Purification and characterization of the invertase from Schizosaccharomyces pombe. A comparative analysis with the invertase from Saccharomyces cerevisiae.

Invertase (EC 3.2.1.26) was purified to homogeneity from exponentially growing cells of Schizosaccharomyces pombe fully de-repressed for synthesis of the enzyme, and was shown to be a high-molecular-mass glycoprotein that can be dissociated in the presence of 8 M-urea/1% SDS into identical subunits with an apparent molecular mass of 205 kDa. The carbohydrate moiety, accounting for 67% of the total mass, is composed of equimolar amounts of mannose and galactose. There is a small amount of glucosamine, which is probably involved in the linkage to the protein moiety, since the enzyme is sensitive to treatment with endoglycosidase H. The composition of the carbohydrate moiety resembles that found in higher-eukaryotic glycoproteins and differs from glycoproteins found in Saccharomyces cerevisiae. The protein portion of each subunit is a polypeptide of molecular mass 60 kDa, very similar to the invertase of Sacch. cerevisiae. Both proteins cross-react with antibodies raised against the protein fractions of the other, indicating that the two enzymes are similar.     

Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1.26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively.These results provide a substantial basis for the further studies of the acid invertases in apple fruit.     

Comparative study of the extracellular laccases from Cerrena unicolor 059 0784 and Pleurotus oastreatus 0432

The laccases of the basidiomycetes Cerrena unicolor 059, C. unicolor 0784, and Pleurotus oastreatus 0432 were assayed comparatively. The laccases were isolated as homogenous preparations with molecular weight 55, 56, and 57 kD, respectively. The three enzymes were found to be glycoproteins. The carbohydrate moiety of the glycoproteins included mannose, galactose, and N-acetylglucosamine. The carbohydrate moiety of the laccases from C. unicolor 059, C. unicolor 0784, and P. oastreatus 0432 accounted for 17, 23, and 24%, respectively. The pH optimum of the enzymes was at 4.0, 3.75, and 5.6, respectively. Thermal stability testing of laccases at 40 degrees C revealed that the C. unicolor 0784 enzyme was characterized by the highest thermal stability (after 172-h incubation the enzyme activity was maintained at a level of 25%). The Michaelis constant (Km) values of the reactions of oxidation of pyrocatechol, hydroquinone, and potassium ferrocyanide catalyzed by the basidiomycete laccases were determined.     

Immobilization of invertase via carbohydrate moiety on chitosan to enhance its thermal stability

A new technique using chitosan as support for covalent coupling of invertase via carbohydrate moiety improved the activity and thermal stability of immobilized invertase. The best preparation of immobilized invertase retained 91% of original specific activity (412 U mg^–1). The half-life at 60°C was increased from 2.3 h (free invertase) to 7.2 h (immobilized invertase). In contrast, the immobilization of invertase via protein moiety on chitosan or using Sepharose as support resulted in less thermostable preparations. Additionally, immobilization of invertase on both supports caused the optimal reaction pH to shift from 4.5 to 2.5 and the substrate (sucrose) concentration for maximum activity to increase from 0.5 M to 1.0 M.     

Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1.26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively.These results provide a substantial basis for the further studies of the acid invertases in apple fruit.     

Extracellular invertase from Aspergillus athecius: Isolation and immobilization

Summary A fungal strain isolated from soil and identified asAspergillus athecius, when grown on moistened wheat bran produced large amounts of extracellular invertase. Most of the invertase from the moldy bran was easily extracted by low ionic strength buffer (0.005 M, pH 5.7). The crude invertase immobilized on DEAE cellulose showed not only increased activity (45%) but also greater thermal and storage stability than the free enzyme. The free and the bound enzymes showed a temperature optimum of 50–55°C and a pH optimum of 5.7 and 4.8 respectively. The K_m app. of the bound enzyme was lower than that of the free enzyme.     

Biochemical Characterization of Soluble Acid and Alkaline Invertases from Shoots of Etiolated Pea Seedlings

Soluble invertase was purified from pea(Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation,DEAE-Sepharose column,Con-A-and Green 19-Sepharose affinity columns,hydroxyapatite column,ultra-filtration,and Sephacryl 300 gel filtration.The purified soluble acid(SAC) and alkaline(SALK) invertases had a pH optimum of 5.3 and 7.3,respectively.The temperature optimum of two invertases was 37 ℃.The effects of various concentrations of Tris-HCl,HgCl2,and CuSO4 on the activities of the two purified enzymes were examined.Tris-HCl and HgCl2 did not affect SAC activity,whereas 10 mM Tris-HCl and 0.05 mM HgCl2 inhibited SALK activity by about 50%.SAC and SALK were inhibited by 4.8 mM and 0.6 mM CuSO4 by 50%,respectively.The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis.The Kms of SAC and SALK were determined to be 1.8 and 38.6 mM,respectively.The molecular masses of SAC shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were 22 kDa and 45 kDa.The molecular mass of SALK was 30 kDa.Iso-electric points of the SAC and SALK were estimated to be about pH 7.0 and pH 5.7,respectively.     

Expression of Pichia anomala INV1 gene in Saccharomyces cerevisiae results in two different active forms of hypoglycosylated invertase

The Pichia anomala invertase gene (INV1) was introduced at different copy numbers into a sucrose-nonfermenting mutant of Saccharomyces cerevisiae and expressed from its own promoter sequences. The level reached in the production of invertase by the transformants (up to 540 units/10(10) cells) was in agreement with the INV1 gene dosage. Two forms of multimeric active and glycosylated invertase displaying different subcellular locations and molecular masses could be detected in the transformants. One was found to be present in the culture medium and in the periplasm, and the other could only be detected inside the cell. Each of the two heterologous forms of invertase was shown to be an oligomer composed of identical subunits. The difference found in the apparent molecular masses of their monomers (81.5 and 78.3 kDa, respectively) seems to be due to the size of their N-linked oligosaccharide chains (on average 2.4 and 1.9 kDa, respectively), since the number of sugar chains (9) and the molecular mass of the protein moiety (60.5 kDa) are identical in both forms. The shorter size of their oligosaccharides must also be the reason for the lower apparent molecular masses of the heterologous invertases when compared with the enzyme purified from P. anomala. The hypoglycosylated invertase accumulated within the cells of the transformants to an unusual level (up to 130 units/10(10) cells). Such accumulation of active enzyme inside the cells, as well as its underglycosylation, could be due to intrinsic properties of the P. anomala invertase that are determined by the particular primary structure of its protein moiety.     

Immobilized, thermostable S- and F-forms of the extracellular invertase from Candida utilis can hydrolyse sucrose up to 100 °C

When grown on a sucrose-containing medium, Candida utilis synthesizes and secretes two invertases: one of molecular size of 280 kDa (the S-form – Slow-migrating) and a new form of Mr of 62 kDa (the F-form – Fast-migrating). Prior to immobilization, purification of S- and F-forms of invertase increased the immobilization yield to 89–100%, in comparison with that of crude invertase preparation (52%). The immobilized purified S- and F-form of invertase remained partially active after 15 min at 100 °C; the F-form retained almost 30% of its maximum activity. The immobilized S-form or F-form of invertase almost completely inverted (95% hydrolysis) 60% (w/v) sucrose over 5 h continuous reaction at 80 °C. Moreover, at 90 °C the immobilized F-form hydrolysed 70% of 60% (w/v) sucrose over 5 h, while the capability of the immobilized S-form of inverting sucrose over 5 h reaction decreased from 80% to 45%.     

Molecular and biochemical characterization of a novel intracellular invertase from Aspergillus niger with transfructosylating activity

A novel subfamily of putative intracellular invertase enzymes (glycoside hydrolase family 32) has previously been identified in fungal genomes. Here, we report phylogenetic, molecular, and biochemical characteristics of SucB, one of two novel intracellular invertases identified in Aspergillus niger. The sucB gene was expressed in Escherichia coli and an invertase-negative strain of Saccharomyces cerevisiae. Enzyme purified from E. coli lysate displayed a molecular mass of 75 kDa, judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Its optimum pH and temperature for sucrose hydrolysis were determined to be 5.0 and 37 to 40 degrees C, respectively. In addition to sucrose, the enzyme hydrolyzed 1-kestose, nystose, and raffinose but not inulin and levan. SucB produced 1-kestose and nystose from sucrose and 1-kestose, respectively. With nystose as a substrate, products up to a degree of polymerization of 4 were observed. SucB displayed typical Michaelis-Menten kinetics with substrate inhibition on sucrose (apparent K(m), K(i), and V(max) of 2.0 +/- 0.2 mM, 268.1 +/- 18.1 mM, and 6.6 +/- 0.2 mumol min(-1) mg(-1) of protein [total activity], respectively). At sucrose concentrations up to 400 mM, transfructosylation (FTF) activity contributed approximately 20 to 30% to total activity. At higher sucrose concentrations, FTF activity increased to up to 50% of total activity. Disruption of sucB in A. niger resulted in an earlier onset of sporulation on solid medium containing various carbon sources, whereas no alteration of growth in liquid culture medium was observed. SucB thus does not play an essential role in inulin or sucrose catabolism in A. niger but may be needed for the intracellular conversion of sucrose to fructose, glucose, and small oligosaccharides.     

Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization

Two carboxylesterases (ME-III and ME-IV) have been purified to apparent homogeneity from the seeds of Mucuna pruriens employing ammonium sulfate fractionation, cation exchange chromatography on CM-cellulose, gel-permeation chromatography on Sephadex G-100 and preparative PAGE. The homogeneity of the purified preparations was confirmed by polyacrylamide gel electrophoresis (PAGE), gel-electrofocussing and SDS–PAGE. The molecular weights determined by gel-permeation chromatography on Sephadex G-200 were 20.89 kDa (ME-III) and 31.62 kDa (ME-IV). The molecular weights determined by SDS–PAGE both in the presence and absence of 2-mercaptoethanol were 21 kDa (ME-III) and 30.2 kDa (ME-IV) respectively, suggesting a monomeric structure for both the enzymes. The enzymes were found to have Stokes radius of 2.4 nm (ME-III) and 2.7 nm (ME-IV). The isoelectric pH values of the enzymes, ME-III and ME-IV, were 6.8 and 7.4, respectively. ME-III and ME-IV were classified as carboxylesterases employing PAGE in conjunction with substrate and inhibitor specificity. The K_m of ME-III and ME-IV with 1-naphthyl acetate as substrate was 0.1 and 0.166 mM while with 1-naphthyl propionate as substrate the K_m was 0.052 and 0.0454 mM, respectively. As the carbon chain length of the acyl group increased, the affinity of the substrate to the enzyme increased indicating hydrophobic nature of the acyl group binding site. The enzymes exhibited an optimum temperature of 45 °C (ME-III) and 37 °C (ME-IV), an optimum pH of 7.0 (ME-III) and 7.5 (ME-IV) and both the enzymes (ME-III and ME-IV) were stable up to 120 min at 35 °C. Both the enzymes were inhibited by organophosphates (dichlorvos and phosphamidon), but resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB).     

Immobilization and stabilization of invertase on Cajanus cajan lectin support

Use of lectins as ligands for the immobilization and stabilization of glycoenzymes has immense application in enzyme research and industry. But their widespread use could be limited by the high cost of their production. In the present study preparation of a novel and inexpensive lectin support for use in the immobilization of glycoenzymes containing mannose or glucose residues in their carbohydrate moiety has been described. Cajanus cajan lectin (CCL) coupled covalently to cyanogen bromide activated Seralose 4B could readily bind enzymes such as invertase, glucoamylase and glucose oxidase. The immobilized and glutaraldehyde crosslinked preparations of invertase exhibited high resistance to inactivation upon exposure to enhanced temperature, pH, denaturants and proteolysis. Binding of invertase to CCL-Seralose was however found to be readily reversible in the presence of 1.0 M methyl alpha-D mannopyranoside. In a laboratory scale column reactor the CCL-Seralose bound invertase was stable for a month and retained more than 80% of its initial activity even after 60 days of storage at 4 degrees C. CCL-Seralose bound invertase exhibited marked stability towards temperature, pH changes and denaturants suggesting its potential to be used as an excellent support for the immobilization of other glycoenzymes as well.     

Diverse properties of external and internal forms of yeast invertase derived from the same gene

It has been shown by genetic analysis that the external and internal invertases from Saccharomyces cerevisiae share a common structural gene [Taussig, R., & Carlson, M. (1983) Nucleic Acids Res. 11, 1943-1954]. However, the only amino acid composition of these two forms of invertase reported to date has revealed extensive differences [Gascon, S., Neumann, N.P., & Lampen, J.O. (1968) J. Biol. Chem. 243, 1573-1577]. We have found from amino acid analyses of both enzymes and sodium dodecyl sulfate-polyacrylamide gel analysis of their cyanogen bromide peptides that they are most likely identical in their amino acid sequence. However, the invertases exhibit dramatically different physical properties, particularly in their stability. The most striking difference was in their renaturation following guanidine treatment where it was shown that inactivated external invertase could be renatured completely. Endo-beta-N-acetylglucosaminidase H treated external invertase was restored to 40% of its original activity while internal invertase remained completely inactive. The observed differences may be attributed to the presence and absence of the oligosaccharide moiety in the external and internal invertases, respectively.     

Function of carbohydrate moiety in acid phosphatase of Rhodotorula glutinis

The properties of native and partially deglycosylated forms of acid phosphatase from Rhodotorula glutinis were compared. The removal of carbohydrate moiety resulted in higher thermostability and resistance to proteolysis whereas specific activity, pH optimum and Km value with p-nitrophenyl phosphate remained unchanged. The role of carbohydrate moiety in stabilization of the enzyme structure and protection against proteolysis is suggested.     

Extracellular Laccases from Cerrena unicolor 059, Cerrena unicolor 0784, and Pleurotus oastreatus 0432: A Comparative Assay

Laccases of the basidiomycetes Cerrena unicolor 059, C. unicolor 0784, and Pleurotus oastreatus 0432 were subjected to a comparative study. The enzymes were isolated as homogeneous preparations with molecular weights of 55, 56, and 57 kD, respectively. The three enzymes were found to be glycoproteins. The carbohydrate moiety of the glycoproteins included mannose, galactose, and N-acetylglucosamine. The carbohydrate moieties of the laccases from C. unicolor 059, C. unicolor 0784, and P. oastreatus 0432 accounted for 17, 23, and 24%, respectively. The pH optima of the enzymes corresponded to 4.0, 3.75, and 5.6, respectively. Thermal stability tests (carried out at 40°C) demonstrated that the laccase of C. unicolor 0784 was characterized by the highest temperature resistance (the enzyme retained 25% activity after 172 h of incubation). The values of the Michaelis constant (K _M) were determined for the reactions of oxidation of pyrocatechol, hydroquinone, and potassium ferrocyanide catalyzed by the laccases of the basidiomycetes.     

Effects of Size of Carbohydrate Chain on Protease Digestion of Aspergillus niger Endo-β-1,4-glucanase

Three different carbohydrate-depleted enzymes were prepared from an endo-β-l,4-glucanase of Aspergillus niger IF031125 by treatment with endo-β-N-acetylglucosaminidase or α-mannosidase. They were purified by Concanavalin A-Sepharose affinity and DEAE ion-exchange column chromatographies. The molecular sizes of these enzymes had been decreased from 40 kDa containing 9.0% carbohydrate to 39, 38, and 37kDa with carbohydrate at 4.5, 1.3, and 0.8% (wt/wt), respectively. The native and these carbohydrate-depleted enzymes were compared in their enzymatic properties, and it was found that they were identical in their catalytic activities and both thermal and pH stabilities. However, the 37-kDa enzyme was more susceptible to proteolysis by Savinase, proteinase K, and Pronase E. On the other hand, the specific protease trypsin showed no such effect on activity of all enzymes. These results suggested that the core structure of the asparagine-linked sugar chain, which consisted of three monosaccharide residues, contributed to the high stability of the endo-β-l,4-glucanase against protease digestion.     

Purification and partial characterization of fructosyltransferase and invertase from Aspergillus niger AS0023

Fructosyltransferase (EC.2.4.1.9) and invertase (EC.3.2.1.26) have been purified from the crude extract of Aspergillus niger AS0023 by successive chromatographies on DEAE-sephadex A-25, sepharose 6B, sephacryl S-200, and concanavalin A-Sepharose 4B columns. On acrylamide electrophoresis the two enzymes, in native and denatured forms, gave diffused glycoprotein bands with different electrophoretic mobility. On native-PAGE and SDS-PAGE, both enzymes migrated as polydisperse aggregates yielding broad and diffused bands. This result is typical of heterogeneous glycoproteins and the two enzymes have proved their glycoprotein nature by their adsorption on concanavalin A lectin. Fructosyltransferase (FTS) on native PAGE migrated as two enzymatically active bands with different electrophoretic mobility, one around 600 kDa and the other from 193 to 425 kDa. On SDS-PAGE, these two fractions yielded one band corresponding to a molecular weight range from 81 to 168 kDa. FTS seems to undergo association-dissociation of its glycoprotein subunits to form oligomers with different degrees of polymerization. Invertase (INV) showed higher mobility corresponding to a molecular range from 82 to 251 kDa, on native PAGE, and from 71 to 111 kDa on SDS-PAGE. The two enzymes exhibited distinctly different pH and temperature profiles. The optimum pH and temperature for FTS were found to be 5.8 and 50 degrees C, respectively, while INV showed optimum activity at pH 4.4 and 55 degrees C. Metal ions and other inhibitors had different effects on the two enzyme activities. FTS was completely abolished with 1 mM Hg(2+) and Ag(2+), while INV maintained 72 and 66% of its original activity, respectively. Furthermore, the two enzymes exhibited distinctly different kinetic constants confirming their different nature. The K(m) and V(m) values for each enzyme were calculated to be 44.38 mM and 1030 micromol ml(-1)min(-1) for FTS and 35.67 mM and 398 micromol ml(-1) min(-1) for INV, respectively. FTS and INV catalytic activity was dependent on sucrose concentration. FTS activity increased with increasing sucrose concentrations, while INV activity decreased markedly with increasing sucrose concentration. Furthermore, INV exhibited only hydrolytic activity producing exclusively fructose and glucose from sucrose, while FTS catalyzed exclusively fructosyltransfer reaction producing glucose, 1-kestose, nystose and fructofuranosyl nystose. In addition, at 50% sucrose concentration FTS produced fructooligosaccharides at the yield of 62% against 54% with the crude extract.     

Comparative study of stability of soluble and cell wall invertase from Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae is the most significant source of enzyme invertase. It is mainly used in the food industry as a soluble or immobilized enzyme. The greatest amount of invertase is located in the periplasmic space in yeast. In this work, it was isolated into two forms of enzyme from yeast S. cerevisiae cell, soluble and cell wall invertase (CWI). Both forms of enzyme showed same temperature optimum (60°C), similar pH optimum, and kinetic parameters. The significant difference between these biocatalysts was observed in their thermal stability, stability in urea and methanol solution. At 60°C, CWI had 1.7 times longer half-life than soluble enzyme, while at 70°C CWI showed 8.7 times longer half-life than soluble enzyme. After 2-hr of incubation in 8?M urea solution, soluble invertase and CWI retained 10 and 60% of its initial activity, respectively. During 22?hr of incubation of both enzymes in 30 and 40% methanol, soluble invertase was completely inactivated, while CWI changed its activity within the experimental error. Therefore, soluble invertase and CWI have not shown any substantial difference, but CWI showed better thermal stability and stability in some of the typical protein-denaturing agents.     

Effect of carbohydrates on fructosyltransferase expression and distribution in Streptococcus mutans GS-5 biofilms

Streptococcus mutans produces a fructosyltransferase (FTF) enzyme, which synthesizes fructan polymers from sucrose. Fructans contribute to the virulence of the biofilm by acting as binding sites for S. mutans adhesion and as extracellular nutrition reservoir for the oral bacteria. Antibodies raised against a recombinant S. mutans FTF were used to test the effect of glucose, fructose, and sucrose on FTF expression in S. mutans GS-5 biofilms. Biofilms formed in the presence of fructose and glucose showed a higher ratio of FTF compared to biofilms formed in the presence of sucrose. Confocal laser scanning microscopy images of S. mutans biofilms indicated a carbohydrate-dependent FTF distribution. The layer adjacent to the surface and those at the liquid interface displayed high amounts cell-free FTF with limited amount of bacteria while the in-between layers demonstrated both cell-free FTF and cells expressing cell-surface FTF. Biofilm of S. mutans grown on hydroxyapatite surfaces expressed several FTF bands with molecular masses of 160, 125, 120, 100, and 50 kDa, as detected by using FTF specific antibodies. The results show that FTF expression and distribution in S. mutans GS-5 biofilms is carbohydrate regulated.     

Stability, quaternary structure, and folding of internal, external, and core-glycosylated invertase from yeast.

The role of carbohydrate chains for the structure, function, stability, and folding of glycoproteins has been investigated using invertase as a model. The protein is encoded by several different genes, and its carbohydrate moiety is heterogeneous. Both properties complicate physicochemical comparisons. Here we used the temperature-sensitive sec18 secretion mutant of yeast with a single invertase gene (SUC2). This mutant produces the carbohydrate-free internal invertase, the core-glycosylated form, and, at the permissive temperature, the fully glycosylated external enzyme, all with identical protein moieties. The core-glycosylated enzyme resembles the nascent glycoprotein chain that folds in the endoplasmic reticulum. Therefore, it may be considered a model for the in vivo folding of glycoproteins. In addition, because of its uniform glycosylation, it can be used to investigate the state of association of native invertase. Glycosylation is found to stabilize the protein with respect to thermal denaturation and chaotropic solvent components; the stabilizing effect does not differ for the external and the core-glycosylated forms. Unlike the internal enzyme, the glycosylated forms are protected from aggregation. Native internal invertase is a dimer (115 kDa) whereas the core-glycosylated enzyme is a mixture of dimers, tetramers, and octamers. This implies that core-glycosylation is necessary for oligomerization to tetramers and octamers. Dimerization is required and sufficient to generate enzymatic activity; further association does not alter the specific activity of core-glycosylated invertase, suggesting that the active sites of invertase are not affected by the association of the dimeric units. Reconstitution of the glycosylated and nonglycosylated forms of the enzyme after preceding guanidine denaturation depends on protein concentration. The maximum yield (approximately 80%) is obtained at pH 6-8 and protein concentrations < or = 4 micrograms/mL for the nonglycosylated and < or = 40 for the glycosylated forms of the enzyme. The lower stability of the internal enzyme is reflected by a narrower pH range of reactivation and enhanced aggregation. As indicated by the sigmoidal reactivation kinetics at low protein concentration both folding and association are rate-determining.