICS
Lysozyme Regeneration Lactoferrinl
L. ANDERSON of Immunology,
THOMAS
AND
Mayo Clinic,
Rochester,
by Transferrin
B. TOMASI, Minnesota
and
JR.2 55901
Received March 7, 1977 Human serum transferrin, bovine lactoferrin, and hen conalbumin were investigated with respect to the ability of the bound metal to catalyze thiol oxidation. All three proteins were able to stimulate the oxidation of thiols in both reduced lysozyme and reduced glutathione. The efficiency of the metal in catalyzing thiol oxidation was not decreased by binding to transferrin, suggesting that transferrin-bound metals are completely available to both low and high molecular weight thiols. A 5 x lo-’ M concentration of transfer-r-in isolated from serum was able to catalyze the formation of 70% of the theoretical lysozyme activity from reduced inactive lysozyme by 60 min. Increased rates of lysozyme activity formation were observed with copper-saturated transferrin. Decreased lysozyme regeneration rates were observed with the iron-saturated molecule compared to native transferrin. The results presented suggest that metalloproteins may aid in the maintenance of the steady-state cellular concentrations of low molecular weight disulfide by catalyzing the autooxidation of thiols.
During the biosynthesis of proteins the growing peptide chain folds into a unique three-dimensional structure. Work cited in several recent reviews (l-3) confirms the idea introduced by Sela et al. (4) that in many cases protein sequence determines structure. However, the pathway by which the final conformation is formed is unclear. In the case of disulfide-containing proteins, folding involves the formation of disulfide bonds but, again, the in uiuo mechanism(s) responsible are not known. The initial work on the folding of reduced RNase (4) used a trace metal-catalyzed air oxidation to form the disulfides. This oxidation system has been successfully employed in the folding of several other disulfide-containing proteins (reviewed in Refs. 5 and 6). An iron-containing protein has been isolated from bovine whey that catalyzes air oxidation (7). Whether or not this or a similar enzyme functions intracellularly to synthesize protein disulfide is not known. * Supported by U.S. Public Health Research Grant AM 17554 and by Fellowship F32 AI 05151 to Dr. Anderson. * To whom requests for reprints should be sent.
A rapid method of stimulating protein disulfide formation has been described by Bradshaw et al. (8) and Saxena and Wetlaufer (9). This system involves thiol disulfide exchanges between protein and low molecular weight thiols and disulfides. Rapid regeneration rates and physiological concentrations of thiols and disulfides (10) make this system an attractive candidate for the physiological mechanism of protein thiol oxidation. All necessary components are present in cell fluids. An enzyme has been isolated that catalyzes the thiol disulfide exchange reaction (11); however, the role of this enzyme during protein folding in uiuo is unclear (1, 12). Poulsen and Zeigler (13) point out that for a thiol disulfide regeneration system to function in uiuo some mechanisms must be present to maintain a constant supply of low molecular weight disulfide. They suggest that microsomal monooxygenase (EC 1.14.13.8) functions in that capacity; however, it is not clear if microsomal monooxygenase is the only source of intracellular disulfide. Are other oxidases capable of thiol oxidation or, as suggested in this study, can protein-bound metal serve as a 705
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
706
ANDERSON
catalyst for nonenzymic thiol oxidation? In this report we examine the possibility that metals specifically bound to protein may catalyze thiol oxidation. The transferrins were chosen as a model for these studies because they are found in significant concentrations in many tissues and physiological fluids and specifically bind several different metals. At present, they have no recognized enzymic activity (reviewed in Refs. 14 and 15). In addition, there are reports of the isolation of intracellular iron transport proteins (16). EXPERIMENTAL
PROCEDURES
Materials. Human serum transferrin was purchased from Schwarz/Mann (Lot y 3179). Additional samples of human transfer-r-in were isolated as a byproduct from a previously reported a,-antitrypsin isolation procedure (17). Bovine lactoferrin was isolated from fresh milk by the method described below. Conalbumin was obtained from the Sigma Chemical Company. Hen egg lysozyme and M. lysodeikitus were purchased from Worthington Biochemical Corp. Trisma base was the ultrapure product of Schwarz/Mann. Glutathione reductase was purchased from Calbiochem. All other reagents were of the purest grades commercially obtainable. The water used in all experiments was glass-distilled deionized water. All glassware was rinsed in dilute HCl and glass-distilled deionized water to minimize metal contamination. Meth.oo!s. Lactoferrin was isolated from the 3366% saturated ammonium sulfate precipitate of fresh bovine whey. The ammonium sulfate precipitate was dissolved in H,O and desalted on a 3 x 50cm column of Sephadex G-25. The column was equilibrated and eluted with 0.01 N T&+-Cl buffer, pH 8.0. The desalted protein fraction was immediately applied to a 4 x 3-cm column of DEAE-cellulose3 equilibrated in 0.01 N Tris-Cl buffer, pH 8.0. The column was eluted with the Tris-Cl buffer and the void volume was collected. This fraction was then titrated to pH 5.5 and applied to a 1 x 25cm column of CM-cellulose equilibrated in 0.004 N sodium acetate, pH 5.5. Proteins were eluted with a 1000~ml sodium acetate gradient from 0.004 to 1.00 N. The red fractions were pooled and concentrated by positive pressure. The concentrated protein was neutralized with 4% Na,CO, and applied to a 2.5 x llOcm column of Sephadex G-200 equilibrated and 3 Abbreviations used: DEAE-cellulose, diethylaminoethyl cellulose; CM-cellulose, carboxymethyl cellulose; TF, transferrin; GSH, glutathione; GSSG, oxidized giutathione; SDS, sodium dodecyl sulfate; DDC, diethyldithiocarbamate.
AND TOMASI eluted with 0.10 M ammonium bicarbonate. The lactoferrin fractions were lyophilized. The dried protein samples were stored desiccated at 4°C. The commercial samples of transferrin were further purified on the CM-cellulose column described above and by gel filtration on Sephadex G-200. The pooled fractions from the gel filtration column were lyophilized. Commercial conalbumin was used without further purification. Apotransferrin was prepared by the method of Johanson (18). Iron- and coppersaturated transferrins were prepared as described by Lehrer (19). Reduced lysozyme was prepared and characterized by the procedures of Ristow and Wetlaufer (20). All samples of reduced lysozyme contained between 7.7 and 8.0 SH groups/molecule as measured by the method of Ellman (21). Reduced lysozyme was refolded in 0.10 N Trisacetate buffer, pH 8.0, at room temperature. A final lysozyme concentration of 1 mg/lOO ml was used in all regenerations. Modifications to this basic regeneration system are described in the text. The progress of the regeneration was monitored with the lysozyme assay of Jolles (22) as modified by Wetlaufer et al. (23). Results are reported as percentage of theoretical lysozyme activity. Alkaline disc electrophoresis was performed by the method of Davis and Ornstein (24) using 7% gels running at pH 9.5 and stacking at pH 8.3. Acid urea electrophoresis was performed on 5% gels containing 5 M urea using a current of 3 mA/gel. Reduced glutathione oxidation was followed enzymically using glutathione reductase. A typical assay mixture contains 4 units/ml of glutathione reductase, 2 x 1O-4 M NADPH, 2.8 x 10e3 M GSH, and the transferrin or CuSO,. In the assay procedure, GSH is added prior to either transferrin or CuSO, and a baseline rate of thiol oxidation is determined. Iron was analyzed by the method of Harris and Aisen (25). Copper was analyzed by mixing 0.50 ml of protein solution, 2.00 ml of 0.10 N acetic acid, and 0.10 ml of 0.10 M diethyldithiocarbamate (DDC). Copper was estimated from the absorbance at 450 nm and a standard curve of CuSO,. Neither lysozyme nor apotransferrin had any effect on this curve. Protein was determined by both lyophilized weight and ultraviolet spectrum using EJa nm = 11.4 for apotransferrin and E&j$,,,, = 14.1 for iron-saturated transferrin (26). Transferrin purity was determined by potential metal binding. Transferrin samples were saturated with iron and excess iron was removed by gel filtration. Both iron and protein were assayed and the purity was determined based on a molecular weight of 7.7 x lo4 for transferrin and a stoichiometry of two atoms of iron per molecule of protein. RESULTS
The results chromatogram
of a typical CM-cellulose of human transfer-r-in iso-
TRANSFERRIN-CATALYZED
OXIDATION
OF
LYSOZYME
THIOLS
707
urated ammonium sulfate precipitate is lated as described by Musiani and Tomasi shown in Fig. 3. Lactoferrin was present (17) are shown in Fig. 1. The chromatoonly in peak 6. Sephadex G-200 gel filtragram of commercial transferrin is similar. Transfer-r-in is found in both peak 2 and tion of this peak reveals only two fractions: peak 3. Peak 2 is reddish in color and is a major lactoferrin peak and a smaller of unknown nature 50% saturated with iron, while fraction 3 is amount of material eluting at a large molecular size. Based on less than 10% iron saturated. If iron-satupotential metal binding, the major peak is rated transfer-r-in is applied to the column, 95100% pure. a similar pattern is obtained, with a small additional transferrin-containing peak obThe polyacrylamide gel electrophoresis served in the void volume representing re- patterns of the transfer+ lactoferrin, and conalbumin samples are shown in Fig. sidual saturated transfer-r-in from which overloaded to the iron had not been removed by the acid 4. All gels were intentionally pick up impurity. Application of 25 pg of during chromatography. Apotransferrin chromatographs in the position of peak 3. the protein sample resulted in a single The material isolated from peak 2 is used zone in both acid urea and alkaline disc gel for the remainder of the experiments. systems. Only one band was observed for all three proteins in the SDS gel system at Figure 2 shows the results of a typical Sephadex G-200 gel filtration of peak 2 all concentrations tested. These results are consistent with the greater than 95% pufrom Fig. 1. Based on potential metal binding, the major peak is 97-100% pure. rity determined by potential iron binding. Table I demonstrates the effect of huThe CM-cellulose chromatography of bovine lactoferrin isolated from a 33-66% sat- man transfer-r-in, bovine lactoferrin, and conalbumin from various sources on lysozyme folding. As can be seen, all of the transferrins were capable of stimulating the formation of lysozyme activity. This effect cannot be attributed to a nonspecific protein effect as equal concentrations of RNase, y-globulin, and cytochrome c were without effect. All stimulatory ability was Fraction number lost when the transfer-r-in sample was boiled for 5 min. However, heating transFIG. 1. CM-cellulose chromatography of human ferrin to 70°C for 5 min did not remove transferrin isolated by Musiani and Tomasi (17). A 1 x 25cm column was eluted with a sodium acetate stimulator-y activity. The activities of gradient, described in the text. The horizontal lines transferrin and lactoferrin were approxiindicate the pooled fractions. The material found mately equal, whereas conalbumin was between peaks 1 and 2 was variable, eluting at significantly less active. different positions and of different magnitude with The effect of different preparations of varying
sources
of transferrin.
FIG. 2. Sephadex
G-200 gel filtration of peak 2 from Fig. 1. A column of 2.5 x 110 cm was eluted with 0.10 M NH,HCO,; 6.7-ml fractions were collected. The material eluted around fraction 50 contained transferrin.
FIG. 3. CM-cellulose chromatography of bovine lactoferrin from the DEAE-cellulose fractionation. The column was eluted with the sodium acetate gradient described in the text. The horizontal lines indicate the pooled fractions. Peak 6 contained lactoferrin.
708
ANDERSON
AND TOMASI TABLE COMPARISON LACTOFERRIN SOURCES
I
OF THE ABILITY , AND CONALBUMIN
TO CATAL~E
Sample
OF TRANSFERRIN, FROM VARIOUS
LYSOZYME
REGENERATION”
Lysozyme activity (%) 30 min
a
b
c e
d
f
FIG. 4. Polyacrylamide gel electrophoresis of transferrin (a, b), lactoferrin (c, d), and conalbumin (e, f). Gels a, c, and e are disc gels run at pH 8.6. Gels b, d, and fare acid urea gels. Aliquots of 100 yg of protein were applied per gel.
purified transferrin on lysozyme regeneration is shown in Fig. 5. As can be seen, all batches of transferrin are similar in their ability to stimulate lysozyme activity formation. Purified lactoferrin has a similar activity. These preparations result in 6080% reactivation by 45 min. This value remains constant to 120 min. The final activity achieved is similar to that using the optimal thiol disulfide reactivation reported by Saxena and Wetlaufer (9) employing the GSH-GSSG system although the rate is slower. The ranges of values reported here are slightly greater than those found using optimal concentrations of GSH and GSSG (23). The dependence on protein concentration is seen in Fig. 6. From this figure it is evident that the lysozyme regeneration rate is dependent on lactoferrin concentration. The curve for the lactoferrin concentration of 5 x lop7 M fits well within the limits of the transfer-r-in-catalyzed regenerations seen in Fig. 5. Both lactoferrin isolated from a 50-75% ammonium sulfate precipitate and human transfer-r-in gener-
No addition Human transferrin (Schwarz/Mann) Human transferrin (purified) Bovine transferrin (Miles, -60% pure) Conalbumin (95% pure) Bovine lactoferrin
l-L1
60 min
22 k 4
222 51 2 5
33 2 2
70 k 5
10 * 2
48 e 5
lkl
20 f 1
35 f 2 70 + 5 a The uncertainty represents the range of values of three or more experiments. Contaminating metal salts were removed from all protein samples by gel filtration on Sephadex G-25. The purity of protein preparations was determined by potential iron bindIng analysis. Transferrin concentration was 38 pg/ ml.
FIG. 5. Effect of purified transferrin on lysozyme regeneration. Lysozyme, 6.9 x lo-’ M transferrin, 4.9 x lo-’ M in 0.10 N Tris-acetate buffer, pH 8.0. Transfer-r-in purified from serum (01, transferrin purified from commercial preparation (A,), regeneration without transferrin (01.
FIG. 6. Effect of lactoferrin concentration on lysozyme regeneration. Regeneration conditions are described in the text. Lactoferrin concentrations were: 0, 0 M; 0, 2.5 x lo-’ M; A, 5.0 x lo-’ M; 0, 7.5 X lo-’ M; 0, 12.5 X IO-’ M.
TRANSFERRIN-CATALYZED
OXIDATION
ate an essentially identical set of curves. The rate of lysozyme regeneration, at the highest lactoferrin concentration, is faster than the optimal rates reported by Yutani et al. (27) using air oxidation. Table II shows the effect of various modifications to the lysozyme regeneration system. Both 1 x 1O-4 M EDTA and N, saturation will effectively inhibit the transferrin-catalyzed lysozyme regeneration. Both nitrogen and EDTA inhibitions are predicted assuming that lysozyme regeneration is stimulated by a metal-catalyzed air oxidation of thiol. Transferrinbound iron, however, is slowly removed only by high concentrations of EDTA (28). The complete inhibition by 1 x low4 M EDTA suggests that something other than iron transfer-r-in is responsible for the stimulation of lysozyme regeneration. To test this possibility, either aliquots of transferrin were saturated with iron or the iron was removed and the aliquots were saturated with CuS04. The effect of the modified transferrin on lysozyme regeneration is also seen in Table II. Saturation with Fe(II1) causes a significant decrease in the stimulatory ability of transferrin. However, in 10 trials with iron-saturated transfer+ there were no cases where lysozyme regeneration was at baseline rate, so that a small but significant stimulatory effect is evident, especially at 60 min. Ironsaturated transferrin treated with acid to produce one-half-saturated Fe-transferrin had an increased activity over the ironTABLE
II
EFFECT OF VARIOUS MODIFICATIONS TO EITHER REGENERATION BUFFER OR THE TRANSFERRIN MOLECULE ON THE LYSOZYME REGENERATIONS Condition
Lysozyme
No addition Transferrin only +l x W4 M EDTA N, saturation Fe(II1) saturation Cu(I1) saturation (50% saturated) a Transferrin was the product and was used at 38 pg/ml. The sents the range of values of three
activity
OF
LYSOZYME
saturated molecule but not to the levels of native transferrin. Copper-saturated transfer-i-in was very effective in stimulating lysozyme regeneration. The metal chelator EDTA completely eliminated the 430-nm absorbance due to Cu-transferrin, while 4 mM GSH treatment caused the absorbance to slowly decrease, stabilizing at 60-70% of the initial value in 15 min. The reason for this decrease is unknown. It may be due to either SH removal of Cu from transfer-i-in or a change in spectra due to GSH binding to the protein. A direct assay of the effect of transferrin on glutathione oxidation is illustrated in Table III. Copper bound to transferrin is capable of catalyzing SH oxidation at a rate equal to that of free copper, while iron-saturated transferrin shows only a slight stimulation of glutathione oxidation. The purified and commercial transferrins have an activity equivalent to that of one-half-saturated CU-transferrin. The Cu-transferrin complex is as active as an equivalent concentration of free CL?+. Doubling the free Cu2+ concentration approximately doubles the rate of thiol oxidation. DISCUSSION
The data presented in this paper suggest that microsomal monooxygenase need not be the sole source of cellular low molecular weight disulfide. The reactions producing low molecular weight disulfide may include microsomal monooxygenase, possibly other oxidases and peroxidases, and, as shown in this paper, catalysis by transferTABLE OXIDATION
(%)
30 min
60 min
l+l 22 2 4 OAO 3 6?2 35 ? 2
2t2 51 ” 5 It1 4 25 -+ 7 53 k 5
of Schwarz/Mann uncertainty repredifferent trials.
709
THIOLS
III
OF REDUCED
(A&,,lmin)
Sample Free CuSO, (6.5582 x 10e5 Free CuSO, (3.279 x IO-” M) CuTF ([Cu2+] = 3.4 x 10-j Commercial SM (y 3179) Native TF Fe saturated Buffer only
GLUTATHIONE”
M) M)
0.052 0.032 0.033
-r- 0.004 L 0.001 ” 0.003
0.038 0.033 0.005 + 1 0.001 + 1
n Thiol oxidation was monitored with glutathione reductase. The procedure is described in the text. The uncertainty represents the range of values found in three trials.
710
ANDERSON
rin-bound metal ions. Other metalloproteins have yet to be tested. We suggest that the steady-state relationship between thiol and disulfide can then be generated by the constant oxidation of thiol, while the intracellular thiol concentration is maintained by enzymes such as glutathione reductase using metabolically produced NADPH as the reducing agent. The above model for the maintenance of the cellular SH/SS concentration ratios has an advantage over the previously proposed enzymic model (13) in that NADPH is required only for the generation of SH. In the microsomal monooxidase model, NADPH is required for the formation of both thiol and disulfide. Unless the enzymes in such a system are well controlled, the net of both disulfide reductase and monoxidase will be cyclic reactions yielding NADPH oxidation. Such control mechanisms have not been described. The transferrins were chosen for these studies as model metal-binding proteins not with the specific thought of their functioning in such a capacity in uiuo but rather because they are metal-binding proteins found in many different tissues and fluids and have no established enzymic activity. The complete availability of metal on the transfer-r-ins even to high molecular weight thiols such as reduced lysozyme and the fact that these proteins were used at less than physiological concentrations suggest that they have the capacity to function in thiol oxidation in uiuo. It is well established that transferrin functions as a source of iron for iron-requiring tissues. A metal transport property and a function in thiol oxidation are not necessarily mutually exclusive functions. Differences in the two transferrin metal-binding (29, 30) sites suggest the possibility of multiple functions (31). The thiol oxidation catalyzed by transfer-r-in may also be related to the mechanism of metal removal from transferrin (38). The metal usually associated with transferrin is Fe(II1) (14, 15). Ferric iron is a poor catalyst for GSH oxidation (32), although it does slowly catalyze thiol oxidation, as does the ferric-saturated transferrin molecule. Since copper is a much
AND TOMASI
more efficient catalyst for thiol oxidation than iron, it is expected that copper transferrin would also be a more efficient catalyst. It is not known, however, if the small but significant thiol oxidation activity of iron-saturated transferrin is due to contamination with copper transferrin. The low concentrations of copper required make this possibility difficult to exclude. Iron-saturated transferrin can only bind copper in a nonspecific manner due to the saturation of the metal-binding sites with iron which is not displaced by copper. Serum transferrin is normally one-third saturated with iron (14, 15). The metals, if any, binding to the remainder of the metal binding sites are unknown. There have been suggestions that Zn2+ (33) is also bound to transferrin in serum. The binding of copper by transferrin in serum has not been demonstrated. If copper is normally bound to transferrin in uiuo this copper would be found in the 2% of serum copper that reacts directly with DDC. A relationship between copper and iron metabolism is, however, well documented (34). The metal composition of intracellular transferrin is unknown. There are reports that support either a transferrin interaction with thiols or transferrin participation in electron transport reactions. Edwards and Felding (35) have shown that thiol blocking agents will inhibit iron transport from transferrin to reticulocytes. This observation has been explained by effects on membrane conformation. Conformational effects are undoubtedly important and could explain their observations. However, such observations are also consistent with the transferrin-thiol interaction reported in this paper and the reported reaction of transferrin and thiolglycolate (38). In addition, the reported ferroxidase activity associated with transferrin (36) adds strong support to the suggestion that transfer-i-in-bound metal catalyzes thiol oxidation. A ferroxidase activity would rapidly reoxidize the ferrous iron formed during an electron transport from thiolate anion to form the thiyl radical (37). It is not known whether transferrin catalyzes a similar Cu(1) oxidation.
TRANSFERRIN-CATALYZED
OXIDATION
Transferrin may be functioning either by an initial release of the metal and rebinding of the metal after oxidation or by oxidation with metal bound to the protein. In either case, transferrin can bind metal in a way that allows interaction with thiol. The complete availability of transferrin copper for thiol oxidation and the observation that absorption at 440 nm remains when copper transferrin is treated with GSH suggest, but do not prove, that the metal may remain bound to transferrin during oxidation. The observation of an iron-transferrimthiol interaction (38) is consistent with the suggestion that the metal functions while bound to protein. While the data presented suggest that metal transferrin complexes will support a thiol oxidation, the physiological significance of this observation has not been demonstrated. It will in fact depend on the quantitative fraction of the total intracellular disulfide formed by the metal protein oxidation reactions. This number is at present unknown. REFERENCES 1. WETLAUFER, D. B., AND RISTOW, S. (1973)Annu. Rev. Biochem. 42, 135-138. 2. ANFINSEN, C. B., AND SCHERAGA, H. A. (1975) Advan. Protein Chem. 29, 205-299. 3. BALDWIN, R. L. (1975)Annu. Rev. Biochem. 44, 453-475. 4. SELA, M., WHITE, F. H., AND ANFINSEN, C. B. (1957) Science 125, 691. 5. ANFINSEN, C. B. (1966) Harvey Lect. 61, 95-116. 6. WHITE, F. H. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, pp. 481-484, Academic Press, New York. 7. JANOLINA, V. G., AND SWAISCOOD, H. E. (1975) J. Biol. Chem. 250, 2532-2538. 8. BRADSHAW, R. A., KAMAREK, L., AND HILL, R. L. (1967) J. Biol. Chem. 242, 3789. 9. SAXENA, V. P., AND WETLAUFER, D. B. (1970) Biochemistry 9, 50155023. 10. TIETZE, F. (1969) Anal. Biochem. 27, 502. 11. GOLDBERGER, R. F., EPSTEIN, C. J., AND ANFINSEN, C. B. (1963) J. Biol. Chem. 238, 628-635. 12. EPSTEIN, C. J. (1972) in Aspects of Protein Synthesis (Anfmsen, C. B., Jr., ed.), p 463. Academic Press, New York. 13. POULSEN, L. L., AND ZIEGLER, D. M. (1976)Fed. Proc. 35, 1653.
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LYSOZYME
THIOLS
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14. AISEN, P., AND BROWN, E. B. (1975) Progr. Hemat&. 9, 25-56. S. K. (1964) in 15. FEENEY, R. E., AND KOMATSU, Structure and Bonding, Vol. 1, p. 149, Springer, Berlin-Heidelberg/New York. 16. WORKMAN, E. F., AND BATES, G. W. (1974) Biothem. Biophys. Res. Commun. 58, 787-794. 17. MUSIANI, P., AND TOMASI, T. B. (1976) Biochemistry 15, 798-804. 18. JOHANSON, B. (1960)Actu Chem. Sand. 14,510. 19. LEHRER, S. S. (1969) J. Biol. Chem. 244, 36133617. 20. RISTOW, S. S., AND WETLAUFER, D. B. (1973) Biochem. Biophys. Res. Commun. 50,544-550. 21. ELLMAN, G. (1959) Arch. B&hem. Biophys. 82, 70. 22. JOLLBS, P. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N.O., eds.), Vol. 5, p. 117, Academic Press, New York. 23. WETLAUFER, D. B., JOHNSON, E. R., AND CLAUSS, L. M. (1962) in Lysozyme (Osserman, E., ed.), pp. 269-280, Academic Press, New York. 24. DAVIS, B. J., AND ORNSTEIN, L. (1968) in Methods in Immunology and Immunochemistry (Williams, C. A., and Chase, M. W., eds.), Vol. 2, pp. 38-47, Academic Press, New York. 25. HARRIS, D. C., AND AISEN, P. (1975) Biochemistry 14, 262-268. 26. AISEN, P., AASA, R., MALSTR~M, B. G., AND V~~NNGJ(RD, T. (1967) J. Biol. Chem. 242,2484. 27. YUTANI, K., YUTANI, A., IMANISHI, A., ANDISEMURA, T. (1968) J. B&hem. (Tokyo) 64, 449455. 28. BATES, G. W., BILLUPS, C., AND SALTMAN, P. (1967) J. Biol. Chem. 242, 2816-2821. 29. DEMING, R. L., AND WOODWORTH, R. C. (1976) Fed. PFOC. 35, 1607. 30. LESTAS, A. N. (1976) Brit. J. Huematol. 32, 341350. 31. FLETCHER, J., AND HUEHNS, E. R. (1968) Nature (London) 218, 1211-1214. 32. TSEN, C. C., AND TAPPEL, A. L. (1958) J. Bill. Chem. 233, 1230. 33. EVANS, G. W., AND WINTER, T. W. (1975) Biothem. Biophys. Res. Commun. 66, 1218-1224. 34. HOLMBERG, C. G., AND LAURELL, C. B. (1947) Actu Chem. Sand. 1, 944-950. 35. EDWARDS, S. A., AND FIELDING, J. (1971) Brit. J. Huemutol. 20, 405-416. 36. BATES, G. W., WORKMAN, E. F., AND SCHLABACH, M. R. (1973) Biochem. Biophys. Res. Commun. 50, 84-90. 37. MISRA, H. P. (1974) J. Biol. Chem. 249, 21512155. 38. FIELDING, J., AND RYALL, R. (1970) Clin. Chem. Actu 28, 423.
OF BIOCHEMISTRY
Stimulation
AND
of Reduced WILLIAM Department
182, 705-711 (1977)
BIOPHYSICS
Lysozyme Regeneration Lactoferrinl
L. ANDERSON of Immunology,
THOMAS
AND
Mayo Clinic,
Rochester,
by Transferrin
B. TOMASI, Minnesota
and
JR.2 55901
Received March 7, 1977 Human serum transferrin, bovine lactoferrin, and hen conalbumin were investigated with respect to the ability of the bound metal to catalyze thiol oxidation. All three proteins were able to stimulate the oxidation of thiols in both reduced lysozyme and reduced glutathione. The efficiency of the metal in catalyzing thiol oxidation was not decreased by binding to transferrin, suggesting that transferrin-bound metals are completely available to both low and high molecular weight thiols. A 5 x lo-’ M concentration of transfer-r-in isolated from serum was able to catalyze the formation of 70% of the theoretical lysozyme activity from reduced inactive lysozyme by 60 min. Increased rates of lysozyme activity formation were observed with copper-saturated transferrin. Decreased lysozyme regeneration rates were observed with the iron-saturated molecule compared to native transferrin. The results presented suggest that metalloproteins may aid in the maintenance of the steady-state cellular concentrations of low molecular weight disulfide by catalyzing the autooxidation of thiols.
During the biosynthesis of proteins the growing peptide chain folds into a unique three-dimensional structure. Work cited in several recent reviews (l-3) confirms the idea introduced by Sela et al. (4) that in many cases protein sequence determines structure. However, the pathway by which the final conformation is formed is unclear. In the case of disulfide-containing proteins, folding involves the formation of disulfide bonds but, again, the in uiuo mechanism(s) responsible are not known. The initial work on the folding of reduced RNase (4) used a trace metal-catalyzed air oxidation to form the disulfides. This oxidation system has been successfully employed in the folding of several other disulfide-containing proteins (reviewed in Refs. 5 and 6). An iron-containing protein has been isolated from bovine whey that catalyzes air oxidation (7). Whether or not this or a similar enzyme functions intracellularly to synthesize protein disulfide is not known. * Supported by U.S. Public Health Research Grant AM 17554 and by Fellowship F32 AI 05151 to Dr. Anderson. * To whom requests for reprints should be sent.
A rapid method of stimulating protein disulfide formation has been described by Bradshaw et al. (8) and Saxena and Wetlaufer (9). This system involves thiol disulfide exchanges between protein and low molecular weight thiols and disulfides. Rapid regeneration rates and physiological concentrations of thiols and disulfides (10) make this system an attractive candidate for the physiological mechanism of protein thiol oxidation. All necessary components are present in cell fluids. An enzyme has been isolated that catalyzes the thiol disulfide exchange reaction (11); however, the role of this enzyme during protein folding in uiuo is unclear (1, 12). Poulsen and Zeigler (13) point out that for a thiol disulfide regeneration system to function in uiuo some mechanisms must be present to maintain a constant supply of low molecular weight disulfide. They suggest that microsomal monooxygenase (EC 1.14.13.8) functions in that capacity; however, it is not clear if microsomal monooxygenase is the only source of intracellular disulfide. Are other oxidases capable of thiol oxidation or, as suggested in this study, can protein-bound metal serve as a 705
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
706
ANDERSON
catalyst for nonenzymic thiol oxidation? In this report we examine the possibility that metals specifically bound to protein may catalyze thiol oxidation. The transferrins were chosen as a model for these studies because they are found in significant concentrations in many tissues and physiological fluids and specifically bind several different metals. At present, they have no recognized enzymic activity (reviewed in Refs. 14 and 15). In addition, there are reports of the isolation of intracellular iron transport proteins (16). EXPERIMENTAL
PROCEDURES
Materials. Human serum transferrin was purchased from Schwarz/Mann (Lot y 3179). Additional samples of human transfer-r-in were isolated as a byproduct from a previously reported a,-antitrypsin isolation procedure (17). Bovine lactoferrin was isolated from fresh milk by the method described below. Conalbumin was obtained from the Sigma Chemical Company. Hen egg lysozyme and M. lysodeikitus were purchased from Worthington Biochemical Corp. Trisma base was the ultrapure product of Schwarz/Mann. Glutathione reductase was purchased from Calbiochem. All other reagents were of the purest grades commercially obtainable. The water used in all experiments was glass-distilled deionized water. All glassware was rinsed in dilute HCl and glass-distilled deionized water to minimize metal contamination. Meth.oo!s. Lactoferrin was isolated from the 3366% saturated ammonium sulfate precipitate of fresh bovine whey. The ammonium sulfate precipitate was dissolved in H,O and desalted on a 3 x 50cm column of Sephadex G-25. The column was equilibrated and eluted with 0.01 N T&+-Cl buffer, pH 8.0. The desalted protein fraction was immediately applied to a 4 x 3-cm column of DEAE-cellulose3 equilibrated in 0.01 N Tris-Cl buffer, pH 8.0. The column was eluted with the Tris-Cl buffer and the void volume was collected. This fraction was then titrated to pH 5.5 and applied to a 1 x 25cm column of CM-cellulose equilibrated in 0.004 N sodium acetate, pH 5.5. Proteins were eluted with a 1000~ml sodium acetate gradient from 0.004 to 1.00 N. The red fractions were pooled and concentrated by positive pressure. The concentrated protein was neutralized with 4% Na,CO, and applied to a 2.5 x llOcm column of Sephadex G-200 equilibrated and 3 Abbreviations used: DEAE-cellulose, diethylaminoethyl cellulose; CM-cellulose, carboxymethyl cellulose; TF, transferrin; GSH, glutathione; GSSG, oxidized giutathione; SDS, sodium dodecyl sulfate; DDC, diethyldithiocarbamate.
AND TOMASI eluted with 0.10 M ammonium bicarbonate. The lactoferrin fractions were lyophilized. The dried protein samples were stored desiccated at 4°C. The commercial samples of transferrin were further purified on the CM-cellulose column described above and by gel filtration on Sephadex G-200. The pooled fractions from the gel filtration column were lyophilized. Commercial conalbumin was used without further purification. Apotransferrin was prepared by the method of Johanson (18). Iron- and coppersaturated transferrins were prepared as described by Lehrer (19). Reduced lysozyme was prepared and characterized by the procedures of Ristow and Wetlaufer (20). All samples of reduced lysozyme contained between 7.7 and 8.0 SH groups/molecule as measured by the method of Ellman (21). Reduced lysozyme was refolded in 0.10 N Trisacetate buffer, pH 8.0, at room temperature. A final lysozyme concentration of 1 mg/lOO ml was used in all regenerations. Modifications to this basic regeneration system are described in the text. The progress of the regeneration was monitored with the lysozyme assay of Jolles (22) as modified by Wetlaufer et al. (23). Results are reported as percentage of theoretical lysozyme activity. Alkaline disc electrophoresis was performed by the method of Davis and Ornstein (24) using 7% gels running at pH 9.5 and stacking at pH 8.3. Acid urea electrophoresis was performed on 5% gels containing 5 M urea using a current of 3 mA/gel. Reduced glutathione oxidation was followed enzymically using glutathione reductase. A typical assay mixture contains 4 units/ml of glutathione reductase, 2 x 1O-4 M NADPH, 2.8 x 10e3 M GSH, and the transferrin or CuSO,. In the assay procedure, GSH is added prior to either transferrin or CuSO, and a baseline rate of thiol oxidation is determined. Iron was analyzed by the method of Harris and Aisen (25). Copper was analyzed by mixing 0.50 ml of protein solution, 2.00 ml of 0.10 N acetic acid, and 0.10 ml of 0.10 M diethyldithiocarbamate (DDC). Copper was estimated from the absorbance at 450 nm and a standard curve of CuSO,. Neither lysozyme nor apotransferrin had any effect on this curve. Protein was determined by both lyophilized weight and ultraviolet spectrum using EJa nm = 11.4 for apotransferrin and E&j$,,,, = 14.1 for iron-saturated transferrin (26). Transferrin purity was determined by potential metal binding. Transferrin samples were saturated with iron and excess iron was removed by gel filtration. Both iron and protein were assayed and the purity was determined based on a molecular weight of 7.7 x lo4 for transferrin and a stoichiometry of two atoms of iron per molecule of protein. RESULTS
The results chromatogram
of a typical CM-cellulose of human transfer-r-in iso-
TRANSFERRIN-CATALYZED
OXIDATION
OF
LYSOZYME
THIOLS
707
urated ammonium sulfate precipitate is lated as described by Musiani and Tomasi shown in Fig. 3. Lactoferrin was present (17) are shown in Fig. 1. The chromatoonly in peak 6. Sephadex G-200 gel filtragram of commercial transferrin is similar. Transfer-r-in is found in both peak 2 and tion of this peak reveals only two fractions: peak 3. Peak 2 is reddish in color and is a major lactoferrin peak and a smaller of unknown nature 50% saturated with iron, while fraction 3 is amount of material eluting at a large molecular size. Based on less than 10% iron saturated. If iron-satupotential metal binding, the major peak is rated transfer-r-in is applied to the column, 95100% pure. a similar pattern is obtained, with a small additional transferrin-containing peak obThe polyacrylamide gel electrophoresis served in the void volume representing re- patterns of the transfer+ lactoferrin, and conalbumin samples are shown in Fig. sidual saturated transfer-r-in from which overloaded to the iron had not been removed by the acid 4. All gels were intentionally pick up impurity. Application of 25 pg of during chromatography. Apotransferrin chromatographs in the position of peak 3. the protein sample resulted in a single The material isolated from peak 2 is used zone in both acid urea and alkaline disc gel for the remainder of the experiments. systems. Only one band was observed for all three proteins in the SDS gel system at Figure 2 shows the results of a typical Sephadex G-200 gel filtration of peak 2 all concentrations tested. These results are consistent with the greater than 95% pufrom Fig. 1. Based on potential metal binding, the major peak is 97-100% pure. rity determined by potential iron binding. Table I demonstrates the effect of huThe CM-cellulose chromatography of bovine lactoferrin isolated from a 33-66% sat- man transfer-r-in, bovine lactoferrin, and conalbumin from various sources on lysozyme folding. As can be seen, all of the transferrins were capable of stimulating the formation of lysozyme activity. This effect cannot be attributed to a nonspecific protein effect as equal concentrations of RNase, y-globulin, and cytochrome c were without effect. All stimulatory ability was Fraction number lost when the transfer-r-in sample was boiled for 5 min. However, heating transFIG. 1. CM-cellulose chromatography of human ferrin to 70°C for 5 min did not remove transferrin isolated by Musiani and Tomasi (17). A 1 x 25cm column was eluted with a sodium acetate stimulator-y activity. The activities of gradient, described in the text. The horizontal lines transferrin and lactoferrin were approxiindicate the pooled fractions. The material found mately equal, whereas conalbumin was between peaks 1 and 2 was variable, eluting at significantly less active. different positions and of different magnitude with The effect of different preparations of varying
sources
of transferrin.
FIG. 2. Sephadex
G-200 gel filtration of peak 2 from Fig. 1. A column of 2.5 x 110 cm was eluted with 0.10 M NH,HCO,; 6.7-ml fractions were collected. The material eluted around fraction 50 contained transferrin.
FIG. 3. CM-cellulose chromatography of bovine lactoferrin from the DEAE-cellulose fractionation. The column was eluted with the sodium acetate gradient described in the text. The horizontal lines indicate the pooled fractions. Peak 6 contained lactoferrin.
708
ANDERSON
AND TOMASI TABLE COMPARISON LACTOFERRIN SOURCES
I
OF THE ABILITY , AND CONALBUMIN
TO CATAL~E
Sample
OF TRANSFERRIN, FROM VARIOUS
LYSOZYME
REGENERATION”
Lysozyme activity (%) 30 min
a
b
c e
d
f
FIG. 4. Polyacrylamide gel electrophoresis of transferrin (a, b), lactoferrin (c, d), and conalbumin (e, f). Gels a, c, and e are disc gels run at pH 8.6. Gels b, d, and fare acid urea gels. Aliquots of 100 yg of protein were applied per gel.
purified transferrin on lysozyme regeneration is shown in Fig. 5. As can be seen, all batches of transferrin are similar in their ability to stimulate lysozyme activity formation. Purified lactoferrin has a similar activity. These preparations result in 6080% reactivation by 45 min. This value remains constant to 120 min. The final activity achieved is similar to that using the optimal thiol disulfide reactivation reported by Saxena and Wetlaufer (9) employing the GSH-GSSG system although the rate is slower. The ranges of values reported here are slightly greater than those found using optimal concentrations of GSH and GSSG (23). The dependence on protein concentration is seen in Fig. 6. From this figure it is evident that the lysozyme regeneration rate is dependent on lactoferrin concentration. The curve for the lactoferrin concentration of 5 x lop7 M fits well within the limits of the transfer-r-in-catalyzed regenerations seen in Fig. 5. Both lactoferrin isolated from a 50-75% ammonium sulfate precipitate and human transfer-r-in gener-
No addition Human transferrin (Schwarz/Mann) Human transferrin (purified) Bovine transferrin (Miles, -60% pure) Conalbumin (95% pure) Bovine lactoferrin
l-L1
60 min
22 k 4
222 51 2 5
33 2 2
70 k 5
10 * 2
48 e 5
lkl
20 f 1
35 f 2 70 + 5 a The uncertainty represents the range of values of three or more experiments. Contaminating metal salts were removed from all protein samples by gel filtration on Sephadex G-25. The purity of protein preparations was determined by potential iron bindIng analysis. Transferrin concentration was 38 pg/ ml.
FIG. 5. Effect of purified transferrin on lysozyme regeneration. Lysozyme, 6.9 x lo-’ M transferrin, 4.9 x lo-’ M in 0.10 N Tris-acetate buffer, pH 8.0. Transfer-r-in purified from serum (01, transferrin purified from commercial preparation (A,), regeneration without transferrin (01.
FIG. 6. Effect of lactoferrin concentration on lysozyme regeneration. Regeneration conditions are described in the text. Lactoferrin concentrations were: 0, 0 M; 0, 2.5 x lo-’ M; A, 5.0 x lo-’ M; 0, 7.5 X lo-’ M; 0, 12.5 X IO-’ M.
TRANSFERRIN-CATALYZED
OXIDATION
ate an essentially identical set of curves. The rate of lysozyme regeneration, at the highest lactoferrin concentration, is faster than the optimal rates reported by Yutani et al. (27) using air oxidation. Table II shows the effect of various modifications to the lysozyme regeneration system. Both 1 x 1O-4 M EDTA and N, saturation will effectively inhibit the transferrin-catalyzed lysozyme regeneration. Both nitrogen and EDTA inhibitions are predicted assuming that lysozyme regeneration is stimulated by a metal-catalyzed air oxidation of thiol. Transferrinbound iron, however, is slowly removed only by high concentrations of EDTA (28). The complete inhibition by 1 x low4 M EDTA suggests that something other than iron transfer-r-in is responsible for the stimulation of lysozyme regeneration. To test this possibility, either aliquots of transferrin were saturated with iron or the iron was removed and the aliquots were saturated with CuS04. The effect of the modified transferrin on lysozyme regeneration is also seen in Table II. Saturation with Fe(II1) causes a significant decrease in the stimulatory ability of transferrin. However, in 10 trials with iron-saturated transfer+ there were no cases where lysozyme regeneration was at baseline rate, so that a small but significant stimulatory effect is evident, especially at 60 min. Ironsaturated transferrin treated with acid to produce one-half-saturated Fe-transferrin had an increased activity over the ironTABLE
II
EFFECT OF VARIOUS MODIFICATIONS TO EITHER REGENERATION BUFFER OR THE TRANSFERRIN MOLECULE ON THE LYSOZYME REGENERATIONS Condition
Lysozyme
No addition Transferrin only +l x W4 M EDTA N, saturation Fe(II1) saturation Cu(I1) saturation (50% saturated) a Transferrin was the product and was used at 38 pg/ml. The sents the range of values of three
activity
OF
LYSOZYME
saturated molecule but not to the levels of native transferrin. Copper-saturated transfer-i-in was very effective in stimulating lysozyme regeneration. The metal chelator EDTA completely eliminated the 430-nm absorbance due to Cu-transferrin, while 4 mM GSH treatment caused the absorbance to slowly decrease, stabilizing at 60-70% of the initial value in 15 min. The reason for this decrease is unknown. It may be due to either SH removal of Cu from transfer-i-in or a change in spectra due to GSH binding to the protein. A direct assay of the effect of transferrin on glutathione oxidation is illustrated in Table III. Copper bound to transferrin is capable of catalyzing SH oxidation at a rate equal to that of free copper, while iron-saturated transferrin shows only a slight stimulation of glutathione oxidation. The purified and commercial transferrins have an activity equivalent to that of one-half-saturated CU-transferrin. The Cu-transferrin complex is as active as an equivalent concentration of free CL?+. Doubling the free Cu2+ concentration approximately doubles the rate of thiol oxidation. DISCUSSION
The data presented in this paper suggest that microsomal monooxygenase need not be the sole source of cellular low molecular weight disulfide. The reactions producing low molecular weight disulfide may include microsomal monooxygenase, possibly other oxidases and peroxidases, and, as shown in this paper, catalysis by transferTABLE OXIDATION
(%)
30 min
60 min
l+l 22 2 4 OAO 3 6?2 35 ? 2
2t2 51 ” 5 It1 4 25 -+ 7 53 k 5
of Schwarz/Mann uncertainty repredifferent trials.
709
THIOLS
III
OF REDUCED
(A&,,lmin)
Sample Free CuSO, (6.5582 x 10e5 Free CuSO, (3.279 x IO-” M) CuTF ([Cu2+] = 3.4 x 10-j Commercial SM (y 3179) Native TF Fe saturated Buffer only
GLUTATHIONE”
M) M)
0.052 0.032 0.033
-r- 0.004 L 0.001 ” 0.003
0.038 0.033 0.005 + 1 0.001 + 1
n Thiol oxidation was monitored with glutathione reductase. The procedure is described in the text. The uncertainty represents the range of values found in three trials.
710
ANDERSON
rin-bound metal ions. Other metalloproteins have yet to be tested. We suggest that the steady-state relationship between thiol and disulfide can then be generated by the constant oxidation of thiol, while the intracellular thiol concentration is maintained by enzymes such as glutathione reductase using metabolically produced NADPH as the reducing agent. The above model for the maintenance of the cellular SH/SS concentration ratios has an advantage over the previously proposed enzymic model (13) in that NADPH is required only for the generation of SH. In the microsomal monooxidase model, NADPH is required for the formation of both thiol and disulfide. Unless the enzymes in such a system are well controlled, the net of both disulfide reductase and monoxidase will be cyclic reactions yielding NADPH oxidation. Such control mechanisms have not been described. The transferrins were chosen for these studies as model metal-binding proteins not with the specific thought of their functioning in such a capacity in uiuo but rather because they are metal-binding proteins found in many different tissues and fluids and have no established enzymic activity. The complete availability of metal on the transfer-r-ins even to high molecular weight thiols such as reduced lysozyme and the fact that these proteins were used at less than physiological concentrations suggest that they have the capacity to function in thiol oxidation in uiuo. It is well established that transferrin functions as a source of iron for iron-requiring tissues. A metal transport property and a function in thiol oxidation are not necessarily mutually exclusive functions. Differences in the two transferrin metal-binding (29, 30) sites suggest the possibility of multiple functions (31). The thiol oxidation catalyzed by transfer-r-in may also be related to the mechanism of metal removal from transferrin (38). The metal usually associated with transferrin is Fe(II1) (14, 15). Ferric iron is a poor catalyst for GSH oxidation (32), although it does slowly catalyze thiol oxidation, as does the ferric-saturated transferrin molecule. Since copper is a much
AND TOMASI
more efficient catalyst for thiol oxidation than iron, it is expected that copper transferrin would also be a more efficient catalyst. It is not known, however, if the small but significant thiol oxidation activity of iron-saturated transferrin is due to contamination with copper transferrin. The low concentrations of copper required make this possibility difficult to exclude. Iron-saturated transferrin can only bind copper in a nonspecific manner due to the saturation of the metal-binding sites with iron which is not displaced by copper. Serum transferrin is normally one-third saturated with iron (14, 15). The metals, if any, binding to the remainder of the metal binding sites are unknown. There have been suggestions that Zn2+ (33) is also bound to transferrin in serum. The binding of copper by transferrin in serum has not been demonstrated. If copper is normally bound to transferrin in uiuo this copper would be found in the 2% of serum copper that reacts directly with DDC. A relationship between copper and iron metabolism is, however, well documented (34). The metal composition of intracellular transferrin is unknown. There are reports that support either a transferrin interaction with thiols or transferrin participation in electron transport reactions. Edwards and Felding (35) have shown that thiol blocking agents will inhibit iron transport from transferrin to reticulocytes. This observation has been explained by effects on membrane conformation. Conformational effects are undoubtedly important and could explain their observations. However, such observations are also consistent with the transferrin-thiol interaction reported in this paper and the reported reaction of transferrin and thiolglycolate (38). In addition, the reported ferroxidase activity associated with transferrin (36) adds strong support to the suggestion that transfer-i-in-bound metal catalyzes thiol oxidation. A ferroxidase activity would rapidly reoxidize the ferrous iron formed during an electron transport from thiolate anion to form the thiyl radical (37). It is not known whether transferrin catalyzes a similar Cu(1) oxidation.
TRANSFERRIN-CATALYZED
OXIDATION
Transferrin may be functioning either by an initial release of the metal and rebinding of the metal after oxidation or by oxidation with metal bound to the protein. In either case, transferrin can bind metal in a way that allows interaction with thiol. The complete availability of transferrin copper for thiol oxidation and the observation that absorption at 440 nm remains when copper transferrin is treated with GSH suggest, but do not prove, that the metal may remain bound to transferrin during oxidation. The observation of an iron-transferrimthiol interaction (38) is consistent with the suggestion that the metal functions while bound to protein. While the data presented suggest that metal transferrin complexes will support a thiol oxidation, the physiological significance of this observation has not been demonstrated. It will in fact depend on the quantitative fraction of the total intracellular disulfide formed by the metal protein oxidation reactions. This number is at present unknown. REFERENCES 1. WETLAUFER, D. B., AND RISTOW, S. (1973)Annu. Rev. Biochem. 42, 135-138. 2. ANFINSEN, C. B., AND SCHERAGA, H. A. (1975) Advan. Protein Chem. 29, 205-299. 3. BALDWIN, R. L. (1975)Annu. Rev. Biochem. 44, 453-475. 4. SELA, M., WHITE, F. H., AND ANFINSEN, C. B. (1957) Science 125, 691. 5. ANFINSEN, C. B. (1966) Harvey Lect. 61, 95-116. 6. WHITE, F. H. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, pp. 481-484, Academic Press, New York. 7. JANOLINA, V. G., AND SWAISCOOD, H. E. (1975) J. Biol. Chem. 250, 2532-2538. 8. BRADSHAW, R. A., KAMAREK, L., AND HILL, R. L. (1967) J. Biol. Chem. 242, 3789. 9. SAXENA, V. P., AND WETLAUFER, D. B. (1970) Biochemistry 9, 50155023. 10. TIETZE, F. (1969) Anal. Biochem. 27, 502. 11. GOLDBERGER, R. F., EPSTEIN, C. J., AND ANFINSEN, C. B. (1963) J. Biol. Chem. 238, 628-635. 12. EPSTEIN, C. J. (1972) in Aspects of Protein Synthesis (Anfmsen, C. B., Jr., ed.), p 463. Academic Press, New York. 13. POULSEN, L. L., AND ZIEGLER, D. M. (1976)Fed. Proc. 35, 1653.
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LYSOZYME
THIOLS
711
14. AISEN, P., AND BROWN, E. B. (1975) Progr. Hemat&. 9, 25-56. S. K. (1964) in 15. FEENEY, R. E., AND KOMATSU, Structure and Bonding, Vol. 1, p. 149, Springer, Berlin-Heidelberg/New York. 16. WORKMAN, E. F., AND BATES, G. W. (1974) Biothem. Biophys. Res. Commun. 58, 787-794. 17. MUSIANI, P., AND TOMASI, T. B. (1976) Biochemistry 15, 798-804. 18. JOHANSON, B. (1960)Actu Chem. Sand. 14,510. 19. LEHRER, S. S. (1969) J. Biol. Chem. 244, 36133617. 20. RISTOW, S. S., AND WETLAUFER, D. B. (1973) Biochem. Biophys. Res. Commun. 50,544-550. 21. ELLMAN, G. (1959) Arch. B&hem. Biophys. 82, 70. 22. JOLLBS, P. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N.O., eds.), Vol. 5, p. 117, Academic Press, New York. 23. WETLAUFER, D. B., JOHNSON, E. R., AND CLAUSS, L. M. (1962) in Lysozyme (Osserman, E., ed.), pp. 269-280, Academic Press, New York. 24. DAVIS, B. J., AND ORNSTEIN, L. (1968) in Methods in Immunology and Immunochemistry (Williams, C. A., and Chase, M. W., eds.), Vol. 2, pp. 38-47, Academic Press, New York. 25. HARRIS, D. C., AND AISEN, P. (1975) Biochemistry 14, 262-268. 26. AISEN, P., AASA, R., MALSTR~M, B. G., AND V~~NNGJ(RD, T. (1967) J. Biol. Chem. 242,2484. 27. YUTANI, K., YUTANI, A., IMANISHI, A., ANDISEMURA, T. (1968) J. B&hem. (Tokyo) 64, 449455. 28. BATES, G. W., BILLUPS, C., AND SALTMAN, P. (1967) J. Biol. Chem. 242, 2816-2821. 29. DEMING, R. L., AND WOODWORTH, R. C. (1976) Fed. PFOC. 35, 1607. 30. LESTAS, A. N. (1976) Brit. J. Huematol. 32, 341350. 31. FLETCHER, J., AND HUEHNS, E. R. (1968) Nature (London) 218, 1211-1214. 32. TSEN, C. C., AND TAPPEL, A. L. (1958) J. Bill. Chem. 233, 1230. 33. EVANS, G. W., AND WINTER, T. W. (1975) Biothem. Biophys. Res. Commun. 66, 1218-1224. 34. HOLMBERG, C. G., AND LAURELL, C. B. (1947) Actu Chem. Sand. 1, 944-950. 35. EDWARDS, S. A., AND FIELDING, J. (1971) Brit. J. Huemutol. 20, 405-416. 36. BATES, G. W., WORKMAN, E. F., AND SCHLABACH, M. R. (1973) Biochem. Biophys. Res. Commun. 50, 84-90. 37. MISRA, H. P. (1974) J. Biol. Chem. 249, 21512155. 38. FIELDING, J., AND RYALL, R. (1970) Clin. Chem. Actu 28, 423.
