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• W1998499505 abstract "Protecting proteins from aggregation is one of the most important issues in both protein science and protein engineering. In this research, the mechanism of enhancing the refolding of guanidine hydrochloride-denatured carbonic anhydrase B by polyvinylpyrrolidone 40 (PVP40) was studied by both kinetic and equilibrium refolding experiments. The reactivation and refolding kinetics indicated that the rate constant of refolding the first refolding intermediate (I1) to the second one (I2) is promoted by the addition of PVP. Fluorescence quenching studies further indicated that PVP could bind to the aggregation-prone species I1, resulting in the protection of the exposed hydrophobic surface, a minimization of the protein surface, and more importantly, an increase of the refolding rate of I1. These properties were quite different from those of poly(ethylene glycol) (PEG), which has been shown to have a strong and stoichiometric binding to I1 and does not interfere with the refolding pathway. Unlike PEG, the binding of PVP to I1 does not block the aggregation pathway directly but decreases the energy barrier for I1 to refold to I2 and thus reduces the accumulation of I1. These results suggested that PVP works by a quite different mechanism from those well established ones in chaperones and chemical promoters. PVP is more like a folding catalyst rather than a chemical chaperone. The distinct mechanism of enhancing protein aggregation by PVP is expected to facilitate the attempt to develop new chemical compounds as well as new strategies to protect proteins from aggregation. Protecting proteins from aggregation is one of the most important issues in both protein science and protein engineering. In this research, the mechanism of enhancing the refolding of guanidine hydrochloride-denatured carbonic anhydrase B by polyvinylpyrrolidone 40 (PVP40) was studied by both kinetic and equilibrium refolding experiments. The reactivation and refolding kinetics indicated that the rate constant of refolding the first refolding intermediate (I1) to the second one (I2) is promoted by the addition of PVP. Fluorescence quenching studies further indicated that PVP could bind to the aggregation-prone species I1, resulting in the protection of the exposed hydrophobic surface, a minimization of the protein surface, and more importantly, an increase of the refolding rate of I1. These properties were quite different from those of poly(ethylene glycol) (PEG), which has been shown to have a strong and stoichiometric binding to I1 and does not interfere with the refolding pathway. Unlike PEG, the binding of PVP to I1 does not block the aggregation pathway directly but decreases the energy barrier for I1 to refold to I2 and thus reduces the accumulation of I1. These results suggested that PVP works by a quite different mechanism from those well established ones in chaperones and chemical promoters. PVP is more like a folding catalyst rather than a chemical chaperone. The distinct mechanism of enhancing protein aggregation by PVP is expected to facilitate the attempt to develop new chemical compounds as well as new strategies to protect proteins from aggregation. Non-native aggregation, an off-pathway product during unfolding/refolding that has been associated with more than 20 serious degenerative diseases (1Dobson C.M. Nature. 2003; 426: 884-890Crossref PubMed Scopus (3809) Google Scholar), is also a challenge in protein industry engineering (2Kopito R.R. Trends Cell Biol. 2000; 10: 524-530Abstract Full Text Full Text PDF PubMed Scopus (1612) Google Scholar). Numerous studies have been carried out to identify the determinants and driving force of protein aggregation. In general, protein molecules are prone to stick together if considerable hydrophobic residues are exposed (3Speed M.A. Wang D.I. Nat. Biotechnol. 1996; 14: 1283-1287Crossref PubMed Scopus (282) Google Scholar), and it has been widely accepted that the intermolecular cross-β motif is the core structure of aggregates (1Dobson C.M. Nature. 2003; 426: 884-890Crossref PubMed Scopus (3809) Google Scholar, 4Tycko R. Curr. Opin. Struct. Biol. 2004; 14: 96-103Crossref PubMed Scopus (354) Google Scholar). Protein folding studies have suggested that the partially denatured states, particularly the “molten globule”-like intermediates, play a crucial role in the formation of aggregation (1Dobson C.M. Nature. 2003; 426: 884-890Crossref PubMed Scopus (3809) Google Scholar, 5Ptitsyn O.B. Pain R.H. Semisotnov G.V. Zerovnik F. Razgulyaev. O.I. FEBS Lett. 1990; 262: 20-24Crossref PubMed Scopus (673) Google Scholar, 6Semisotnov G.V. Uversky V.N. Sololovsky I.V. Gutin A.M. Razgulyaev O.I. Rodionova N.A. J. Mol. Biol. 1990; 213: 561-568Crossref PubMed Scopus (48) Google Scholar, 7Fields G. Alonso D. Stiger D. Dill K. J. Phys. Chem. 1992; 96: 3974-3981Crossref Scopus (86) Google Scholar, 8Li S. Bai J.H. Park Y.D. Zhou H.M. Int. J. Biochem. Cell Biol. 2001; 33: 279-286Crossref PubMed Scopus (35) Google Scholar). The occurrence of aggregation, which facilitates non-native intermolecular interactions, competes with the refolding pathway, which facilitates the correct assembly of intramolecular interactions to the native state (9Ptitsyn O.B. Protein Folding.in: Creighton T.E. Freeman, New York1992: 243-300Google Scholar). In this case, the yield of correctly folded proteins is gradually affected by the nonproductive off-pathway aggregation. The tendency of a protein to aggregate in aqueous solution is affected by its physicochemical properties (10Chiti F. Taddei N. Baroni F. Capanni C. Stefani M. Ramponi G. Dobson C.M. Nat. Struct. Biol. 2002; 9: 137-143Crossref PubMed Scopus (377) Google Scholar), the existence of chaperones (11Martin J. Hartl F.U. Curr. Opin. Struct. Biol. 1997; 7: 41-52Crossref PubMed Scopus (165) Google Scholar), co-solutes (12Yancey P.H. Clark M.E. Hand S.C. Bowlus R.D. Somero G.N. Science. 1982; 217: 1214-1222Crossref PubMed Scopus (3030) Google Scholar), and environmental conditions (13Chi E.Y. Krishnan S. Randolph T.W. Carperter J.F. Pharm. Res. (N. Y.). 2003; 20: 1325-1336Crossref PubMed Scopus (1131) Google Scholar). Various strategies have been developed to stabilize the native protein (13Chi E.Y. Krishnan S. Randolph T.W. Carperter J.F. Pharm. Res. (N. Y.). 2003; 20: 1325-1336Crossref PubMed Scopus (1131) Google Scholar) or to enhance protein refolding from inclusion bodies in vitro (14Rudolph R. Lilie H. FASEB J. 1996; 10: 49-56Crossref PubMed Scopus (574) Google Scholar). Among these, the utilization of chemical additives, including osmolytes, surfactants, and water-soluble polymers, is of particular interest because of their good biocompatibility and wide applicability (15Tandon S. Horowitz P.M. J. Biol. Chem. 1986; 261: 15615-15618Abstract Full Text PDF PubMed Google Scholar, 16Charman S.A. Mason M.L. Charman W.N. Pharm. Res. (N. Y.). 1993; 10: 954-962Crossref PubMed Scopus (115) Google Scholar, 17Van Gelder P. De Cock H. Tommassen J. Eur. J. Biochem. 1994; 226: 783-787Crossref PubMed Scopus (27) Google Scholar, 18Wetlaufer D.B. Xie Y. Protein Sci. 1995; 4: 1535-1543Crossref PubMed Scopus (143) Google Scholar, 19Rozema D. Gellman S.H. J. Am. Chem. Soc. 1995; 117: 2373-2374Crossref Scopus (227) Google Scholar, 20Rozema D. Gellman S.H. Biochemistry. 1996; 35: 15760-15771Crossref PubMed Scopus (211) Google Scholar, 21Maa Y.F. Nguyen P.A. Hsu S.W. J. Pharm. Sci. 1998; 87: 152-159Abstract Full Text PDF PubMed Scopus (186) Google Scholar, 22Meng F.G. Park Y.D. Zhou H.M. Int. J. Biochem. Cell Biol. 2001; 33: 701-709Crossref PubMed Scopus (117) Google Scholar, 23Ou W.B. Park Y.D. Zhou H.M. Int. J. Biochem. Cell Biol. 2002; 34: 136-147Crossref PubMed Scopus (75) Google Scholar). The mechanism by which these chemical additives prevent protein aggregation has been well established (24Bolen D.W. Methods Mol. Biol. 2001; 168: 17-36PubMed Google Scholar, 25Timasheff S.N. Adv. Protein Chem. 1998; 51: 355-432Crossref PubMed Google Scholar, 26Randolph T.W. Jones L.S. Carpenter J.F. Mannings M.C. Rational Design of Stable Protein Formulations, Theory and Practice. Kluwer Academic/Plenum Publishers, New York2002: 159-175Google Scholar, 27Meng F.G. Hong Y.K. He H.W. Lyubarev A.E. Kurganov B.I. Yan Y.B. Zhou H.M. Biophys. J. 2004; 87: 2247-2254Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and most of these additives are thought to act as chemical chaperones to prevent aggregation-prone species from sticking together during protein refolding. For example, poly(ethylene glycol) (PEG), 3The abbreviations used are: PEG, poly(ethylene glycol); PVP, polyvinylpyrrolidone; CAB, carbonic anhydrase B; GdnHCl, guanidine hydrochloride; SEC, size exclusion chromatography; MG, molten globule. one of the most widely used water-soluble polymers in assisting protein refolding in vitro (28Lee J.C. Lee S.N. Biochemistry. 1987; 26: 7813-7819Crossref PubMed Scopus (202) Google Scholar), was found to specifically bind to the first refolding intermediate of bovine carbonic anhydrase B (CAB) and to perturb the self-association of the aggregation-prone intermediate (29Cleland J.L. Randolph T.W. J. Biol. Chem. 1992; 267: 3147-3153Abstract Full Text PDF PubMed Google Scholar). The stoichiometric interaction between PEG and the first molten globule (MG) intermediate of CAB results in a PEG·protein complex, which reduces the non-native dimers but does not affect the refolding rate of the first MG intermediate (30Cleland J.F. Hedgepeth C. Wang D.I. J. Biol. Chem. 1992; 267: 13327-13334Abstract Full Text PDF PubMed Google Scholar). These properties are thought to coincide with the mechanisms in chaperonin-mediated protein folding. Although blocking the off-pathway process can effectively enhance the refolding yield, the efficiency of various chemical additives is usually different from case to case and is protein- and solution condition-dependent (18Wetlaufer D.B. Xie Y. Protein Sci. 1995; 4: 1535-1543Crossref PubMed Scopus (143) Google Scholar, 22Meng F.G. Park Y.D. Zhou H.M. Int. J. Biochem. Cell Biol. 2001; 33: 701-709Crossref PubMed Scopus (117) Google Scholar, 23Ou W.B. Park Y.D. Zhou H.M. Int. J. Biochem. Cell Biol. 2002; 34: 136-147Crossref PubMed Scopus (75) Google Scholar, 31Singh R. Haque I. Ahmad F. J. Biol. Chem. 2005; 280: 11035-11042Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Thus the development of new strategies is still a challenge in the protein aggregation problem. In this research, the mechanism of enhancing refolding of guanidine hydrochloride (GdnHCl)-denatured CAB by polyvinylpyrrolidone (PVP) was studied by both kinetic and equilibrium experiments. PVP, a water-soluble polymer, has been widely used as in pharmaceutics because of its low toxicity (32Ravin H.A. Seligman A.M. Fine J. N. Engl. J. Med. 1952; 247: 921-929Crossref PubMed Scopus (123) Google Scholar, 33Polson A. Potgieter G.M. Largier J.F. Mears G.E. Joubert F.J. Biochim. Biophys. Acta. 1964; 82: 463-475Crossref PubMed Scopus (280) Google Scholar). PVP is a linear polymer with a structure similar to the structure of PEG but with more hydrophobic side chains. Considering that protein aggregation comes from intermolecular hydrophobic interactions, PVP should have the ability to bind the exposed hydrophobic parts of protein refolding intermediate by nonspecific interactions, which might protect the protein from aggregation and facilitate protein refolding. Consistent with this analysis, several studies have shown that the existence of PVP can protect proteins from thermal aggregation (34Townsend M.W. DeLuca P.P. J. Parenter. Sci. Technol. 1988; 42: 190-199PubMed Google Scholar, 35Harrison R.A. Biochem. J. 1988; 252: 875-882Crossref PubMed Scopus (25) Google Scholar, 36Gombotz W.R. Pankey S.C. Phan D. Drager R. Donaldson K. Antonsen K.P. Hoffman A.S. Raff H.V. Pharm Res. (N. Y.). 1994; 11: 624-632Crossref PubMed Scopus (52) Google Scholar, 37Remmele Jr., R.L. Nightlinger N.S. Srinivasan S. Gombotz W.R. Pharm. Res. (N. Y.). 1998; 15: 200-208Crossref PubMed Scopus (124) Google Scholar). However, it was also found that PVP could promote heat-induced aggregation in some cases (38Vrkljan M. Foster T.M. Powers M.E. Henkin J. Porter W.R. Staack H. Carpenter J.F. Manning M.C. Pharm. Res. (N. Y.). 1994; 11: 1004-1008Crossref PubMed Scopus (49) Google Scholar). These properties led us to the hypothesis that PVP might interact with the protein intermediate(s) in a different way than PEG. In this research, the effect of PVP on protein refolding was evaluated using the well studied CAB as a model protein. In both kinetic and equilibrium studies, the refolding of CAB has been found to involve two intermediates (6Semisotnov G.V. Uversky V.N. Sololovsky I.V. Gutin A.M. Razgulyaev O.I. Rodionova N.A. J. Mol. Biol. 1990; 213: 561-568Crossref PubMed Scopus (48) Google Scholar, 39Cleland J.F. Wang D.I. Biochemistry. 1990; 29: 11072-11078Crossref PubMed Scopus (147) Google Scholar). The first MG refolding intermediate (I1) is prone to aggregate and can self-associate to yield off-pathway dimer, but the second refolding intermediate (I2) is not aggregationprone (40Cleland J.L. Wang D.I. Am. Chem. Soc. Symp. Ser. 1991; 470: 169-179Crossref Google Scholar). Similar to the study concerning the PEG·CAB complex (30Cleland J.F. Hedgepeth C. Wang D.I. J. Biol. Chem. 1992; 267: 13327-13334Abstract Full Text PDF PubMed Google Scholar), it was found that PVP could also bind to the first MG refolding intermediate. The binding of PVP to the first MG refolding intermediate resulted in protection of the exposed hydrophobic surface, a decrease of the protein volume, and more importantly, an increase of the refolding rate of the first MG refolding intermediate. The mechanism by which PVP enhances protein refolding is quite different from those well established ones in osmolyte-, surfactant-, or chaperonin-mediated protein refolding. The present research sheds new light on further development of new strategies in assisting protein refolding by organic compounds. Materials—Bovine CAB (EC 4.2.1.1), GdnHCl, polyvinylpyrrolidone with a molecular mass of 40 kDa (PVP40), Tris base, sulfuric acid, p-nitrophenol acetate, N-acetyltryptophan, and acrylamide were purchased from Sigma-Aldrich. All buffers and protein solutions were prepared by using ultrapure water from a MilliQ water purification system (Millipore Corp., Bedford, MA). Preparation of the Denatured CAB—Denatured CAB samples for rapid dilution refolding were prepared by incubating the native CAB in denaturation buffer (containing 100 mm Tris sulfate, pH 7.5, and 5 m GdnHCl) for 6 h at 25 °C. Aggregation Experiments—The aggregation experiments were carried out at 25 °C through rapid dilution of GdnHCl-denatured CAB into Tris sulfate buffer with various concentrations of PVP40. The final concentration of CAB was 0.3 mg/ml. It has been proposed that the light scattering intensity is proportional to the amount of the protein in the aggregated form (41Finke J.M. Roy M. Zimm B.H. Jennings P. Biochemistry. 2000; 39: 575-583Crossref PubMed Scopus (64) Google Scholar, 42Kurganov B.I. Biochemistry (Mosc.). 2002; 67: 409-422Crossref PubMed Scopus (119) Google Scholar), and thus the aggregation of CAB was monitored by measuring the light absorption at 400 nm with an Ultraspec 4300 pro UV-visible spectrophotometer from Amersham Biosciences. Esterase Activity and Reactivation Kinetics—The CAB activity was determined by using the esterase activity assay described by Pocker and Stone (43Pocker Y. Stone J.T. Biochemistry. 1967; 6: 668-678Crossref PubMed Scopus (462) Google Scholar). The refolding samples for activity measurements were prepared by rapid dilution of the denatured CAB into dilution buffer containing different concentrations of PVP40 at a mixing ratio of 1:10. The activities of refolding samples were measured after 1 h of equilibration. The details regarding the kinetic experiments have been described elsewhere (44Pan J.C. Yu Z.H. Su X.Y. Sun Y.Q. Rao X.M. Zhou H.M. Protein Sci. 2004; 13: 1892-1901Crossref PubMed Scopus (19) Google Scholar). In brief, standard buffer was added to the GdnHCl-denatured CAB to initiate the refolding of the protein. Samples were removed at given times, and the activity of the samples was recorded immediately using a cell with a 1-cm light path length on an Ultraspec 4300 pro UV-visible spectrophotometer as a function of time. The rate of the reaction was obtained by recording the intensity increase in the absorption at 348 nm for 1 min using the maximum linear rate. The dead time before measurement was 5 s. As a control, no significant difference of native CAB activity was found between samples with or without the addition of PVP40. Stopped-flow Measurements—The samples for stopped-flow measurements were prepared in 100 mm Tris sulfate buffer, pH 7.5, with or without 1% PVP40. The changes in fluorescence upon refolding were monitored using a stopped-flow apparatus π *-180 (Applied Photophysics Ltd., Surrey, United Kingdom) at 25 °C. The mixing ratio of the GdnHCl-denatured CAB and the standard buffer was 1:10 (v/v), and the final protein concentration was 0.1 mg/ml. The fluorescence emission intensity was monitored at wavelengths above 320 nm using a 320-nm cut-off filter by excitation at 296 nm with a slit width of 1 nm. The dead time of the instrument was determined to be about 10 ms. Equilibrium Intrinsic Fluorescence—The denatured CAB was diluted into 100 mm Tris sulfate buffer containing certain concentrations of GdnHCl by a mixing ratio of 1:100 with or without 1% PVP40. The final protein concentration was 0.1 mg/ml. The samples were equilibrated for 15 h before measurement. Then the intrinsic fluorescence of each solution was measured on an F-2500 fluorescence spectrophotometer (Hitachi Ltd., Tokyo, Japan) with a 5-nm slit width for both excitation and emission. The excitation and emission wavelengths were 296 and 340 nm, respectively (45Stein P.J. Henkens R.W. J. Biol. Chem. 1978; 253: 8016-8018Abstract Full Text PDF PubMed Google Scholar, 46Rodionova N.A. Semisotnov G.V. Kutyshenko V.P. Uverskii V.N. Bolotina I.A. Bychkova V.E. Ptitsyn O.B. Mol. Biol. (Mosc.). 1989; 23: 683-692PubMed Google Scholar). The ratio of the fluorescence intensity at 340 nm of the samples with given concentrations of GdnHCl (F) to that of the native protein (F0) was defined as the relative fluorescence (F/F0) (29Cleland J.L. Randolph T.W. J. Biol. Chem. 1992; 267: 3147-3153Abstract Full Text PDF PubMed Google Scholar). The fluorescence of N-acetyltryptophan with different concentrations of GdnHCl and PVP40 in Tris sulfate buffer was also measured as control experiments. The effect of PVP40 on the fluorescence of the first MG refolding intermediate (I1) of CAB was carried out by diluting the denatured CAB into a buffer containing 2.0 m GdnHCl with different concentrations of PVP40 (0.05–4%) in the refolding solution, and the fluorescence of each solution was measured after equilibration for 6 h. The final protein concentration was 0.05 mg/ml. The relative fluorescence was defined as the variation in the fluorescence between the samples containing PVP40 (F) and the sample without PVP40 (F0). F0 was taken as 100% to normalize all data to show the percentage of change caused by different concentrations of PVP40. The effect of PVP on CAB fluorescence and the number of binding sites between PVP40 and the first MG intermediate of CAB were analyzed using the method described by Pesce et al. (47Pesce A.J. Orsen C.G. Pasby T.L. Fluorescence Spectoscopy: An Introduction for Biology and Medicine. Marcel Dekker, Inc., New York1971: 20-217Google Scholar) (see also “Results”). CAB Fluorescence Quenched by Acrylamide—The samples were prepared by diluting the denatured CAB into 100 mm Tris sulfate buffer containing different concentrations of GdnHCl and 1% PVP40. After 15 h of equilibration, each refolding sample was divided equally into two samples. The two samples were mixed with stock solutions with or without acrylamide, respectively. The final concentration of acrylamide was 0.5 m. The mixed solutions were equilibrated for an additional 2 h. In consideration of the possible volume change caused by the addition of acrylamide, the protein concentration was corrected using the samples without acrylamide. The ratio of the fluorescence without acrylamide to that in the presence of acrylamide (F0/F) was used to monitor the quenching effect of acrylamide. The concentration-dependent effect of acrylamide was evaluated by samples with or without PVP at three GdnHCl concentrations: 1.0, 1.4, and 2.0 m. The concentration range of acrylamide was 0.05–0.5 m. Size Exclusion Chromatography—Size exclusion chromatography (SEC) was performed on a Tricorn high performance column (10/300 GL) attached to an AKTA purifier (Amersham Biosciences) at 4 °C with the fractionation range for globular proteins between 10,000 and 200,000. The column was first equilibrated with 2 column volumes of elution buffer (2.0 m GdnHCl, 0.1 m Tris sulfate with or without 1% PVP40) to characterize the impact of PVP40 on the elution volume of the first intermediate present in 2 m GdnHCl. When analyzing the transient forming dimer during the refolding of denatured CAB in 5 m GdnHCl by rapid dilution to 1 m GdnHCl, the Tricorn high performance column was pretreated with 2 column volumes of elution buffer (1.0 m GdnHCl, 0.1 m Tris sulfate with or without 1% PVP40). Aliquots were taken from the refolding solution after 10 min of dilution, and then the refolding solutions were applied to the column and eluted at a flow rate of 1.0 ml/min. All solutions prepared for fast protein liquid chromatography analysis were passed through a 0.2-μm nylon filter before being measured. Effect of PVP40 on CAB Reactivation and Aggregation—To characterize the effect of PVP on CAB refolding, the aggregation and reactivation of CAB in the presence of various concentrations of PVP40 was investigated first. Consistent with previous studies (18Wetlaufer D.B. Xie Y. Protein Sci. 1995; 4: 1535-1543Crossref PubMed Scopus (143) Google Scholar), serious aggregation was observed during the refolding of CAB even at a final concentration of 0.3 mg/ml (Fig. 1A). We were surprised to find that aggregation of CAB during refolding was inhibited by the presence of PVP40 in the refolding buffer with concentrations lower than 2%, but aggregation was promoted with PVP40 concentrations higher than 2%. The optimal concentration of PVP to prevent aggregation is 1%. Consistent with the turbidity measurements, which showed that 1% PVP40 had the best capability to inhibit aggregation, 1% PVP40 was also found to be the most beneficial in assisting the reactivation of GdnHCl-denatured CAB. The optimal concentration of PVP40 for the reactivation of CAB was dependent on the final concentration of the protein in the refolding buffer. When the CAB concentration was 0.1 mg/ml in the refolding buffer, the best concentration of PVP was about 1.8%, whereas an optimal concentration was about 0.2% when the CAB concentration was 3 mg/ml (Fig. 1B). These distinct effects of PVP on protein refolding had not been observed in other osmolytes, polymers, or surfactants (18Wetlaufer D.B. Xie Y. Protein Sci. 1995; 4: 1535-1543Crossref PubMed Scopus (143) Google Scholar, 22Meng F.G. Park Y.D. Zhou H.M. Int. J. Biochem. Cell Biol. 2001; 33: 701-709Crossref PubMed Scopus (117) Google Scholar, 26Randolph T.W. Jones L.S. Carpenter J.F. Mannings M.C. Rational Design of Stable Protein Formulations, Theory and Practice. Kluwer Academic/Plenum Publishers, New York2002: 159-175Google Scholar, 27Meng F.G. Hong Y.K. He H.W. Lyubarev A.E. Kurganov B.I. Yan Y.B. Zhou H.M. Biophys. J. 2004; 87: 2247-2254Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 48Ou W.B. Park Y.D. Zhou H.M. Eur. J. Biochem. 2001; 268 (Biochemistry 21, 5918–5923): 5901-5911Crossref PubMed Scopus (25) Google Scholar), which suggested that PVP might assist protein refolding by a mechanism different from the well established one of PEG. To verify this hypothesis, the effect of PVP on CAB refolding was studied by both kinetic and equilibrium studies using a final CAB concentration in the refolding buffer of 0.1 mg/ml. This concentration was chosen because it has been shown that no aggregation will occur at this concentration (40Cleland J.L. Wang D.I. Am. Chem. Soc. Symp. Ser. 1991; 470: 169-179Crossref Google Scholar). Effect of PVP40 on CAB Reactivation Kinetics—The kinetics of reactivation was investigated by diluting the denatured CAB into the refolding buffer, and the recovery of activity was measured as a function of refolding time. The results of the control experiment showed that the presence of PVP40 would not affect the esterase activity of native CAB. As presented in Fig. 2, the reactivation of denatured CAB shows a typical biphasic process, which is quite consistent with previous studies (6Semisotnov G.V. Uversky V.N. Sololovsky I.V. Gutin A.M. Razgulyaev O.I. Rodionova N.A. J. Mol. Biol. 1990; 213: 561-568Crossref PubMed Scopus (48) Google Scholar). The final recovery of the enzyme activity was about 90% of the native enzyme for samples both with and without the addition of PVP. However, the activity recovery of the sample with PVP40 was much faster than that of the sample without PVP40. To further characterize whether the fast phase or the slow phase was affected by the addition of PVP in the dilution buffer, the kinetic parameters of the reactivation were obtained by fitting the data using a biphasic model. The rate constant of the fast phase for the sample with PVP40 in the dilution buffer ((3.0 ± 0.1) × 10–3 s–1) was somewhat faster than that of the sample without PVP40 ((1.4 ± 0.1) × 10–3 s–1), whereas that of the slow phase of the sample with PVP40 ((2.8 ± 0.2) × 10–4 s–1) showed no significant difference or was slightly slower than that of the sample without PVP40 ((4.2 ± 0.3) × 10–4 s–1). These results suggested that the refolding of the first refolding intermediate (I1) might be affected by the presence of PVP40, but the second refolding intermediate (I2) was not. To further characterize this deduction, the refolding kinetics was studied by stopped-flow experiments. Effect of PVP40 on CAB Refolding Kinetics by Stopped-flow Experiments—To further investigate whether the formation of the I1 was affected by the presence of PVP40, the refolding of CAB was monitored by the change of fluorescence using a stopped-flow apparatus. As shown in Fig. 3, three refolding phases could be observed: a major fast phase, which was completed within 100 ms after the mixing in the stopped-flow apparatus; and two minor slower phases, which had a time scale ranging from seconds to hours. The kinetic parameters were obtained by fitting the kinetic data to a 2 or 3 exponential decay function. The rate constants of CAB refolding without PVP40 were quite consistent with those found in the literature (Table 1). In the presence of PVP40, both ki1 and kn were within the experimental error of the samples without the addition of PVP40, whereas a significant increase (about 11-fold) of the refolding rate was found for ki2. These results from stopped-flow experiments clearly indicated that the presence of PVP40 affected only the refolding rate of I1 but did not affect either the formation rate of I1 from the unfolded state or the refolding of I2 to the native state. Furthermore, this specific effect of PVP40 on I1 suggested that interactions between PVP40 and I1 might exist.TABLE 1Refolding rate constants of CAB measured by stopped-flow kineticsRate constantIn literatureaThe rate constants were from the data reported by Cleland and Wang (39) with the unit of measure converted from min-1 to s-1.Without PVPWith 1% PVPki1bThe rate constants were obtained by fitting the 5-s stopped-flow data using double exponential kinetics. (s-1)23.131.6 ± 0.430.0 ± 0.2ki2cThe rate constants were obtained by fitting the 500-s stopped-flow data using triple exponential kinetics. (×10-2 s-1)2.321.58 ± 0.0617.6 ± 0.1kncThe rate constants were obtained by fitting the 500-s stopped-flow data using triple exponential kinetics. (×10-3 s-1)1.213.4 ± 0.39.16 ± 0.01a The rate constants were from the data reported by Cleland and Wang (39Cleland J.F. Wang D.I. Biochemistry. 1990; 29: 11072-11078Crossref PubMed Scopus (147) Google Scholar) with the unit of measure converted from min-1 to s-1.b The rate constants were obtained by fitting the 5-s stopped-flow data using double exponential kinetics.c The rate constants were obtained by fitting the 500-s stopped-flow data using triple exponential kinetics. Open table in a new tab Effect of PVP40 on CAB Equilibrium Refolding—The possible binding of PVP40 to I1 was explored by equilibrium refolding studies. The denatured CAB was diluted to a given GdnHCl concentration with or without 1% PVP40. A time course study indicated that the sample did not reach equilibrium after 4–6 h equilibration, and a 15-h equilibration was necessary for further experiments. The intrinsic fluorescence of each sample was measured to reflect the state of the sample. The fluorescence data were normalized by the fluorescence intensity of native CAB at 340 nm (F0); the normalized data F/F0 are shown in Fig. 4. Consistent with a previous study (39Cleland J.F. Wang D.I. Biochemistry. 1990; 29: 11072-11078Crossref PubMed Scopus (147) Google Scholar), the relative fluorescence curve of the equilibrium refolding of CAB had a plateau between 1.5 and 2.0 m GdnHCl. The slight increase of the intrinsic fluorescence of samples with a GdnHCl concentration between 0 and 1.5 m might arise from the occurrence of small aggregates after a long incubation time. With the addition of 1% PVP40" @default.
• W1998499505 created "2016-06-24" @default.
• W1998499505 creator A5025615610 @default.
• W1998499505 creator A5066441869 @default.
• W1998499505 creator A5083781460 @default.
• W1998499505 date "2006-04-01" @default.
• W1998499505 modified "2023-10-17" @default.
• W1998499505 title "Polyvinylpyrrolidone 40 Assists the Refolding of Bovine Carbonic Anhydrase B by Accelerating the Refolding of the First Molten Globule Intermediate" @default.
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