Refolding of Triose Phosphate Isomerase by STEPHEN G

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.

Load more