Int. J . Peptide Protein Res. 7 , 1975, 179-184 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s

MECHANISM OF GLUTATHIONE REGENERATION OF REDUCED PANCREATIC RIBONUCLEASE A

STEPHEN W. SCHAFFER*

Lehigh University, Department of Chemistry, Bethlehem, Pennsylvania, U.S.A.

Received 5 August 1974

A comparative study was made of the kinetics of glutathione regeneration of reduced pancreatic ribonuclease A , as determined by circular dichroism, sulfhydryl oxidation and the kinetics of reactivation. Four sulfhydryls were reoxidized prior to any large circular dichroic changes or recovery of enzymatic activity. The helical and 8 segments in ribonuclease were shown to reform at approximately the same rate. The results are discussed in terms of a regeneration mechanism for ribonuclease involving ( I ) nuclea- tion, (2) polypeptide backbone refolding, and (3) reshufling of incorrectly paired disulfide bonds.

.

Three systems have been shown to facilitate regeneration of disulfide-reduced proteins : (a) metal catalyzed air oxidation, (b) a microsomal enzyme catalyzed system, and (c) a nonenzymic oxidation and shuffling catalyzed by a mixture of reduced and oxidized glutathione. Numerous proteins have been shown to be regenerated by one or more of these systems (1-3).

Regeneration of these disulfide-containing proteins may conceptually be divided into three processes : (1) nucleation; (2) polypeptide back- bone refolding; (3) reshuffling of incorrectly paired disulfide bonds. Several studies have appeared recently which indicate that air re- generation of proteins proceeds by a pathway containing a limited number of structures (4-6). The refolding process leading to such structures is determined by certain nucleation processes preceding it.

In a previous study we examined the gluta- thione-facilitated regeneration of reduced pan- creatic ribonuclease A (7). It was shown that air regeneration of reduced ribonuclease A proceeded

*This work was carried out at the University of Minnesota, Department of Biochemistry, Minneapolis, Minnesota.

by a different mechanism than glutathione- catalyzed regeneration. The experiments reported here focus primarily upon structural changes associated with disulfide bond formation and recovery of enzymic activity. These results indi- cate that glutathione regeneration of protein also proceeds via a preferred pathway.

EXPERIMENTAL PROCEDURES

Materials Bovine pancreatic ribonuclease A was purchased from Sigma Chemical Co., type X1-A, lot 106B-8600. Both reduced and oxidized gluta- thione were also purchased from Sigma: GSH 78B-1650 and GSSG 23B-762. Tris (hydroxy- methyl) aminomethane was also from Sigma. Yeast RNA was obtained from Boehringer & Soehne, and was dialyzed against 0.1 M NHIOH before use. Urea (AR) was a Mallinckrodt pro- duct. Eastman Organic Chemicals provided the 8-mercaptoethanol, which was redistilled before use.

Methods

Reduction of ribonuclease A . The method of White (8) was employed to prepare reduced

179

STEPHEN W. SCHAFFER

ribonuclease. The protein was reduced in 8 M urea, 0.01 M pH 8.5 tris buffer solution contain- ing a 500-fold excess of /3-mercaptoethanol. Re- duction was allowed to proceed for 4 h, after which the solution was cooled to 0°C and pre- cipitated with 10 volumes of a cold mixture of acetone and 1 N HCI (39: 1). The precipitate was collected by centrifugation, washed twice with cold acetone: 1 N HCl, followed by two washes with ether. The precipitate was dissolved in deoxygenated water, lyophilized and stored at - 12°C in a desiccator. The reduced protein con- tained 8.0 & 0.2 sulfhydryls, as determined by the method of Ellman (9).

Ribonuclease assay. Ribonuclease activity was determined by the method of Kalnitsky et al. (10). Control assays, containing the same concentra- tion of reagents (GSH, GSSG, etc.) as the re- generated mixture, were carried out with each experiment.

Regeneration of reduced ribonuclease A. A modi- fied method of Ahmed et al. (7) was used to carry out the regeneration. Three ml of reduced ribonuclease solution containing approximately 1 mg ribonuclease/ml H 2 0 was added to 6 ml 0.1 M tris-perchlorate buffer (pH 8.2 at 30°C), which contained 2 x lo-, GSHand 3 x M GSSG. The glutathione concentration was decreased 10-fold to minimize the contribution it would make to the circular dichroic spectra. At appropriate times, 0.100 ml aliquots were with- drawn for assay. Protein concentration of re- duced ribonuclease was determined by ultra- violet absorbance ( E ~ ~ ~ . ~ = 9300).

SurfhVdryl determination. Regeneration was car- ried out as described above. The partially re- generated sample was precipitated and washed as described previously. The sample was dissolved in water and its sulfhydryl content was deter- mined by Ellman's reagent (9). Control experi- ments with reduced ribonuclease revealed essen- tially no sulfhydryl loss by this method.

Circular dichroic measurements. Measurements were made on a Durrum-Jasco J-10 circular dichrometer, calibrated according to Cassim & Yang (11). Regeneration was carried out as described previously. At appropriate times, a

0.200 ml sample was withdrawn and quenched with 0.01 M HCI04, lowering the pH to 3.3. Spectra were taken using 0.50 cm silica cells. The protein concentration was determined by ultra- violet absorbance.

RESULTS AND DISCUSSION

Circular dichroism A reliable circular dichroism (CD) reference state for unordered proteins is presently not available. The CDs of several reduced proteins in 6 M guanidinium chloride (GuCI) were shown to all be similar (12), and to differ significantly from the spectra observed for random polypeptides (13) and from the calculated spectra of Chen et al. (14). Utilizing the CD of reduced ribonuclease A in GuCl as the reference state, reduced ribonuclease A dissolved in 0.01 M KCIO, (pH 3.3) appears to have a residual amount of structure. The major difference between the two spectra is that re- duced ribonuclease A in GuCl lacks the broad negative shoulder in the 215-240 nm region, which is characteristic of reduced ribonuclease in Kclo , (Fig. 1). In addition, the trough of reduced ribonuclease in KCIO, is red shifted in respect to that of other random structures (l3,14). Young & Potts (1 5) , based upon fluorescent depolarization studies of reduced ribonuclease-5-dimethy lamino- 1 -naphthalene sulfonyl conjugates, also concluded that reduced ribonuclease contained some struc- ture.

The rate of structural refolding of reduced ribonuclease A during the process of regeneration was estimated by monitoring the CD spectral changes in the far U.V. To minimize the contribu- tion of glutathione to the CD spectra, regenera- tions were carried out at a glutathione concentra- tion lower than the optima! system for regenera- tion of proteins (7). At appropriate time intervals the reaction was quenched by lowering the pH to 3.3 with 0.01 M HCIO,. Reduced ribonuclease exposed to the regenerating medium for 15 sec exhibited a CD spectrum identical with reduced ribonuclease. This would suggest that the initial events in regeneration of ribonuclease are con- siderably different from initial events in lysozyme refolding. A similar experiment with reduced lysozyme indicated that 70% of the original helical content of the native enzyme was reformed immediately after dilution (4).

180

GLUTATHIONE REGENERATION OF REDUCED RIBONUCLEASE

0

2

4

6

8

El 10

12

14

16

l? 0

x n

I

v

FIGURE 1

\ \

A \ -. \ i *. ~ -47

-* i \ I

i \ - *\ '. ,' \ *

I I I 1 I I I 1

200 210 220 230

X,nm

Circular dichroism changes accompanying glutathione regeneration of ribonuclease A. Regenerations were carried out at 30°C in 0.1 M tris-perchlorate buffer, pH 8.2, containing 2 x M GSSG and 0.33 mg/ml ribonuclease. At appropriate times a 200 pI sample was withdrawn and quenched with 0.01 M HC104, lowering the pH to 3.3. Spectra were taken using 0.50 cm silica cells.

M GSH, 3 x

- - . - - . - - - . - . - - - . . . . . .

reduced ribonuclease 30 min regeneration 120 min regeneration 240 min regeneration native ribonuclease

Fig. 1 shows the results of 30, 120 and 240 min of regeneration. As regeneration proceeds there is a progressive change of the CD spectra from the reduced to the native state. Characteristic of this change is a decrease in intensity and red shift in the position of the trough, an enhancement of the 21 5-240 Cotton effect, and a red shift in the cross- over position. The similar rate at which all of these changes occur indicates that reformation of helical and /l structure occurs at the same rate. Regenerations allowed to proceed overnight generated a CD spectrum nearly identical to native ribonuclease.

Sulfhydryf oxidation and enzymatic activity During regeneration four sulfhydryls are oxidized rapidly, whereas the remaining four sulfhydryls

are oxidized much slower (Fig. 2). The slow re- oxidation of the last three sulfhydryls probably represents reshuffling of incorrectly paired disul- fide bonds.

Negligible enzymatic activity could be ob- served prior to reoxidation of about half of the eight sulfhydryl groups (Fig. 3). Enzymatic activity increases hyperbolically to a maximal yield of 70-85%. Half of the enzymatic activity was recovered by 4-112 h, at which time most of the three-dimensional structure and disulfide bonds are reformed.

Model Hauschka & Harrington (16) have suggested that gelation of collagen proceeds by three steps: nucleation, growth, and correction of incorrectly

181

STEPHEN W. SCHAFFER

I I 1 I 1 I I I

- -

6 .. -

-

- FIQURE 2 Kinetics of glutathione catalyzed I sulfhydryl reoxidation of reduced a 3 ribonuclease A. Regeneration was carried out at 30°C in 0.1 M tris-perchlorate buffer,pH8.2,containing2 x - M GSH, 3 x M GSSG and 0.33 mg/ml ribonuclease. At

enzyme was precipitated with 10 volumes of cold acetone: 1 N HCI (39:l). After two washes with acetone acid and twice with

-

fi - appropriate time intervals the 1 , -

0, I 1 I 1 I I I I

formed structure. Similarly, regeneration of disul- fide-containing proteins presumably proceeds by three related processes: (1) nucleation; (2) growth, or polypeptide backbone refolding; (3) reshuffling of incorrectly paired disulfide bonds.

I t is becoming increasingly apparent that the first process in protein regeneration is a “nuclea- tion” step (3, 1619). Nagano (18) views this process as the interaction among hydrophobic side chains of helical or candidates. Formation of such nuclei would assure rapid formation of specific disulfide bonds in the region of nuclea- tion. Fig. 4 shows that three-four sulfhydryls are reoxidized prior to any large change in the pep- tide CD spectrum, or any recovery of enzymatic activity. A nucleation-directed disulfide bond formation step could best explain these results.

The second process is polypeptide backbone

formation. During this process most of the pro- tein structure is recovered (Fig. 4). Also associa- ted with this phase of refolding is partial recovery of enzymatic activity and further sulfhydryl oxida- tion. One important feature of refolding is that formation of the helical and B segments in ribo- nuclease A occurs at approximately the same rate. This conclusion is based upon the observation that recovery of [el,,, and [O]200 proceed at the same rate (Fig. 4). Chen et al. (14) have found that the helical content of proteins is pro- portional to Likewise p structure and ran- dom coil can be reasonably defined by changes in [el2 , and [elzoo respectively.

The last process of refolding is shuffling of incorrectly paired disulfide bonds. It has been suggested that this process is the rate-limiting step in refolding (21). Two lines of evidence

182

GLUTATHIONE REGENERATION OF REDUCED RIBONUCLEASE

TIME (hours 1

support that hypothesis. (1) Although sulfhydryl- disulfide interchange generally proceeds rapidly, the last three sulfhydryls of ribonuclease A are reoxidized very slowly (Fig. 4). (2) The positive band at 240 nm of ribonuclease A has been associated with reformation of the correct di- sulfide bond conformation (22). This band is formed only during the latter stages of regenera- tion.

Thus the initial event in regeneration of ribo- nuclease A appears to be one or more nucleation steps, which permit rapid formation of two di- sulfide bonds. This is followed by a growth phase in which both helical and B segments reform structure at the same rate. Reshuffling of in- correctly paired disulfide bonds occurs during the latter phase of regeneration. It appears to be the rate limiting step of regeneration and accounts for the slow reoxidation of the last two sulfhydryl groups.

F~GURE 3 Kinetics of enzymatic reactiva- tion of ribonuclease A. Regenerations were carried out as described in Fig. 1. At appro- priate times a 100 ,d sample was withdrawn and enzymatic activity was determined by the method of Kalnitsky et al. (10).

ACKNOWLEDGMENTS

The author thanks Professor Donald B. Wetlaufer for stimulating discussions and for the use of his labora- tory. I am also indebted to Mrs. Jeanne Loosbrock for excellent secretarial assistance.

1 . 2.

3.

4.

5. 6.

7.

8.

REFERENCES

ANFINSEN, C. B. (1966) Harvey Lect. 61,95-116. WHITE, F. H. (1967) Methods Enzymol. 11, 481- 484. WETLAUFER, D. B. & &STOW, S. S. (1973) Ann. Rev. Biochem. 42,135-158. YIJTANI, K., YUTANI, A., IMANISHI, A. & ISEMURA, T. (1968) J. Biochem. 64,449-455. SAXENA, V. P. (1971) Fed. Proc. 30, 1287 (Abst.). RISTOW, S. S. & WETLAUFER, D. B. (1973) Bio- chem. Biophys. Res. Comm. 50,544-550. AHMED, A. K., SCHAFFER, S. W. & WETLAUFER, D. B., J. Biol. Chem., in press. WHITE, F. H., JR. (1961) J. Biol. Chem. 236, 1353-1360.

183

STEPHEN W. SCHAFFER

I

W c3 z 0

s I

FIGURE 4 Kinetics comparison of activity recovery, sulfhydryl reoxidation and circular dichroism changes during regeneration of ribonu- clease A. The CD and sulfhydryl re- oxidation data were obtained from Figs. 1 and 2 respectively. Activity recovery was obtained under identical conditions to Figs. 1 and 2. 0 sulfhydryl reoxidation 0 change in [O],,, u change in [B], I A change in [O]200

'0°"''''"1 t 1 80 - -

-

-

-

-

-

-

-

0 60 120 180 240

<> recovery of enzymatic activity.

9. ELLMAN, G. L. (1959) Arch. Biochem. Biophys. 82,

10. KALNITSKY, G., HUMMEL, J. P. & DIERKS, C. (1959) J. Biol. Chem. 234, 1512-1516.

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184

TIME (minutes)

18. NAGANO, K. (1974) J. Mol. Biol. 84,337-372. 19. ANDERSON, W. L. & WETLAUFER, D. B. (1974)

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Address: Stephen W. Schufer Lehigh University Department of Chemistry Bethlehem Pennsylvania 18015 U.S.A.