- Email: [email protected]
Denaturation by urea and renaturation of 20β-hydroxysteroid dehydrogenase studied by high-performance size exclusion chromatography
AN.ALYTICAL
HIO(‘HEMISTRY
180, 181-185
(1989)
Denaturation by Urea and Renaturation of 20/3Hydroxysteroid Dehydrogenase Studied by HighPerformance Size Exclusion Chromatography Giacomo
Carrea,
Zstituto di Chimica
Received
January
Renato de&i Ormoni
Longhi,
Giorgio
Mazzola,
de1 C.N.R., Via Mario
Bianco
20@-Hydroxysteroid dehydrogenase from Streptomyces hydrogenans is an NAD-dependent enzyme that catalyzes the reversible reduction of 20-ketosteroids (1). The enzyme, which consists of four identical subunits, has a molecular weight of 110,000 (2). Previous studies have shown that immobilized subunits of 20P-hydroxysteroid dehydrogenase can fold independently, but that subunit association is essential for enzymatic activity (3,4). In a study of denaturation of this enzyme with urea the noncoincidence of transition curves obtained from different measurements (activity, intrinsic fluorescence, and circular dichroism) suggested the existence of intermediates in the unfolding process (5). In fact, transient compounds may accumulate to detectable levels when they precede a rate-limiting step in the unfolding or refolding of proteins (6-14). Among the characteristics that can be used to monitor unfolding and refolding of proteins there are the changes in size associated with the folded, unfolded, and the partially structured forms, too, when they occur as stable Copyright All rights
and Giuseppe
Vecchio
Italy
30.1989
The denaturation by urea and renaturation of 20& hydroxysteroid dehydrogenase, a tetrameric enzyme consisting of four identical subunits, were followed by high-performance size exclusion chromatography to detect intermediates in the processes. During the denaturation process no intermediate form (structured monomers or dimers) between the tetramer and the denatured monomer was observed. During the renaturation process, carried out either with or without NADH, high molecular weight aggregates, native tetramers, and low molecular weight intermediates were evidenced and quantified. The contemporaneous measurement of recovery of activity unambiguously demonstrated that the tetrameric structure is essential for enzymatic activity. 0 1989 Academic PPZSS, 1~.
0003-2697/89
Piero Pasta, 9, 20131 Milan,
$3.00
0 1989 by Academic Press, Inc. of reproduction in any form reserved.
intermediates. One of the methods used to determine such size changes is gel permeation, first introduced by Fish et al. (15) to study protein denaturation. Since suitable rigid matrices, such as silica-based packing materials resistant to high flow rates, were developed, high-performance size exclusion chromatography (HPSEC)’ has been adopted to measure hydrodynamic changes over short times (16-20). Some of the significant applications of this chromatographic technique to monomeric proteins include the studies of the denaturation process of sperm whale myoglobin and ribonuclease (6,7,21) and of the refolding process of bovine trypsinogen (22) and thioredoxin (23). We know of only one HPSEC study about oligomeric proteins, restricted to the denaturation process (7). In the present study, we used HPSEC to monitor the denaturation of 20/3-hydroxysteroid dehydrogenase by urea and its renaturation, with the aim of detecting intermediates and obtaining deeper insight into the unfolding and refolding of this oligomeric enzyme. MATERIALS
Crystalline 20P-hydroxysteroid dehydrogenase (17p, 20,21 - trihydroxysteroid: NAD+ oxidoreductase, EC 1.1.1.53), with a specific activity of 10 U/mg, was obtained from Sigma. Before use, the enzyme was dialyzed at 4°C against 0.05 M potassium phosphate buffer (pH 7) with 1 mM dithiothreitol. NADH was obtained from Boehringer, urea (for biochemistry) and sodium azide were from Merck, and Dextran Blue T 2000 was from Pharmacia. The HPSEC calibration proteins, cytochrome c, chymotrypsinogen A, hen egg albumin, bovine serum albumin, rabbit muscle aldolase, beef liver catalase, and ferritin, were purchased as the Combithek (gel 1 Abbreviation chromatography.
used:
HPSEC,
high-performance
size
exculsion
181
182
CARREA
ET AL
chromatography) kit from Boehringer. Bovine pancreatic ribonuclease A was from Serva. Horse heart myoglobin (type III), porcine stomach pepsin, and bovine milk P-lactoglobulin were obtained from Sigma. All other reagents and compounds were of analytical grade.
‘0
. 1
08
‘--A-
06 t
_
i
2 j--
:
5
‘2 . . . ‘f
METHODS
Analyses. The concentration of soluble 20@-hydroxysteroid dehydrogenase was calculated from Ea& = 9.33 cm-’ (3). Enzyme activity was determined spectrophotometrically in 0.05 M potassium phosphate buffer (pH 7), 0.12 mM NADH, and0.1 mM cortisone (3). HPLC apparatus. The HPLC system was a JASCO liquid chromatograph and the column was a IO-pm TSK-G 3000 SW (7.5 X 300 mm) obtained from LKB. The eluant flow rate was 1 ml/min and readings were made at 280 nm. The peak areas were integrated by a Hewlett-Packard Model HP-3390 integrator. Denaturation and renaturation of 20P-hydroxysteroid dehydrogenase. BOP-Hydroxysteroid dehydrogenase was incubated (0.1 mg/ml) in urea solutions of various concentrations (0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 8 M), in 0.1 M potassium phosphate buffer (pH 7) containing 1 mM dithiothreitol, at 25”C, for 30 min. At this time, the enzyme activity was measured and contemporaneously an aliquot (100 ~1, 10 pg protein) was injected into the HPSEC column, equilibrated with solutions of urea of concentrations identical to those used for the denaturation. 20/l-Hydroxysteroid dehydrogenase, fully denatured by incubation (4 mg/ml) in 6 M urea in 0.1 M potassium phosphate buffer (pH 7) containing 1 mM dithiothreitol, at 25°C for 30 min, was renatured by 40 times dilution with 0.1 M phosphate buffer (pH 7) containing 1 mM dithiothreitol. Renaturation was performed both with and without 1 mM NADH added. Enzyme activity measurements and injections (100 ~1, 10 pgprotein) into HPSEC column were performed contemporaneously at various renaturation times. The HPSEC column was equilibrated and eluted with 0.1 M phosphate buffer, 0.2 M NaCl (pH 7). RESULTS
Calibration
of HPSEC
Column
Several nondenaturing conditions were used to calibrate the column with standard proteins of different molecular weights and the best eluting system was found to be 0.1 M potassium phosphate buffer (pH 7), 0.2 M NaCl. In Fig. 1, the upper line shows the dependence of the distribution coefficient (&) on molecular weight for native proteins. A linear relationship between Kd and logarithm of molecular weights was obtained, with a correlation coefficient 0.96 (least-squares method). Our results and those of other investigators (l&20,24,25) show that not all the commonly used standard proteins fit well on
FIG. 1.
Dependenceof distribution coefficients (I&) on protein molecular weight. Native proteins (upper line): eluant 0.1 M potassium phosphate buffer (pH 7), 0.2 M NaCl; flow rate 1 ml/min; injected volume 100 11; protein concentration 0.1 mg/ml in 0.1 M potassium phosphate buffer (pH 7). Denatured proteins (lower line): eluant 0.1 M potassium phosphate buffer (pH 7), 8 M urea. Protein standards and 20@-hydroxysteroid dehydrogenase were incubated, before injection for 24 hr at 25°C with 0.1 M potassium phosphate buffer (pH 7), 10 mM dithiothreitol, 8 M urea. The elution volume of 20/Shydroxysteroid dehydrogenase did not change after 30 min of incubation without dithiothreitol. (X) 200.Hydroxysteroid dehydrogenase; (1) cytochrome c (molecular weight 12,500); (2) ribonuclease (13,700); (3) myoglobin (16,900); (4) /?-lactoglobulin monomer (18,400); (5) chymotrypsinogen A (25,000); (6) pepsin (34,700); (7) fl-lactoglobulin dimer (36,800); (8) ovalbumin (45,000); (9) bovine serum albumin (68,000); (10) aldolase (158,000); (11) catalase (240,000); (12) ferritin (450,000). Kd = (V, - V,)/( V, - V,), in which V, is the protein elution volume, V, (void volume) is the elution volume of Dextran Blue T 2000, and V, (total volume) is the elution volume of sodium azide.
the calibration curve. This could be due to matrix interactions (24,26,27) or, with oligomeric proteins, to dissociation phenomena (21). In any case, the molecular weight of native 20/!?-hydroxysteroid dehydrogenase we determined (109,000) is in good agreement with the values obtained by Blomquist (2) by agarose gel filtration (111,000) and density gradient centrifugation (106,000) under nondenaturing conditions. In Fig. 1, the lower line is the calibration curve for denatured standard proteins, using as eluant 0.1 M potassium phosphate buffer (pH 7), 8 M urea. The Kd values for denatured standard proteins plotted versus the logarithm of molecular weight show very good linearity, with a correlation coefficient 0.999. Under these conditions, we obtained a molecular weight for monomeric 20@-hydroxysteroid dehydrogenase of 27,000, identical to that obtained by Blomquist (2) by gel electrophoresis in sodium dodecyl sulfate. A linear correlation between the peak areas and the amounts of injected proteins (0.5-10 pg) was found both in denaturing and in nondenaturing conditions. Denaturation
of 20P-Hydroxysteroid
Dehydrogenase
20@-Hydroxysteroid dehydrogenase was treated with various concentrations of urea in 0.1 M potassium phosphate buffer (pH 7) for 30 min. The 30-min incubation allowed the system to come close to equilibrium. Longer
CHROMATOGRAPHIC
STUDY
OF
ENZYME
183
DENATURATION-RENATURATION
70-a 0
2 UREA
4
6
8
It.41
FIG. 3. Effects of urea concentration on the elution volume of native tetramers (0) and denatured monomers (A) of 20@-hydroxysteroid dehydrogenase. One hundred-microliter (10 fig protein) aliquots were injected.
L 0
FIG. 2. Chromatograms of 20@-hydroxysteroid dehydrogenase (100 ~1, 10 pg protein) at various concentrations of urea. (0) Peaks corresponding to native tetramers and (v) denatured monomers of 20/?hydroxysteroid dehydrogenase. (1) Phosphate buffer (pH 7); (2) 1 M urea; (3) 1.5 M urea; (4) 2 M urea; (5) 3 M urea; (6) 4 M urea; (7) 6 M urea.
incubation times were not used because, at intermediate concentrations of urea, these induced extensive formation of aggregates that could not be reactivated (5). After urea treatment the enzyme was chromatographed and some of the HPSEC chromatograms obtained are shown in Fig. 2. Up to 1.5 M urea, only the tetrameric form was present, even though there was a small decrease in elution volume as a function of urea concentration. At 2 M urea, the coexistence of the tetrameric peak with a new peak with a larger elution volume was observed. At 3 M urea and over, there was only this second peak, attributable to the denatured monomer, and its elution volume also diminished with increasing urea concentration. Figure 3 shows the changes in elution volumes of tetrameric and denatured monomeric forms, as a function of urea concentration. The percentage of 20@-hydroxysteroid dehydrogenase present as native tetramer or denatured monomer was determined (Fig. 4). Comparing the relative areas of the peaks for monomers and tetramers (Fig. 4), it can be seen that the decrease in tetramer concentration is balanced by the appearance of denatured monomers only at a high urea concentration. There is a deficit in eluted protein ascribed to unspecific adsorption of denatured monomers to column material at low urea concentrations. In any case, no peak with a hydrodynamic volume
corresponding to that of a structured dimer or monomer was evidenced. Enzyme activity was measured contemporaneously to HPLC and it can be seen that it decreases along with the decrease in the native tetrameric form (Fig. 4). Renaturation
of 20/SHydroxysteroid
Dehydrogenase
BOO-Hydroxysteroid dehydrogenase denatured in 6 M urea was renatured by diluting 40 times with 0.1 M potassium phosphate buffer (pH 7) either with or without 1 mM NADH which was found to favor the renaturation of the enzyme (3). In Fig. 5 some HPSEC chromatograms for different renaturation times without NADH are shown. At zero time a large peak of molecular weight lower than that of the tetramer is present almost exclusively. As renaturation progresses, there is an accumula-
FIG. 4. Effects of urea concentration on enzyme activity (X) and on percentage of 20fl-hydroxysteroid dehydrogenase present as native tetramers (0) or denatured monomers (A). One hundred-microliter (10 pg protein) aliquots were injected. The areas of denatured monomers were corrected taking into account the fact that the absorbance at 280 nm of the denatured enzyme, measured in 8 M urea, is 94% that of the native enzyme.
184
CARREA
5 ELUTION
10 VOLUME
,ml)
FIG. 5. Chromatograms of ZOO-hydroxysteroid dehydrogenase at different renaturation times. The enzyme, denatured in 6 M urea for 30 min, was renatured by diluting 40 times with 0.1 M potassium phosphate buffer (pH 7). Renaturation time: 0 min (l), 60 min (2), and 360 min (3). One hundred-microliter (10 pg protein) aliquots were injected.
tion of tetramer and high molecular weight aggregates, which are eluted in the void volume of the column. When NADH was added, renaturation was faster and the percentage of tetramer formed was higher. This different behavior is illustrated better in Fig. 6, which shows the percentage of the various enzyme species at different renaturation times. Figure 6 also shows the recovery of enzymatic activity. It can be seen that, when NADH is present (Fig. 6b), enzymatic activity and native tetramer recover faster and to a greater degree than in its absence where the amount of formed aggregates is high (Fig. 6a). It should be emphasized that there was a strict correlation between activity recovery and formation of tetramer.
ET
AL.
havior to a urea-induced swelling of the protein over the whole range of denaturant concentrations. It was interesting to see that, above 3 M urea, the size of the monomer exceeded that of the tetramer (which disappeared above 2 M urea). The quantitative HPSEC analysis of denaturation showed the midpoint of the tetramer decrease to be close to that of the decrease in enzyme activity (about 1 M urea) (Fig. 4). This is in agreement with circular dichroism and intrinsic fluorescence studies (5) that indicated that the loss of secondary and tertiary structure takes place at urea concentrations close to those that induce inactivation of enzyme. The deficit in eluted protein observed at low urea concentrations (Fig. 4) and ascribed by us to adsorption of denatured monomers to column material is a limiting factor in the use of HPSEC for the study of protein denaturation. Unspecific adsorptions had already been noted by Saito and Wada (7) with other denatured oligomeric enzymes and this phenomenon could be favored by the low solubility of several proteins at low concentrations of denaturant (14,28). When analyzing renaturation by HPSEC, we clearly saw high molecular weight aggregates, native tetramers, and low molecular weight intermediates and we quantified them. The peak corresponding to intermediates (approximate molecular weight of 40,000) is quite broad and asymmetric and this suggests the coexistence of mono-
100 Ia)
DISCUSSION
In this work, we used HPSEC to follow the denaturation of BOO-hydroxysteroid dehydrogenase by urea and its renaturation, to detect intermediates of the processes. The HPSEC chromatograms obtained during denaturation evidenced only the denatured monomer, without any intermediates such as structured monomers or dimers. The decrease in elution volume of the tetramer and the denatured monomer at increasing urea concentrations (Fig. 3) has already been reported by Corbett and Roche (21) for myoglobin. They attributed this be-
I J‘4 FIG. 6. Relationship of renaturation time to the recovery of activity (X) and to the percentage of 20&hydroxysteroid dehydrogenase present as partially refolded species (A), tetramers (o), and aggregates (D). Renaturation was carried out without (a) and with (b) NADH. One hundred-microliter (10 pg protein) aliquots were injected.
CHROMATOGRAPHIC
STUDY
OF
mers and dimers in rapid equilibration during the run. In any case these species are structured because previous spectroscopic investigations (29) showed that during renaturation the recovery of the secondary structure and the intrinsic fluorescence is extremely fast. When renaturation was carried out without NADH (Fig. 6a), after a certain time (about 180 min), the intermediates gave rise almost exclusively to aggregates, with no increase in the native enzyme. This suggests that renaturation is a multipathway process (see scheme).
U+M*
M** -
REFERENCES
2. Blomquist,
H. J., and Sahrholz, C. H. (1973)
Arch.
F. G. (1960) Biochem.
Biochem.
Biophys.
2. 333,
159,590-595.
95-
R., and Antonini,
E. (1980)
Biochim.
4. Carrea, G., and Pasta, P. (1987) in Methods in Enzymology bath, K., Ed.), Vol. 135, pp. 475-483, Academic Press, New 5. Vecchio, G., Pasta, chim. Biophys. Acta 6. Saito, 7. Saito,
Y., and Wada, Y., and Wada,
P., Mazzola,
G., and
Carrea,
(MosYork.
G. (1987)
Bio-
914,122-126. A. (1983) A. (1983)
Biopolymers Biopolymers
22,2105-2122. 22,2123-2132.
8. Desmadril, M., and Yon, J. M. (1984) Biochemistry 23,11-19. 9. McCoy, L. F., Rowe, E. S., and Wong, K. P. (1980) Biochemistry
19,473s4743.
12. Havel, (1986)
aggregates
The denatured monomer (U) refolds to a partially structured monomer (M*) which, in turn, can give the correctly folded monomer (M) leading to the native tetramer (M4) or can irreversibly reshuffle to an incorrect monomer (M**) which yields inactive aggregates. The presence of NADH, which stabilizes the native enzyme (final product) (3), favors reconstitution by pulling the equilibrium along the pathway leading to the tetramer. The analysis time (about 10 min) is not very short but, since renaturation of 20P-hydroxysteroid dehydrogenase takes quite a long time (see Fig. 6), it does not substantially affect the results. Moreover the dilution of the protein in the column further slows down the reassociation process, which is concentration dependent for oligomerit enzymes (14). In conclusion, HPSEC has made it possible to obtain fairly simply a “picture” of the relative concentrations of aggregates, tetramers, and intermediates in the renaturation of 20/?-hydroxysteroid dehydrogenase. Furthermore it has unambiguously demonstrated that the tetramerit structure is essential for enzymatic activity. The method appears, therefore, to be extremely promising for the study of denat,uration and, even more, of renaturation of other oligomeric enzymes, because it can furnish information similar to those obtained with methods based on the trapping of intermediates by crosslinking with bifunctional reagents (14,30) or by hybridization procedures (31,32).
1. Hubener, 105.
G., Longhi, 3. Pasta, P., Carrea, Biophys. Acta 616,143-152.
10. Mitchinson, C., and Pain, 11. Tsou, C. L. (1986) Trends
M+-+M, c
185
DENATURATION-RENATURATION
ENZYME
184,331-342.
R. H. (1985) J. Mol. Biol. Biochem. Sci. 11,427-429.
H. A., Kauffman, E. W., Plaisted, Biochemistry 25,6533-6538.
S. M., and Brems,
D. N.
13. Ghelis, C., and Yon, J. (1982) Protein Folding, pp. 374-418, demic Press, New York. 14. Jaenicke, R. (1987) Prog. Biophys. Mol. Biol. 49, 117-237.
Aca-
15. Fish, W. S., Reynolds, J. A., and Tanford, C. (1970) J. Biol. Chem. 245,5166-5168. 16. Kato, Y., Komiya, K., Sasaki, H., and Hashimoto, T. (1980) J. Chromatogr. 190,297-303. 17. Imamura, T., Konishi, K., Yokoyama, M., and Konishi, K. (1981) J. Liq. Chromatogr. 4,613-627. 18. Himmel,
M. E., and Squire,
P. G. (1981)
Znt. J. Pept.
Protein
Res.
17,365-373. 19. Ui, N. (1979) 20.
Montelaro,
Anal.
Biochem.
R. C., West,
97,65-71.
M., and Issel,
C. J. (1981)
Anal.
Biochem.
114,398p406. 21. Corbett, R. J. T., and Roche, R. S. (1984) Biochemistry 23,18881894. 22. Light, A., and Higaki, J. N. (1987) Biochemistry 26.5556-5564. 23. Shalongo, W., Ledger, R., Jagannadham, M. V., and Stellwagen, E. (1987) Biochemistry 26,3135-3141. 24. Rokushika, S., Ohkawa, T., and Hatano, H. (1979) J. Chromatogr.
176,456-461. 25. 26.
Hefti, F. (1982) Anal. Biochem. Kopaciewiecz, W., and Regnier, S-16.
121,378-381. F. E. (1982)
Anal.
Biochem.
126,
27. Regnier, F. E. (1983) in Methods in Enzymology (Hirs, C. H. W. and Timasheff, S. N., Eds.), Vol. 91, pp. 137-190, Academic Press, New York. 28. Ghelis, C., and Yon, Y. (1982) Protein Folding, pp. 470-497, Academic Press, New York. 29. Carrea, G., Pasta, P., and Vecchio, G. (1984) Biochim. Biophys. Acta 784,16-23. 30. Hermann, R., Rudolph, R., and Jaenicke, R. (1979) Nature don) 277,243-244. 31. Burns, D. L., and Schachman, H. K. (1982) J. Biol. Chem. 8638-8647. 32. Burns, D. L., and Schachman, H. K. (1982) J. Biol. Chem. 8648-8654.
(Lon257, 257,
HIO(‘HEMISTRY
180, 181-185
(1989)
Denaturation by Urea and Renaturation of 20/3Hydroxysteroid Dehydrogenase Studied by HighPerformance Size Exclusion Chromatography Giacomo
Carrea,
Zstituto di Chimica
Received
January
Renato de&i Ormoni
Longhi,
Giorgio
Mazzola,
de1 C.N.R., Via Mario
Bianco
20@-Hydroxysteroid dehydrogenase from Streptomyces hydrogenans is an NAD-dependent enzyme that catalyzes the reversible reduction of 20-ketosteroids (1). The enzyme, which consists of four identical subunits, has a molecular weight of 110,000 (2). Previous studies have shown that immobilized subunits of 20P-hydroxysteroid dehydrogenase can fold independently, but that subunit association is essential for enzymatic activity (3,4). In a study of denaturation of this enzyme with urea the noncoincidence of transition curves obtained from different measurements (activity, intrinsic fluorescence, and circular dichroism) suggested the existence of intermediates in the unfolding process (5). In fact, transient compounds may accumulate to detectable levels when they precede a rate-limiting step in the unfolding or refolding of proteins (6-14). Among the characteristics that can be used to monitor unfolding and refolding of proteins there are the changes in size associated with the folded, unfolded, and the partially structured forms, too, when they occur as stable Copyright All rights
and Giuseppe
Vecchio
Italy
30.1989
The denaturation by urea and renaturation of 20& hydroxysteroid dehydrogenase, a tetrameric enzyme consisting of four identical subunits, were followed by high-performance size exclusion chromatography to detect intermediates in the processes. During the denaturation process no intermediate form (structured monomers or dimers) between the tetramer and the denatured monomer was observed. During the renaturation process, carried out either with or without NADH, high molecular weight aggregates, native tetramers, and low molecular weight intermediates were evidenced and quantified. The contemporaneous measurement of recovery of activity unambiguously demonstrated that the tetrameric structure is essential for enzymatic activity. 0 1989 Academic PPZSS, 1~.
0003-2697/89
Piero Pasta, 9, 20131 Milan,
$3.00
0 1989 by Academic Press, Inc. of reproduction in any form reserved.
intermediates. One of the methods used to determine such size changes is gel permeation, first introduced by Fish et al. (15) to study protein denaturation. Since suitable rigid matrices, such as silica-based packing materials resistant to high flow rates, were developed, high-performance size exclusion chromatography (HPSEC)’ has been adopted to measure hydrodynamic changes over short times (16-20). Some of the significant applications of this chromatographic technique to monomeric proteins include the studies of the denaturation process of sperm whale myoglobin and ribonuclease (6,7,21) and of the refolding process of bovine trypsinogen (22) and thioredoxin (23). We know of only one HPSEC study about oligomeric proteins, restricted to the denaturation process (7). In the present study, we used HPSEC to monitor the denaturation of 20/3-hydroxysteroid dehydrogenase by urea and its renaturation, with the aim of detecting intermediates and obtaining deeper insight into the unfolding and refolding of this oligomeric enzyme. MATERIALS
Crystalline 20P-hydroxysteroid dehydrogenase (17p, 20,21 - trihydroxysteroid: NAD+ oxidoreductase, EC 1.1.1.53), with a specific activity of 10 U/mg, was obtained from Sigma. Before use, the enzyme was dialyzed at 4°C against 0.05 M potassium phosphate buffer (pH 7) with 1 mM dithiothreitol. NADH was obtained from Boehringer, urea (for biochemistry) and sodium azide were from Merck, and Dextran Blue T 2000 was from Pharmacia. The HPSEC calibration proteins, cytochrome c, chymotrypsinogen A, hen egg albumin, bovine serum albumin, rabbit muscle aldolase, beef liver catalase, and ferritin, were purchased as the Combithek (gel 1 Abbreviation chromatography.
used:
HPSEC,
high-performance
size
exculsion
181
182
CARREA
ET AL
chromatography) kit from Boehringer. Bovine pancreatic ribonuclease A was from Serva. Horse heart myoglobin (type III), porcine stomach pepsin, and bovine milk P-lactoglobulin were obtained from Sigma. All other reagents and compounds were of analytical grade.
‘0
. 1
08
‘--A-
06 t
_
i
2 j--
:
5
‘2 . . . ‘f
METHODS
Analyses. The concentration of soluble 20@-hydroxysteroid dehydrogenase was calculated from Ea& = 9.33 cm-’ (3). Enzyme activity was determined spectrophotometrically in 0.05 M potassium phosphate buffer (pH 7), 0.12 mM NADH, and0.1 mM cortisone (3). HPLC apparatus. The HPLC system was a JASCO liquid chromatograph and the column was a IO-pm TSK-G 3000 SW (7.5 X 300 mm) obtained from LKB. The eluant flow rate was 1 ml/min and readings were made at 280 nm. The peak areas were integrated by a Hewlett-Packard Model HP-3390 integrator. Denaturation and renaturation of 20P-hydroxysteroid dehydrogenase. BOP-Hydroxysteroid dehydrogenase was incubated (0.1 mg/ml) in urea solutions of various concentrations (0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 8 M), in 0.1 M potassium phosphate buffer (pH 7) containing 1 mM dithiothreitol, at 25”C, for 30 min. At this time, the enzyme activity was measured and contemporaneously an aliquot (100 ~1, 10 pg protein) was injected into the HPSEC column, equilibrated with solutions of urea of concentrations identical to those used for the denaturation. 20/l-Hydroxysteroid dehydrogenase, fully denatured by incubation (4 mg/ml) in 6 M urea in 0.1 M potassium phosphate buffer (pH 7) containing 1 mM dithiothreitol, at 25°C for 30 min, was renatured by 40 times dilution with 0.1 M phosphate buffer (pH 7) containing 1 mM dithiothreitol. Renaturation was performed both with and without 1 mM NADH added. Enzyme activity measurements and injections (100 ~1, 10 pgprotein) into HPSEC column were performed contemporaneously at various renaturation times. The HPSEC column was equilibrated and eluted with 0.1 M phosphate buffer, 0.2 M NaCl (pH 7). RESULTS
Calibration
of HPSEC
Column
Several nondenaturing conditions were used to calibrate the column with standard proteins of different molecular weights and the best eluting system was found to be 0.1 M potassium phosphate buffer (pH 7), 0.2 M NaCl. In Fig. 1, the upper line shows the dependence of the distribution coefficient (&) on molecular weight for native proteins. A linear relationship between Kd and logarithm of molecular weights was obtained, with a correlation coefficient 0.96 (least-squares method). Our results and those of other investigators (l&20,24,25) show that not all the commonly used standard proteins fit well on
FIG. 1.
Dependenceof distribution coefficients (I&) on protein molecular weight. Native proteins (upper line): eluant 0.1 M potassium phosphate buffer (pH 7), 0.2 M NaCl; flow rate 1 ml/min; injected volume 100 11; protein concentration 0.1 mg/ml in 0.1 M potassium phosphate buffer (pH 7). Denatured proteins (lower line): eluant 0.1 M potassium phosphate buffer (pH 7), 8 M urea. Protein standards and 20@-hydroxysteroid dehydrogenase were incubated, before injection for 24 hr at 25°C with 0.1 M potassium phosphate buffer (pH 7), 10 mM dithiothreitol, 8 M urea. The elution volume of 20/Shydroxysteroid dehydrogenase did not change after 30 min of incubation without dithiothreitol. (X) 200.Hydroxysteroid dehydrogenase; (1) cytochrome c (molecular weight 12,500); (2) ribonuclease (13,700); (3) myoglobin (16,900); (4) /?-lactoglobulin monomer (18,400); (5) chymotrypsinogen A (25,000); (6) pepsin (34,700); (7) fl-lactoglobulin dimer (36,800); (8) ovalbumin (45,000); (9) bovine serum albumin (68,000); (10) aldolase (158,000); (11) catalase (240,000); (12) ferritin (450,000). Kd = (V, - V,)/( V, - V,), in which V, is the protein elution volume, V, (void volume) is the elution volume of Dextran Blue T 2000, and V, (total volume) is the elution volume of sodium azide.
the calibration curve. This could be due to matrix interactions (24,26,27) or, with oligomeric proteins, to dissociation phenomena (21). In any case, the molecular weight of native 20/!?-hydroxysteroid dehydrogenase we determined (109,000) is in good agreement with the values obtained by Blomquist (2) by agarose gel filtration (111,000) and density gradient centrifugation (106,000) under nondenaturing conditions. In Fig. 1, the lower line is the calibration curve for denatured standard proteins, using as eluant 0.1 M potassium phosphate buffer (pH 7), 8 M urea. The Kd values for denatured standard proteins plotted versus the logarithm of molecular weight show very good linearity, with a correlation coefficient 0.999. Under these conditions, we obtained a molecular weight for monomeric 20@-hydroxysteroid dehydrogenase of 27,000, identical to that obtained by Blomquist (2) by gel electrophoresis in sodium dodecyl sulfate. A linear correlation between the peak areas and the amounts of injected proteins (0.5-10 pg) was found both in denaturing and in nondenaturing conditions. Denaturation
of 20P-Hydroxysteroid
Dehydrogenase
20@-Hydroxysteroid dehydrogenase was treated with various concentrations of urea in 0.1 M potassium phosphate buffer (pH 7) for 30 min. The 30-min incubation allowed the system to come close to equilibrium. Longer
CHROMATOGRAPHIC
STUDY
OF
ENZYME
183
DENATURATION-RENATURATION
70-a 0
2 UREA
4
6
8
It.41
FIG. 3. Effects of urea concentration on the elution volume of native tetramers (0) and denatured monomers (A) of 20@-hydroxysteroid dehydrogenase. One hundred-microliter (10 fig protein) aliquots were injected.
L 0
FIG. 2. Chromatograms of 20@-hydroxysteroid dehydrogenase (100 ~1, 10 pg protein) at various concentrations of urea. (0) Peaks corresponding to native tetramers and (v) denatured monomers of 20/?hydroxysteroid dehydrogenase. (1) Phosphate buffer (pH 7); (2) 1 M urea; (3) 1.5 M urea; (4) 2 M urea; (5) 3 M urea; (6) 4 M urea; (7) 6 M urea.
incubation times were not used because, at intermediate concentrations of urea, these induced extensive formation of aggregates that could not be reactivated (5). After urea treatment the enzyme was chromatographed and some of the HPSEC chromatograms obtained are shown in Fig. 2. Up to 1.5 M urea, only the tetrameric form was present, even though there was a small decrease in elution volume as a function of urea concentration. At 2 M urea, the coexistence of the tetrameric peak with a new peak with a larger elution volume was observed. At 3 M urea and over, there was only this second peak, attributable to the denatured monomer, and its elution volume also diminished with increasing urea concentration. Figure 3 shows the changes in elution volumes of tetrameric and denatured monomeric forms, as a function of urea concentration. The percentage of 20@-hydroxysteroid dehydrogenase present as native tetramer or denatured monomer was determined (Fig. 4). Comparing the relative areas of the peaks for monomers and tetramers (Fig. 4), it can be seen that the decrease in tetramer concentration is balanced by the appearance of denatured monomers only at a high urea concentration. There is a deficit in eluted protein ascribed to unspecific adsorption of denatured monomers to column material at low urea concentrations. In any case, no peak with a hydrodynamic volume
corresponding to that of a structured dimer or monomer was evidenced. Enzyme activity was measured contemporaneously to HPLC and it can be seen that it decreases along with the decrease in the native tetrameric form (Fig. 4). Renaturation
of 20/SHydroxysteroid
Dehydrogenase
BOO-Hydroxysteroid dehydrogenase denatured in 6 M urea was renatured by diluting 40 times with 0.1 M potassium phosphate buffer (pH 7) either with or without 1 mM NADH which was found to favor the renaturation of the enzyme (3). In Fig. 5 some HPSEC chromatograms for different renaturation times without NADH are shown. At zero time a large peak of molecular weight lower than that of the tetramer is present almost exclusively. As renaturation progresses, there is an accumula-
FIG. 4. Effects of urea concentration on enzyme activity (X) and on percentage of 20fl-hydroxysteroid dehydrogenase present as native tetramers (0) or denatured monomers (A). One hundred-microliter (10 pg protein) aliquots were injected. The areas of denatured monomers were corrected taking into account the fact that the absorbance at 280 nm of the denatured enzyme, measured in 8 M urea, is 94% that of the native enzyme.
184
CARREA
5 ELUTION
10 VOLUME
,ml)
FIG. 5. Chromatograms of ZOO-hydroxysteroid dehydrogenase at different renaturation times. The enzyme, denatured in 6 M urea for 30 min, was renatured by diluting 40 times with 0.1 M potassium phosphate buffer (pH 7). Renaturation time: 0 min (l), 60 min (2), and 360 min (3). One hundred-microliter (10 pg protein) aliquots were injected.
tion of tetramer and high molecular weight aggregates, which are eluted in the void volume of the column. When NADH was added, renaturation was faster and the percentage of tetramer formed was higher. This different behavior is illustrated better in Fig. 6, which shows the percentage of the various enzyme species at different renaturation times. Figure 6 also shows the recovery of enzymatic activity. It can be seen that, when NADH is present (Fig. 6b), enzymatic activity and native tetramer recover faster and to a greater degree than in its absence where the amount of formed aggregates is high (Fig. 6a). It should be emphasized that there was a strict correlation between activity recovery and formation of tetramer.
ET
AL.
havior to a urea-induced swelling of the protein over the whole range of denaturant concentrations. It was interesting to see that, above 3 M urea, the size of the monomer exceeded that of the tetramer (which disappeared above 2 M urea). The quantitative HPSEC analysis of denaturation showed the midpoint of the tetramer decrease to be close to that of the decrease in enzyme activity (about 1 M urea) (Fig. 4). This is in agreement with circular dichroism and intrinsic fluorescence studies (5) that indicated that the loss of secondary and tertiary structure takes place at urea concentrations close to those that induce inactivation of enzyme. The deficit in eluted protein observed at low urea concentrations (Fig. 4) and ascribed by us to adsorption of denatured monomers to column material is a limiting factor in the use of HPSEC for the study of protein denaturation. Unspecific adsorptions had already been noted by Saito and Wada (7) with other denatured oligomeric enzymes and this phenomenon could be favored by the low solubility of several proteins at low concentrations of denaturant (14,28). When analyzing renaturation by HPSEC, we clearly saw high molecular weight aggregates, native tetramers, and low molecular weight intermediates and we quantified them. The peak corresponding to intermediates (approximate molecular weight of 40,000) is quite broad and asymmetric and this suggests the coexistence of mono-
100 Ia)
DISCUSSION
In this work, we used HPSEC to follow the denaturation of BOO-hydroxysteroid dehydrogenase by urea and its renaturation, to detect intermediates of the processes. The HPSEC chromatograms obtained during denaturation evidenced only the denatured monomer, without any intermediates such as structured monomers or dimers. The decrease in elution volume of the tetramer and the denatured monomer at increasing urea concentrations (Fig. 3) has already been reported by Corbett and Roche (21) for myoglobin. They attributed this be-
I J‘4 FIG. 6. Relationship of renaturation time to the recovery of activity (X) and to the percentage of 20&hydroxysteroid dehydrogenase present as partially refolded species (A), tetramers (o), and aggregates (D). Renaturation was carried out without (a) and with (b) NADH. One hundred-microliter (10 pg protein) aliquots were injected.
CHROMATOGRAPHIC
STUDY
OF
mers and dimers in rapid equilibration during the run. In any case these species are structured because previous spectroscopic investigations (29) showed that during renaturation the recovery of the secondary structure and the intrinsic fluorescence is extremely fast. When renaturation was carried out without NADH (Fig. 6a), after a certain time (about 180 min), the intermediates gave rise almost exclusively to aggregates, with no increase in the native enzyme. This suggests that renaturation is a multipathway process (see scheme).
U+M*
M** -
REFERENCES
2. Blomquist,
H. J., and Sahrholz, C. H. (1973)
Arch.
F. G. (1960) Biochem.
Biochem.
Biophys.
2. 333,
159,590-595.
95-
R., and Antonini,
E. (1980)
Biochim.
4. Carrea, G., and Pasta, P. (1987) in Methods in Enzymology bath, K., Ed.), Vol. 135, pp. 475-483, Academic Press, New 5. Vecchio, G., Pasta, chim. Biophys. Acta 6. Saito, 7. Saito,
Y., and Wada, Y., and Wada,
P., Mazzola,
G., and
Carrea,
(MosYork.
G. (1987)
Bio-
914,122-126. A. (1983) A. (1983)
Biopolymers Biopolymers
22,2105-2122. 22,2123-2132.
8. Desmadril, M., and Yon, J. M. (1984) Biochemistry 23,11-19. 9. McCoy, L. F., Rowe, E. S., and Wong, K. P. (1980) Biochemistry
19,473s4743.
12. Havel, (1986)
aggregates
The denatured monomer (U) refolds to a partially structured monomer (M*) which, in turn, can give the correctly folded monomer (M) leading to the native tetramer (M4) or can irreversibly reshuffle to an incorrect monomer (M**) which yields inactive aggregates. The presence of NADH, which stabilizes the native enzyme (final product) (3), favors reconstitution by pulling the equilibrium along the pathway leading to the tetramer. The analysis time (about 10 min) is not very short but, since renaturation of 20P-hydroxysteroid dehydrogenase takes quite a long time (see Fig. 6), it does not substantially affect the results. Moreover the dilution of the protein in the column further slows down the reassociation process, which is concentration dependent for oligomerit enzymes (14). In conclusion, HPSEC has made it possible to obtain fairly simply a “picture” of the relative concentrations of aggregates, tetramers, and intermediates in the renaturation of 20/?-hydroxysteroid dehydrogenase. Furthermore it has unambiguously demonstrated that the tetramerit structure is essential for enzymatic activity. The method appears, therefore, to be extremely promising for the study of denat,uration and, even more, of renaturation of other oligomeric enzymes, because it can furnish information similar to those obtained with methods based on the trapping of intermediates by crosslinking with bifunctional reagents (14,30) or by hybridization procedures (31,32).
1. Hubener, 105.
G., Longhi, 3. Pasta, P., Carrea, Biophys. Acta 616,143-152.
10. Mitchinson, C., and Pain, 11. Tsou, C. L. (1986) Trends
M+-+M, c
185
DENATURATION-RENATURATION
ENZYME
184,331-342.
R. H. (1985) J. Mol. Biol. Biochem. Sci. 11,427-429.
H. A., Kauffman, E. W., Plaisted, Biochemistry 25,6533-6538.
S. M., and Brems,
D. N.
13. Ghelis, C., and Yon, J. (1982) Protein Folding, pp. 374-418, demic Press, New York. 14. Jaenicke, R. (1987) Prog. Biophys. Mol. Biol. 49, 117-237.
Aca-
15. Fish, W. S., Reynolds, J. A., and Tanford, C. (1970) J. Biol. Chem. 245,5166-5168. 16. Kato, Y., Komiya, K., Sasaki, H., and Hashimoto, T. (1980) J. Chromatogr. 190,297-303. 17. Imamura, T., Konishi, K., Yokoyama, M., and Konishi, K. (1981) J. Liq. Chromatogr. 4,613-627. 18. Himmel,
M. E., and Squire,
P. G. (1981)
Znt. J. Pept.
Protein
Res.
17,365-373. 19. Ui, N. (1979) 20.
Montelaro,
Anal.
Biochem.
R. C., West,
97,65-71.
M., and Issel,
C. J. (1981)
Anal.
Biochem.
114,398p406. 21. Corbett, R. J. T., and Roche, R. S. (1984) Biochemistry 23,18881894. 22. Light, A., and Higaki, J. N. (1987) Biochemistry 26.5556-5564. 23. Shalongo, W., Ledger, R., Jagannadham, M. V., and Stellwagen, E. (1987) Biochemistry 26,3135-3141. 24. Rokushika, S., Ohkawa, T., and Hatano, H. (1979) J. Chromatogr.
176,456-461. 25. 26.
Hefti, F. (1982) Anal. Biochem. Kopaciewiecz, W., and Regnier, S-16.
121,378-381. F. E. (1982)
Anal.
Biochem.
126,
27. Regnier, F. E. (1983) in Methods in Enzymology (Hirs, C. H. W. and Timasheff, S. N., Eds.), Vol. 91, pp. 137-190, Academic Press, New York. 28. Ghelis, C., and Yon, Y. (1982) Protein Folding, pp. 470-497, Academic Press, New York. 29. Carrea, G., Pasta, P., and Vecchio, G. (1984) Biochim. Biophys. Acta 784,16-23. 30. Hermann, R., Rudolph, R., and Jaenicke, R. (1979) Nature don) 277,243-244. 31. Burns, D. L., and Schachman, H. K. (1982) J. Biol. Chem. 8638-8647. 32. Burns, D. L., and Schachman, H. K. (1982) J. Biol. Chem. 8648-8654.
(Lon257, 257,