Refolding of Triose Phosphate Isomerase by STEPHEN G

Home , Dithiothreitol, Guanidinium chloride

Biochem. J. (1973) 135, 165-172 165 Printed in Great Britain

Refolding of Triose Phosphate Isomerase By STEPHEN G. WALEY Sir William Dunn School ofPathology, University ofOxford, South Parks Road, Oxford OX1 3RE, U.K. (Received 11 April 1973)

The refolding and reactivation of the glycolytic enzyme triose phosphate isomerase (EC 5.3.1.1) has been studied. The enzyme, which is a dimer, is disaggregated and unfolded in solutions of guanidinium chloride. Unfolding, followed by changes in E233, took place quite rapidly in 3 M-guanidinium chloride (i.e. with a half-life of about 1 min). Refolding also took place rapidly when the solution was diluted about tenfold; two first- order processes could be resolved. Regain of enzymic activity was followed by diluting the solution of the denatured enzyme in guanidinium chloride into assay mixture. The half-life (i.e. the time when the activity was half the final activity) depended markedly on the concentration of protein at low concentrations (about 1O0ng/mi), but at higher concentrations the half-life became independent of concentration. Thus at low concentrations dimerization was a rate-determining step and this is taken to indicate that the monomers showed little or no activity under these conditions. The rate of regain of enzymic activity was the same as the rate of the slower process of refolding, which was de- tected spectroscopically. The native enzyme was resistant to proteolysis; high concentrations ofsubtilisin prevented regain ofactivity, but at lower concentrations refolding com- peted with proteolysis.

The last step in protein biosynthesis is the folding the subunits of aldolase can be catalytically active of newly formed protein. Chain folding in vivo is (Chan, 1970, 1972; Chan & Mawer, 1972; Teipel, completed within, at the most, a few minutes, and 1972). Kinetic evidence suggests that the subunits of presumably gives exclusively active, functional pro- yeast enolase are not active (Gawronski & Westhead, tein. Chain folding in vitro, on the other hand, is often 1969; Brewer & De Sa, 1972), but active monomers slow and seldom leads exclusively to active product. can apparently be formed at high dilutions and There may thus be important differences in the temperatures above 40°C (Keresztes-Nagy & Orman, routes of folding. Nevertheless, there is too little 1971) and so presumably the monomer can exist in known about either process to be sure of this, and so active or inactive conformations. there is much current work on the refolding of pro- The present paper describes experiments on the re- teins. Single-subunit proteins lend themselves to folding and regain of enzymic activity of triose studies on unfolding and refolding; experiments on phosphate isomerase (EC 5.3.1.1). The aim of the the kinetics in both directions are especially helpful in work was to see whether kinetic studies would throw selecting mechanisms (Ikai & Tanford, 1973; Ikai any light on the nature and timing of the events that et al., 1973; Tanford et al., 1973). Multi-subunit pro- occur during this enigmatic process. teins (oligomers) are less easy to study, but have addi- There are two subunits in triose phosphate iso- tional features of interest. The kinetics of unfolding merase, and the crystallographic evidence shows the and refolding of oligomers may be difficult to study subunits to be related by a twofold rotation axis in both directions (Green & Toms, 1972), and this (Johnson & Waley, 1967). The sequence of the rabbit was found in the work to be described here. It is the muscle enzyme (Corran & Waley, 1973) shows that 'retrieving errors' in folding that take the time; with the molecular weight of the dimer is 53 257; the indi- an oligomeric protein, an incorrectly folded oligomer vidual polypeptide chains contain 248 amino acid may have to dissociate again before correct folding residues. can be achieved. This may be the main reason why regain ofactivity in oligomeric enzymes is often slow, and ifsome ofthe incorrectly folded oligomers do not Materials and Methods dissociate at all during the time of the experiment, Materials recovery of activity will also be incomplete. It is not in Ohieral known whether the isolated sub- Rabbit muscle triose phosphate isomerase was units of oligomneric enzymes can function as catalysts from Boehringer Corp. (London) Ltd., London (Frieden, 1971). Several lines of evidence show that W5 2TZ, U.K.; chicken triose phosphate isomerase Vol. 135 166 S. G. WALEY was prepared by the methods of Putman et al. (1972) dinium chloride and 0.19ml of 0.1 M-dithiothreitol in and McVittie et a!. (1972) in the Oxford Enzyme 0.1 M-triethanolamine hydrochloride, pH7. After Group Laboratory. Dihydroxyacetone phosphate 5 min at room temperature, a portion (2-10tul) of the was from Sigma Chemical Co. Kingston-on-Thames, solution was added, with an 'adder-mixer' (Boyer, Surrey, U.K. the dehydrogenases and the coenzymes 1954), to 3ml of assay solution at 14°C; measure- were from Boehringer Corp. (London) Ltd. Subtilisin ments of E340 were started within about 10s (Fig. 1). was from Novo Terapeutisk, Copenhagen, Denmark. The 'controls' were not exposed to 5M-guanidinium chloride. The activity was found from the rate of Methods change ofE340 with time; the half-life was the time at which the slope was half the final slope. The rates in the assays for triose phosphate isomerase were followed at 340nm in a Unicam SP. 800 Results spectrophotometer with expansion and recording on and Discussion a Servoscribe potentiometric recorder. The assays Denaturation in guanidinium chloride with dihydroxyacetone phosphate as substrate contained 110Hg of D-glyceraldehyde 3-phosphate de- The change in extinction in the ultraviolet when hydrogenase/ml, 6mM-sodium arsenate, 1 mM-NAD+ proteins are denatured is convenient for following and 1 mM-dihydroxyacetone phosphate, in 0.1 M- unfolding and refolding. The largest peak in the triethanolamine hydrochloride, pH 7. The assay with difference spectrum is often at about 230nm (Glazer glyceraldehyde phosphate as substrate contained & Smith, 1961), and is mainly due to tryptophan 10tg of a-glycerophosphate dehydrogenase/mI, (Donovan, 1968); the peak was here at 233 nm. At a 170,uM-NADH and 0.4mM-DL-glyceraldehyde 3- concentration of 200,ug/ml, E233 = 0.9 and XE233 = phosphate, in the same buffer as above. The dehydrogenases were freed from (NH4)2SO4 by dialysis. The regain of enzymic activity after dilution of the denaturant was assayed thus: a portion (usually 1 ,ul) ofthe stock suspension oftriose phosphate isomerase in about 3 M-(NH4)2SO4 was added to IOO,lI of 'unfolding solution prepared from 0.15g of guani-

I 10

L LO

Time (min) Time (s) Fig. 1. Regain of enzymic activity after dilution of Fig. 2. Rate ofunfolding oftriosephosphate isomerase denaturant in guanidinium chloride The change of E340 with time is shown; the E340 is A portion (20,il) ofstock suspension ofrabbit muscle recorded in arbitrary units on the Servoscribe re- enzyme was added to 0.98 ml ofguanidinium chloride corder, and the intervals on the time-scale show 1 min. (2-3M) in 0.02M-triethanolamine hydrochloride, The procedure is described in the Materials and pH7, at 14°C. The change in E233 was followed, and Methods section; 10,tl of the solution of chicken the ordinate gives Ea,-E, (on a logarithmic scale) muscle enzyme in guanidinium chloride was added to where E and E, are the amplified readings from the 3 ml of assay solution, pH7, 14°C; dihydroxyacetone Servoscribe recorder at time infinity and time t phosphate was the substrate. The left-hand trace is a respectively. The value for Fo was obtained by the 'control', not exposed to 5 M-guanidinium chloride; method of Freedman & Radda (1968) for the slower the right-hand trace shows the increase of activity runs. Guanidine hydrochloride concentrations: o, with time. 2M; A, 2.5M; O, 3M. 1973 REFOLDING OF TRIOSE PHOSPHATE ISOMERASE 167

0.2, where AE refers to the difference between native soo rate protein and denatured protein. The of unfolding 400 of rabbit triose phosphate isomerase is rapid at high lz

concentrations ofguanidinium chloride, and depends ,., 300 sharply on the concentration of denaturant (Fig. 2). 1- In the experiments described below, denaturation ,q 200 =--@- was carried out in 5 M-guanidinium chloride for at 0 least 2min. The protein is apparently completely denatured; the intrinsic viscosity was 25±4ml/g at 0 5 10 IS 20 25°C in 5.2M-guanidinium chloride, which is about Concn. of protein (nM) the value expected for a random coil (Tanford et al., 1967) containing 248 amino acid residues (Corran & Fig. 3. Variation of half-life for regain of enzymic Waley, 1973). At low concentrations of guanidinium activity with concentration ofprotein chloride unfolding is relatively slow; there is a co- operative transition from native protein to unfolded The time when the slope in the assay reaches a value protein at about 0.8M-guanidiniumchloride (McVittie half the final value is the half-life. The concentration et al., 1972). There may be hysteresis in the unfolding of the protein was calculated on the approximate of oligomeric proteins (Green & Toms, 1972); at basis that 25ng/ml = nrm. Q, Chicken enzyme as- about 1 M-guanidinium chloride the protein is sayed with glyceraldehyde phosphate as substrate; (thermodynamically) unstable in that refolding and o, chicken enzyme assayed with dihydroxyacetone reassociation do not take place, but unfolding is phosphate as substrate; oi, rabbit enzyme assayed relatively slow. with dihydroxyacetone phosphate as substrate. The At still lower concentrations ofguanidinium chlor- buffer was 0.1 M-triethanolamine hydrochloride, pH7. ide another effect is seen: the denaturant appears to ---, 5°C; , 14°C. act as a competitive inhibitor. Somewhat similar effects have been observed with other proteins, e.g. cytochrome c (Ikai et al., 1973). It has been suggested that ligand-binding sites may often be the main sites 1970) was present; these experiments are discussed of attack by dissociating agents (Green & Toms, below. Refolding could be carried out at concentra- 1972). The denaturing effect of guanidinium chloride tions of guanidinium chloride of less than 0.25M. has been ascribed to interaction with exposed pep- The striking feature in Fig. 3 is that the half-life tide bonds and amide groups (Robinson & Jencks, depends on the concentration at low concentrations, 1965). Competitive inhibition, however, does not but becomes independent ofconcentration at concen- show that the inhibitor is binding at the active site trations above, say, IOnM (250ng/ml). A second-order (Ogston, 1955), merely that the substrate and inhibitor reaction whose rate constant is 10m-I s-I will have interfere with each other's binding. At the higher a half-life of 1Os when the concentration ofreactant is concentrations of guanidinium chloride, moreover, lOnM. Since the expected diffusion-controlled limit there was little effect of substrate (or other ligands) for dimerization is probably about 107M-1 S-1 (von on the rate of denaturation. Perhaps there are several Hippel & McGhee, 1972), it is reasonable that sites on the protein that interact with the denaturant dimerization should at least partly determine the in 3M-guanidinium chloride and so ligand bound at rate at the lower concentrations. The curve (Fig. 3) the active site makes little difference. suggests that the dimer is required for enzymic activity, and seems to exclude the possibility that the subunits are fully active under these conditions. For Variation of rate of regain of enzymic activity with ifthe monomer were fully active thenregainofactivity concentration would be first-order even in dilute solution: there The rate of reactivation was followed by adding would be no need for two molecules of monomer to denatured enzyme to the assay medium. The time at collide before activity was acquired. In outline, then, which the slope of the trace was half the final slope the course of reactivation is envisaged as: was taken as the half-life. Measurements were started 10-15s after mixing. In the 'control' experiments with Unfolded monomer -* folded monomer native enzyme the traces were linear. Two folded monomers -- dimer The range of concentrations that can be used is limited by the assay, but this limitation was eased by The chains (monomers) will have to fold to form being able to assay the enzymic reaction in either characteristic 'recognition sites' for dimerization. Of direction. The range shown in Fig. 3 is from 0.2 to course, this folding may well be incomplete, and 16nM; higher concentrations could be used when the there may be further changes in conformation after reversible inhibitor phosphoglycollate (Wolfenden, refolding. The simplest scheme that illustrates the Vol. 135 168 S. G. WALEY interplay of unimolecular and bimolecular step is This value should not depend on the viscosity of the thus: solution because at high concentrations dimerization k+1 is fast and the unimolecular folding determines the k-1 rate. 2Y k+2 z Reactivation where X represents unfolded monomer, Y represents In the presence of phosphoglycollate. Phospho- folded monomer and Z represents dimer. If glycollate is the most powerful competitive inhibitor k_[Y] >k+2[y]2, then [Y] =(k+1/k_1)[X], and d[Z]/ of triose phosphate isomerase (Wolfenden, 1970), dt=k+2[Y]2 = k+2(k+1/k-1)2[X]2, and in this limit- and was used here to slow the enzymic reaction. This ing case the reaction is second-order. If k.1 [Y] < enabled refolding to be carried out at higher concen- k+2 Y]2, then, approximately, d[Y]/dt = 0, and trations of enzyme without the catalysed reaction d[Z]/dt = k+I [X], and the reaction is first-order. Thus (the assay) being too fast. Here there was little or no this scheme represents the required feature of being variation of half-life with concentration, and the ex- second-order at low concentrations and first-order at tent of regain of activity was high (70-80%). The high enough concentrations. This is more-or-less the values for the half-life (Table 1) were similar to those situation in Fig. 3; at low concentrations the half-life at the highest concentration of protein in Fig. 2; at tends to be inversely proportional to the concentra- the lowest concentration of protein in Table 1, the tion; at high concentrations the reaction is clearly half-life was greater when phosphoglycollate was first-order. Thus the shape of thecurve shownin Fig. 3 present. Ligands often accelerate reactivation suggests that the monomer (the subunit) is inactive. and increase recovery (Teipel & Koshland, 1971; The same situation holds over a range ofpH values, Levitzki, 1972). as shown in Fig. 4, and at several temperatures. The Effect of temperature. The rate of reactivation half-life for regain of activity is higher when glycerol depends on the temperature (Fig. 2); the half-life is (8 %, v/v) is present (Fig. 5); this is consistent with the increased when the temperature is lowered by 10°C rate of dimerization being diffusion-controlled, as is by two to three times. the fact, brought out by the reciprocal plot of Fig. 5, Extent of reactivation. The extent of regain of en- that there is little or no effect of glycerol on the value zymic activity in the kinetic runs varied over the of the extrapolated half-life at infinite concentration. range 35-85 %. The factors affecting the extent of the

300 r 300 (a) (b) 200 cn 200 F 0 c4 4. 4- °\ 1= '4 100F la- 100t 0

0 5 10 15 20 0 5 10 15 20

300 r 300 r o (c) (d) ~200 200 4) 100 CI- 1001 '0 0

5 10 15 20 0 20 40 60 80 Concn. of protein (nM) Concn. of protein (nM) Fig. 4. Variation ofhalf-life with concentration at (a) pH6.2, (b) pH6.6, (c)pH8.0 and (d) pH9.2 The assay was with dihydroxyacetone phosphate as substrate; the temperature was 14°C. The buffers at pH6.2 and 6.6 were 0.1 M-imidazole hydrochloride; at pH8 the buffer was 0.075M-triethanolamine hydrochloride; at pH9.2 the buffer was 0.075M-Na2CO3-NaHCO3. Chicken muscle enzyme was used. 1973 REFOLDING OF TRIOSE PHOSPHATE ISOMERASE 169

200 Table 2. Regain of activity in 'subtilisin-quenched' experiments

100 A portion (1-lO,lI) of stock chicken muscle triose phosphate isomerase (1.94mg/ml) was added to 50 ,ul of denaturing solution (0.15g of guanidine hydro- I 0 chloride in 0.19 ml of O.1 m-dithiothreitol in 0.02M- 0 0.1 0.2 0.3 0.4 triethanolamine hydrochloride, pH 7.8, containing 1/Concn. of protein (nM-') 2mg of EDTA/ml); after 0min at 200C, 1.95 ml of 0.1 M-triethanolamine hydrochloride, pH7, chilled to Fig. 5. Variation ofhalf-life with concentration in the 0°C, was added. The regain of activity (at 0°C) was presence ofglycerol followed by withdrawing at timed intervals of 10-20s portions of20,l and diluting with 60FI of'quenching' The assay was with dihydroxyacetone phosphate as solution (20,ug ofsubtilisin/ml, in the 0.1 M-triethanol- substrate, at pH7 and 14°C. o, No additions; El, in amine hydrochloride, pH7, buffer). Portions of this 8.33 % (v/v) glycerol; A, in phosphoglycollate solution were then assayed; for the control, the de- (2.21mM); A , in phosphoglycollate (0.37mM). naturing solution and the refolding solution were Chicken muscle enzyme was used. The abscissa gives mixed, and the isomerase was then added. The the reciprocal of the protein concentration. efficiency of the 'quenching' procedure was tested as follows. The denatured isomerase was diluted with a mixture ofrefolding solution and quenching solution: Table 1. Reactivation oftriose phosphate isomerase in only 3 % of activity was regained (in the control, the the presence ofphosphoglycollate isomerase was added last). The half-life is from four The value given is the half-life as the mean of four experiments, and the standard deviations are given. experiments; the standard deviation is also given. The concn. of phosphoglycollate was 0.37mM for the Concn. of Recovery of experiments at 5°C, and the two lower concentrations isomerase Temp. Half-life activity at 14°C and 2.21 mm for those at 14°C. The reactions (nM) (OC) (s) (% of control) were assayed with glyceraldehyde phosphate as sub- 388 0 24± 2 84 strate. The extent of regain of activity was 70-80%; 78 0 35±9 71 chicken muscle enzyme was used. 39 5 38 ± 9 55 39* 5 61±8 50 Concn. of Half-life (s) protein * The refolding solution contained 1 mM-dihydroxy- (nM) Temp. (°C) 5 14 acetone phosphate. 53 109 ± 10 40±8 74 99±8 42±13 106 93±4 39±4 Refolding in the presence ofjproteolytic enzymes 21.2 57±5 10.6 89±9 Native rabbit muscle or chicken muscle triose 10.6* 51±2 phosphate isomerase is quite resistant to proteolytic enzymes: there is no loss in activity (or evidence for * Without added phosphoglycollate. smaller fragments on electrophoresis) after prolonged incubation with subtilisin, papain, chymotrypsin and trypsin. When the denatured isomerase refolds in regain are not clear; in the initial runs the regain was solutions containing subtilisin, the recovery of acti- higher at higher concentrations of enzyme, but in vity is decreased (Table 2). The effect is greater the later runs this no longer held true. The average regain larger the amount of subtilisin (Fig. 6). Clearly, re- was about 55 %. The extent of regain was similar for folding and proteolysis are competing. The results in the rabbit muscle and chicken muscle enzymes, and Fig. 6 suggest that the effect of subtilisin is best ex- was not increased by the presence of bovine serum pressed as depending on the ratio of the concentra- albumin. The highest extents of regain of activity tions of isomerase to subtilisin. This would be (about 75 %) were found when the inhibitor, phospho- accounted for ifa monomer either reacts with another glycollate, was present. monomer, or with subtilisin. Chicken and rabbit muscle enzymes. No difference was found between the reactivation of chicken and Rate ofreactivation at concentrations rabbit enzyme; some ofthe experiments on unfolding, higher ofprotein and on refolding, were done only with the rabbit The procedure of direct dilution (of the denatured enzyme. protein in 5M-guanidine hydrochloride) into assay Vol. 135 170 S. G. WALEY

(a) slower and parallels the regain of enzymic activity. A 90- The results given in Table 2 show that the half-life ° 80- A for regain of activity was about 40s at 5°C in the 0 'subtilisin-quenched' experiments, compared with 'S 70- a 1 about lOOs for the earlier '(denaturant dilution') 60- experiments. >,40- jOA The shorter half-life in the subtilisin-quenched 30 experiments may possibly be explained as follows. c 20 A correctly refolded monomer may be associated with .< IA 1- an incorrectly folded one; the subtilisin may hydro- 2 4 6 8 10 12 14 16 18 20 22.24 26 lyse the incorrectly folded one, thus freeing the correctly folded monomer and allowing it to di- (b) Isomerase/subtilisin (w/w) merize with a second monomer. The results were not precise enough to be sure whether the half-life was independent of the concentration. The reactivation may be somewhat slower in the presence of substrate (Table 2), but the difference is not enough to account for the difference between the results here and those from the denaturant dilution experiments. When the 'quenched' solution was assayed, there was no increase in activity, but a very slow decrease (1 %/min), and this excludes the possi- that slower reactivation could go on in the 0 l 2 3 4 5 6 7 8 9 loll bility quenching solution. Wt. of subtilisin (Fig) A few experiments were carried out in which 0.5M- Fig. 6. Competition between reactivation and proteo- guanidine hydrochloride was used for 'quenching'. lysis The results were essentially similar to those from the 'subtilisin-quenched' experiments; Chan (1972) used Rabbit muscle enzyme [5-1O,ul of(NH4)2SO4 suspen- assays containing 2.3 M-urea in studying the refolding sion] was added to 50p' of 5.2M-guanidine hydro- of aldolase, and found that intermediates were un- chloride in 0.02M-triethanolamine hydrochloride, stable and only the final tetramer was stable. A com- pH7.8, containing 0.1 M-dithiothreitol. After 15min parable effect is not seen here, in that the activity does at room temperature, 1.95ml of O.1 M-sodium phos- not fall in 0.5M-guanidine hydrochloride. phate, pH6.6, containing subtilisin (Novo) was added at 4°C. The values are the percentage recovery of activity; the controls lacked subtilisin. In (a) the Rate of refolding from changes in extinction abscissa gives the ratio of isomerase to subtilisin, by The progress ofrefolding ofrabbit triose phosphate weight. In (b) the abscissa gives the weight of sub- isomerase, followed by the change in E233, is shown in tilisin; [o, 18.3,ug of isomerase; A, 25.6,ug of isomer- Fig. 7. The slow change may be instrumental drift, ase; o, 36.6,ug of isomerase. and when the values are subtracted in the usual way two phases can be resolved. The time-constants from such plots are expressed in Table 3 as half-lives, for comparison with the results obtained from regain of buffer sets a limit to the concentration of protein, activity. At the lower concentrations, the phases although the experiments with added phosphoglycol- could not be resolved. The concentrations used here, late partially offset this limitation. If the process of about 2-8 M or 50-200ug/ml, are much higher than refolding could be 'quenched' then the rate could be those used in experiments on the regain of activity, followed at higher concentrations of protein. The and about 60% of the activity was regained in the experiments on the effect of subtilisin on the recovery absence of added thiols. The values in Table 3 are of activity suggested that subtilisin could be used to imprecise, and the rates are probably independent of 'quench' refolding; unfolded chains would be split the concentration of the protein. Refolding was two and lose the ability to yield active enzyme, whereas to three times more rapid at the higher temperature. fully folded, active enzyme would be inert to subtilisin. The effect of temperature on refolding was thus This procedure was derived from experiments with comparable with its effect on reactivation, mentioned aldolase (Chan, 1972); although chain-folding of above. aldolase, as judged by physical methods, is complete The longer half-life, at both temperatures, corre- within 30s (Teipel & Koshland, 1971; Teipel, 1972) sponds to the half-life obtained for regain ofenzymic the rate of regain of stability towards trypsin was activity. Although the experiments on reactivation 1973 REFOLDING OF TRIOSE PHOSPHATE ISOMERASE 171

Table 3. Rate of refolding from changes in extinction at 233nm1 Portions (10-20jud) of stock suspension of rabbit muscle triose phosphate isomerase were centrifuged, and the pellet was taken up in 50Opl of 5M-guanidine hydrochloride in 0.02M-triethanolamine hydrochloride, pH7.8. After 2min the solution was added to 0.95 ml of 0.1 M-triethanolamine hydrochloride, pH7, at 5°C in the reference-cell position of the Unicam SP.800 spectrophotometer; the sample-cell position contained a corresponding amount of native protein in previously mixed denaturing solution and q 0 refolding solution. The E233 was followed on a Servo- scribe recorder; readings could be started about 15s 0 x after refolding started. The results are averages of duplicates; for the two experiments at higher concen- at (ACu I0 trations at 5°C and the three such experiments 9 14°C, the results showed two processes (Fig. 7) and so 7 two half-lives are given. 8 Half-life (s) 6 Concn. 5 (nM) S°C 140C 2000 64 26 4 4000 94 10 21 6000 34 83 15 59 8000 38 107 9 57 13 52* 27 95*

* Values for regain of enzymic activity, obtained as 0 500 600 100 200 300 400 described earlier from dilution into assay mixture; rabbit Time (s) muscle triose phosphate isomerase was used here for com- the results from in extinction. Fig. 7. Refolding of triose phosphate isomerase parison with changes The solid from a portion (10,ul) of stock (NH4)2SO4 suspension of rabbit enzyme was kept in 50,ul of SM- guanidine hydrochloride in 0.02M-triethanolamine phase of reassociation; refolding was much more hydrochloride, pH 7, for at least 2min and then rapid than reactivation. added to 0.95 ml of buffer in the spectrophotometer The shorter half-life obtained from the changes in ('reference-cell' position); the 'sample-cell' position extinction corresponds to the half-life for reactivation contained the same amount of enzyme added to obtained from the 'subtilisin-quenched' experiments. 0.25M-guanidine hydrochloride. The change in E233 It does not seem clear what this signifies. was followed. Three phases are shown, in order of The presence of substrate apparently increased the decreasing rate: A, o, *. For further details see the half-life for regain of activity (Table 2); similarly the text. half-life was higher (at 10.6nM-enzyme) when phosphoglycollate was present (Table 1). If the active site has to be completely assembled for ligands to be bound, these results suggest that when one of the were carried out at much lower concentrations of active sites on the dimer is occupied the other is protein, the concentrations were high enough for formed more slowly. Another possibility is that a there to be little or no variation of the rate with concentration of the protein (Fig. 3). Thus the half-life liganded dimer, in which only one monomer was correctly folded, would dissociate more slowly than for reactivation may be half-life compared with the unliganded dimer. Ligands could thus delay reactiva- for as refolding, judged by changes in extinction. It tion; this explanation is related to that put forward seems reasonable that the appearance of enzymic to account for the effect of subtilisin. activity should accompany the final changes in conformation during refolding. Teipel (1972) found that The support of the Medical Research Council is grate- reactivation of aldolase accompanied the second fully acknowledged, as is the technical assistance of Mrs. Vol. 135 172 S. G. WALEY

C. Moss. This paper is a contribution from the Oxford Ikai, A. & Tanford, C. (1973) J. Mol. Biol. 73, 145- Enzyme Group. 163 Ikai, A., Fish, W. W. & Tanford, C. (1973) J. Mol. Biol. References 73, 165-184 Johnson, L. N. & Waley, S. G. (1967) J. Mol. Biol. 29, Boyer, P. D. (1954) in Mechanism of Enzyme Action 321-322 (McElroy, W. D. & Glass, B., eds.), pp. 520-524, The Keresztes-Nagy, S. & Orman, R. (1971) Biochemistry 10, Johns Hopkins Press, Baltimore 2506-2508 Brewer, J. M. & De Sa, R. J. (1972) J. Biol. Chem. 247, Levitzki, A. (1972) FEBS Lett. 24, 301-304 7941-7947 McVittie, J. D., Esnouf, M. P. & Peacocke, A. R. (1972) Chan, W. W.-C. (1970) Biochem. Biophys. Res. Commun. Eur. J. Biochem. 29, 67-73 41, 1198-1204 Ogston, A. G. (1955) Discuss. Faraday Soc. 20, 161-167 Chan, W. W.-C. (1972) Abstr. FEBS Meet. 8th Abstr. 390 Putman, S. J., Coulson, A. F. W., Farley, I. R. T., Chan, W. W.-C. & Mawer, H. M. (1972) Arch. Biochem. Riddleston, B. & Knowles, J. R. (1972) Biochem. J. 129, Biophys. 149, 136-145 301-310 Corran, P. H. & Waley, S. G. (1973) FEBS Lett. 30, 97-99 Robinson, D. R. & Jencks, W. P. (1965) J. Amer. Chem. Donovan, J. W. (1968) Fed. Proc. Fed. Amer. Soc. Exp. Soc. 87, 2462-2470 Biol. 27, 337 Tanford, C., Kawahara, K. & Lapanje, S. (1967) J. Amer. Freedman, R. B. & Radda, G. K. (1968) Biochem. J. 108, Chem. Soc. 89, 729-736 383-391 Tanford, C., Aune, K. C. & Ikai, A. (1973)J. Mol. Biol. 73, Frieden, C. (1971) Annu. Rev. Biochem. 40, 653-696 185-197 Gawronski, T. H. & Westhead, E. W. (1969) Biochemistry Teipel, J. W. (1972) Biochemistry 11, 4100-4107 8, 4261-4270 Teipel, J. W. & Koshland, D. E. (1971) Biochemistry 10, Glazer, A. N. & Smith, E. L. (1961) J. Biol. Chem. 236, 792-798 2942-2947 von Hippel, P. H. & McGhee, J. .D (1972) Annu. Rev. Green, N. M. & Toms, E. J. (1972) Biochem. J. 130, 707- Biochem. 41, 231-300 711 Wolfenden, R. (1970) Biochemistry 9, 3404-3407

1973