Titration Of Sulfhydryl Groups Research Articles

Bcl-2-mediated potentiation of neocarzinostatin-induced apoptosis: requirement for caspase-3, sulfhydryl groups, and cleavable Bcl-2

Overexpression of antiapoptotic Bcl-2 family members is thought to contribute to chemotherapeutic resistance of neural crest tumors. Paradoxical potentiation by Bcl-2 of apoptosis induced by the antineoplastic prodrug, neocarzinostatin (NCS), has been observed in PC12 pheochromocytoma cells. Prior studies have indicated that the cleavage of Bcl-2 to its proapoptotic counterpart mediated by caspase-3 is responsible for this potentiation of apoptosis. This has led to the hypothesis that induction of caspase-3 expression in bcl-2-transfected, caspase-3-deficient MCF-7 cells, will result in Bcl-2 cleavage and Bcl-2-dependent potentiation of NCS-induced apoptosis. These studies have further led to the hypothesis that both cleavable Bcl-2 and sulfhydryl groups are required for the activity of caspase-3 in this regard. As hypothesized, co-transfection of bcl-2-transfected MCF-7 cells with a caspase-3 expression construct results in cleavage of Bcl-2 and potentiation of dose-dependent, NCS-mediated cell death. Furthermore, PC12 cells transfected with an expression construct for cleavage-resistant Bcl-2 demonstrated attenuated potentiation of apoptosis relative to their counterparts transfected with wild-type bcl-2. Finally, irreversible oxidative titration of sulfhydryl groups resulted in concentration-dependent attenuation of apoptosis in PC12 cells, along with prevention of caspase-3 activation and Bcl-2 cleavage. These results definitively demonstrate the requirement for caspase-3, cleavable Bcl-2, and available sulfhydryl groups (separate from those required for NCS activation) in potentiation of NCS-induced apoptosis by Bcl-2.

Reactivity of the Cysteine Residues in the Protein Splicing Active Center of the Mycobacterium tuberculosis RecA Intein

Protein splicing involves the self-catalyzed excision of an intervening polypeptide segment, an intein, from a precursor protein. The first two steps in the protein splicing process lead to the formation of ester intermediates through nucleophilic attacks by the side chains of cysteine, serine, or threonine residues adjacent to the splice junctions. Since both nucleophilic residues in the Mycobacterium tuberculosis RecA intein are cysteine, their reactivities could be compared by sulfhydryl group titration. This was accomplished by using fusion proteins containing a truncated RecA intein modified by mutation to prevent protein splicing, in which the cysteines at the splice junctions were the only sulfhydryl groups. The ability to undergo hydroxylamine-induced cleavage at the upstream splice junction showed that the modified intein was not impaired in the ability to form ester intermediates. Sulfhydryl titration with iodoacetamide, monitored by quantitating the residual thiols after reaction with a maleimide derivative of biotin, revealed a striking difference in the apparent pKa values of the cysteines at the two splice junctions. The apparent pKa of the cysteine at the upstream splice junction, which initiates the N–S acyl rearrangement leading to the linear ester intermediate, was approximately 8.2, whereas that of the cysteine residue at the downstream splice junction, which initiates the transesterification reaction converting the linear ester to the branched ester intermediate, was about 5.8. This suggests that the transesterification step is facilitated by an unusually low pKa of the attacking thiol group. Comparison of the rates of cleavage of the linear ester intermediates derived from the M. tuberculosis RecA and the Saccharomyces cerevisiae VMA inteins by dithiothreitol and hydroxylamine revealed that the former reacted relatively more slowly with dithiothreitol, suggesting that the RecA intein has diverged in the course of evolution to react preferentially with thiolate anions and thus lacks the basic groups that may facilitate nucleophilic attack by thiols in other inteins.

Effect of cysteine residues on the activity of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA synthetase (ArgRS) from Escherichia coli (E. coli) contains four cysteine residues. In this study, the role of cysteine residues in the enzyme has been investigated by chemical modification and site-directed mutagenesis. Titration of sulfhydryl groups in ArgRS by 5, 5'-dithiobis(2-nitro benzoic acid) (DTNB) suggested that a disulfide bond was not formed in the enzyme and that, in the native condition, two DTNB-sensitive cysteine residues were located on the surface of ArgRS, while the other two were buried inside. Chemical modification of the native enzyme by iodoacetamide (IAA) affected only one DTNB-sensitive cysteine residue and resulted in 50% loss of enzyme activity, while modification by N-ethylmeimide (NEM) affected two DTNB-sensitive residues and caused a complete loss of activity. These results, when combined with substrate protection experiments, suggested that at least the two cysteine residues located on the surface of the molecule were directly involved in substrates binding and catalysis. However, changing Cys to Ala only resulted in slight loss of enzymatic activity and substrate binding, suggesting that these four cysteine residues in E. coli ArgRS were not essential to the enzymatic activity. Moreover, modifications of the mutant enzymes indicated that the two DTNB- and NEM-sensitive residues were Cys(320) and Cys(537) and the IAA-sensitive was Cys(320). Our study suggested that inactivation of E. coli ArgRS by sulfhydryl reagents is a result of steric hindrance in the enzyme.

Mechanism for the inhibition of aldehyde dehydrogenase by nitric oxide

The inhibition of Saccharomyces cerevisiae aldehyde dehydrogenase (AlDH) by gaseous nitric oxide (NO) in solution and by NO generated from diethylamine nonoate was time and concentration dependent. The presence of oxygen significantly reduced the extent of inhibition by NO, indicating that NO itself rather than an oxidation of NO such as N 2O 3 is the inhibitory species under physiological conditions. A cysteine residue at the active site of the enzyme was implicated in this inhibition based on the following observations: a) NAD + and NADP +, but not reduced cofactors, significantly enhanced inhibition of AlDH by NO; b) the aldehyde substrate, benzaldehyde, blocked inhibition; and c) inhibition was accompanied by loss of free sulfhydryl groups on the enzyme. Activity of the NO-inactivated enzyme was readily restored by treatment with dithiothreitol (DTT), but not with GSH. This difference was attributed, in part, to a redox process leading to the formation of a cyclic DTT disulfide. Based on the chemistry deduced from model systems, the reaction of NO with AlDH sulfhydryls was shown to produce intramolecular disulfides and N 2O. These disulfides were shown to be intrasubunit disulfides by nonreducing SDS-PAGE analysis of the NO-inhibited enzyme. Following complete inhibition of AlDH by NO, four of the eight titratable (Ellman's reagent) sulfhydryl groups of AlDH were found to be oxidized to disulfides. These results suggest that a) the sulfhydryl group of active site Cys-302 and a proximal cysteine are oxidized to form an intrasubunit disulfide by NO; b) only two of the four subunits of AlDH are catalytically active; and c) NO preferentially oxidizes sulfhydryl groups of the catalytically active subunits. A detailed mechanisms for the inhibition of AlDH by NO is presented.

Physical, biochemical, and immunological characterization of a thermostable amidase from Klebsiella pneumoniae NCTR 1

An amidase capable of degrading acrylamide and aliphatic amides was purified to apparent homogeneity from Klebsiella pneumoniae NCTR 1. The enzyme is a monomer with an apparent molecular weight of 62,000. The pH and temperature optima of the enzyme were 7.0 and 65 degrees C, respectively. The purified amidase contained 11 5,5-dithiobis(2-nitrobenzoate) (DTNB)-titratable sulfhydryl (SH) groups. In the native enzyme 1.0 SH group readily reacted with DTNB with no detectable loss of activity. Titration of the next 3.0 SH groups with DTNB resulted in a loss of activity of more than 70%. The remaining seven inaccessible SH groups could be titrated only in the presence of 8 M guanidine hydrochloride. Titration of SH groups was strongly inhibited by carboxymethylation and KMnO4, suggesting the presence of SH groups at the active site(s). Inductively coupled plasma-atomic emission spectrometry analysis indicated that the native amidase contains 0.33 mol of cobalt and 0.33 mol of iron per mol of the native enzyme. Polyclonal antiserum against K. pneumoniae amidase was raised in rabbits, and immunochemical comparisons were made with amidases from Rhodococcus sp., Mycobacterium smegmatis, Pseudomonas chlororaphis B23, and Methylophilus methylotrophus. The antiserum immunoprecipitated and immunoreacted with the amidases of K. pneumoniae and P. chlororaphis B23. The antiserum failed to immunoreact or immunoprecipitate with other amidases.

Reconsideration of the catalytic center and mechanism of mammalian paraoxonase/arylesterase.

For three decades, mammalian paraoxonase (A-esterase, aromatic esterase, arylesterase; PON, EC 3.1.8.1) has been thought to be a cysteine esterase demonstrating structural and mechanistic homologies with the serine esterases (cholinesterases and carboxyesterases). Human, mouse, and rabbit PONs each contain only three cysteine residues, and their positions within PON have been conserved. In purified human PON, residues Cys-41 and Cys-352 form an intramolecular disulfide bond and neither could function as an active-center cysteine. Highly purified, enzymatically active PON contains a single titratable sulfhydryl group. Thus, Cys-283 is the only probable candidate for an active-center cysteine. Through site-directed mutagenesis of the human cDNA, Cys-283 was replaced with either serine (C283S) or alanine (C283A). The expressed C283 (wild-type) enzyme was inactivated by para-hydroxymercuribenzoate, but the C283S and C283A mutant enzymes were not inactivated. C283A and C283S mutant enzymes retained both paraoxonase and arylesterase activities, and the Km values for paraoxon and phenyl acetate were similar to those of the wild type. Clearly, residue Cys-283 is free in active PON, but a free sulfhydryl group is not required for either paraoxonase or arylesterase activities. Consequently, it is necessary to examine other models for the active-site structure and catalytic mechanism of PON.

Evidence for the Existence of Two Essential and Proximal Cysteinyl Residues in NADP-Malic Enzyme from Maize Leaves

Incubation of maize (Zea mays) leaf NADP-malic enzyme with monofunctional and bifunctional N-substituted maleimides results in an irreversible inactivation of the enzyme. Inactivation by the monofunctional reagents, N-ethylmaleimide (NEM) and N-phenylmaleimide, followed pseudo-first-order kinetics. The maximum inactivation rate constant for phenylmaleimide was 10-fold higher than that for NEM, suggesting a possible hydrophobic microenvironment of the residue(s) involved in the modification of the enzyme. In contrast, the inactivation kinetics with the bifunctional maleimides, ortho-, meta-, and para-phenylenebismaleimide, were biphasic, probably due to different reactivities of the groups reacting with the two heads of these bifunctional reagents, with a possible cross-linking of two sulfhydryl groups. The inactivation by mono and bifunctional maleimides was partially prevented by Mg(2+) and l-malate, and NADP prevented the inactivation almost totally. Determination of the number of reactive sulfhydryl groups of the native enzyme with [(3)H]NEM in the absence or presence of NADP showed that inactivation occurred concomitantly with the modification of two cysteinyl residues per enzyme monomer. The presence of these two essential residues was confirmed by titration of sulfhydryl groups with [(3)H]NEM in the enzyme previously modified by o-phenylenebismaleimide in the absence or presence of NADP.

Oxidation of cysteines activates cGMP-dependent protein kinase.

The functional significance of the oxidation/reduction state of sulfhydryl groups of cGMP-dependent protein kinase (cGMP kinase) was studied at 30 degrees C using different metal ions as oxidizing agents. Mn2+, Zn2+, Fe2+, Ni2+, and Co2+ failed to activate cGMP kinase, whereas Cu2+, Cu+, Fe3+, Hg2+, and Ag+ activated cGMP kinase by oxidation with an activity ratio (-cGMP/+cGMP) of about 0.7. The activation was not caused by degradation of the enzyme to a cGMP-independent constitutively active form. Reduction of the Cu(2+)-activated and gel-filtered enzyme with dithiothreitol lowered the activity ratio in the absence of cGMP to 0.17. Oxidation did not change the kinetic and binding parameters of cGMP kinase significantly but reduced the number of titratable sulfhydryl groups from 9.5 +/- 0.7 to 6.0 +/- 0.4 cysteines/75-kDa subunit. The free cysteinyl residues of the native and Cu(2+)-oxidized cGMP kinase were labeled with 4-dimethylaminoazobenzene-4'-iodoacetamide or N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. Tryptic peptides of the labeled proteins were isolated and sequenced. The cysteinyl residues oxidized by Cu2+ were identified as disulfide bonds between Cys-117 and Cys-195 and Cys-312 and Cys-518, respectively. Cu2+ activation of cGMP kinase was prevented by mild carboxymethylation of the reduced enzyme with iodoacetamide, which apparently modified these four cysteinyl groups. The results show that cGMP kinase is activated by the formation of at least one intrachain disulfide bridge.

.beta. Subunit of sodium-potassium ATPase contains three disulfide bonds

Previous studies of titratable (Na+ + K+)-ATPase sulfhydryl groups have indicated the presence of one disulfide bond per mole of holoenzyme. This single disulfide cross-link was assigned to the beta subunit on the basis of the difference between the number of titrated "free" sulfhydryl groups and the total number of titrated sulfhydryl groups for each subunit [Esmann, M. (1982) Biochim. Biophys. Acta 688, 251; Kawamura, M., & Nagano, K. (1984) Biochim. Biophys. Acta 694, 27]. In the present study, beta-subunit tryptic peptides containing disulfide cross-links were identified and purified by HPLC. Two new peptides were generated from each disulfide-bonded peptide by reduction with dithiothreitol, and the amino acid compositions of these reduced peptides were determined. The data demonstrate that there are three disulfide bonds in the native beta subunit: 125Cys-148Cys, 158Cys-174Cys, and 212Cys-275Cys. The number of disulfide bonds in the beta subunit was also estimated by titration of sulfhydryl groups with [14C]iodoacetamide. Six sulfhydryl groups were identified: two sulfhydryl groups were titrated without prior reduction, and four were identified only after reduction of the protein with dithiothreitol. These data, suggesting that the beta subunit contains two disulfide bonds, are inconsistent with the peptide isolation experiments, which directly identified three disulfide bonds in the beta subunit. This inconsistency was resolved by demonstrating that approximately 20% of each disulfide bond in the beta subunit was reduced prior to the start of the experiment, resulting in an underestimation of the number of disulfide-bonded sulfhydryl groups in the beta subunit from the titration experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

The removal of exogenous thiols from proteins by centrigugal column chromatography

Centrifugal column chromatography was shown to provide a rapid, efficient, and useful means of separation of various low molecular weight thiols from proteins. The single chromatographic step procedure employed standard 5 ml plastic syringes containing Sephadex G-25 as the bed matrix and required less than 5 min to procedure average dilutions of 5000-, 980-, and 25-fold, respectively, from 5 to 200 mM initial concentrations of 2-mercaptoethanol, dithiothreitol, and reduced glutathione in the sample as measured by titration with 5,5′-dithiobis-(2-nitrobenzoic acid). Dihydrofolate reductase solutions of 0.07–0.08 mM were separated from 50 mM 2-mercaptoethanol, dithiothreitol, or reduced glutathione with a minimum 16 500-fold dilution of the thiol after centrifugal chromatography on two consecutive columns. Thymidylate synthase solutions of 0.06 mM were effectively separated from mM 2-mercaptoethanol or dithiothreitol with a minimum average 5900-fold dilution of the thiol after consecutive column chromatography. There was no change in either the physical or chemical properties of the enzyme throughout the course of the experiments as determined by activity, active site sulfhydryl group titration, and binding assays. Recoveries of protein obtained in the load fraction were usually in excess of 70% of the protein with virtually no dilution from the initial concentration. This method was developed in order to facilitate the study of the active site sulfhydryl groups in enzymes.

Ferredoxin from clostridium pasteurianum: Preparation and properties of the cobalt derivative

The two native (4Fe-4S)-clusters of ferredoxin from Clostridium pasteurianum were replaced with cobalt to give ferredoxin containing 2 atoms of cobalt per protein molecule. The cobalt derivative had optical absorption maxima at 270 nm (ϵ = 20,000 M −1 cm −1), 400 nm(ϵ = 12,100 M −1 cm −1),474nm (ϵ = 11,000 M −1 cm −1) and a shoulder around 600 nm. The visible spectrum of the Co-substituted protein was stable in the pH region from 4.8 to 10 at 4°C for hours and did not change when stored aerobically at 4°C at pH 7.5 for several days. There were no EPR signals at 77 K even after addition of excess CN −. The titration of sulfhydryl groups with PMPS revealed that there were no free titratable cysteine residues, while in native ferredoxin 24 mole PMPS react per mole protein. Reduction of the cobalt substituted protein with sodium dithionite gave a decrease in absorbance of approximately 30% of the initial value. The cobalt was not removed from the protein by EDTA.

Sulfhydryl groups and peroxidase-like activity of albumin as scavenger of organic peroxides

The concentration, the reactivity of sulfhydryl (SH) groups and the peroxidase-like activity (PLA) of bovine serum albumin (BSA) have been determined in vitro after treatment with peroxides. Tert-butylhydroperoxide (tBOOH), cumene hydroperoxide (CuOOH), benzoyl hydroperoxide (BOOH) and hydrogen peroxide reacted with BSA, decreasing the titratable SH group concentration and increasing the value of the ratio between the reaction rate and the concentration of albumin SH groups in the sulfhydryl-disulfide exchange reaction. This value was defined as reaction constant (Kr). PLA of albumin was independent of the presence of the SH group, as SH depleted BSA maintained the same activity as the control. From our findings it derives that albumin may have two possibilities of scavenging peroxides: PLA and the SH group. The plasma SH concentration, Kr and PLA of albumin were also determined in carrageenan paw edema and in experimental adjuvant-arthritis in rats. A decrease in SH concentration, an increase in Kr and PLA of rat plasma albumin were observed in both inflammatory processes.

Identification of a monoclonal antibody specific for a neoantigenic determinant on .alpha.2-macroglobulin: use for the purification and characterization of binary proteinase-inhibitor complexes

A monoclonal antibody was obtained from the fusion of spleen cells of mice, immunized with methylamine-treated alpha 2-macroglobulin (alpha 2M), with the myeloma cell line P3-X63-Ag8.653. A competitive binding assay demonstrated that the antibody was specific for a neoantigen expressed on alpha 2M when the inhibitor reacts with proteinases or with methylamine. When immobilized, the monoclonal antibody retained its ability to specifically bind alpha 2M-proteinase complexes or methylamine-treated alpha 2M, both of which could be quantitatively recovered from the immunoaffinity column by lowering the pH to 5.0. Binary alpha 2M-proteinase complexes of trypsin, plasmin, and thrombin, prepared by incubating large amounts of alpha 2M with a small amount of enzyme, were isolated by immunoaffinity chromatography. Each purified complex was characterized with regard to proteinase content, extent of alpha 2M subunit cleavage, extent of thiol ester hydrolysis, and extent of conformational change. Each complex contained 0.8-0.9 mol of proteinase/mol of inhibitor. In the alpha 2M-thrombin, alpha 2M-plasmin, and alpha 2M-trypsin complexes, approximately 50%, 60%, and 75% of the subunits are cleaved, respectively. Titration of sulfhydryl groups revealed that all purified binary complexes contained 2 +/- 0.5 mol of thiol/mol of complex, suggesting that each complex retains two intact thiol ester bonds. When the purified complexes were incubated with excess trypsin or with methylamine, an additional 1-2 mol of sulfhydryl/mol of complex could be titrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Control of Photorespiration: Steady-State Kinetic and Conformational Changes of Ribulose-1,5-bisphosphate Carboxylase/ Oxygenase Obtained with O-p-Nitrophenylhydroxylamine

Spinach RuBP carboxylase-oxygenase (Rubisco) purified from the commercially available enzyme, underwent kinetic changes when micromolar concentrations of O-p-nitrophenylhy-droxvlamine (NPHA) were applied on fully activated enzyme. Under steady-state conditions, a decrease of Km CO2 was observed without changes in Vmax. Double reciprocal plots showed a competitive stimulation of carboxylase function. The substrate specificity factor k = Vc Ko/Vo Kc was increased from approximatively 15-20% upon treatment with NPHA, using both parallel and simultaneous determinations of RuBP-carboxylase and oxygenase activities. Such a limited change in partition between Rubisco functions actually resulted into an increase in CO2 fixation of intact soybeans leaves treated with NPHA. arising from a substantial alleviation of O2-inhibition of photosynthesis. From spectrophotometric and sulfhydryl group titration data, evidence was accumulated for a conformational change of the native enzyme, induced by NPHA. bringing about an increased ratio of carboxylase to oxygenase activities. Direct interaction of NPHA with a cysteine residue of Rubisco near the catalytic site, appears as a candidate for such chemically-induced conformational modification.

Chemical Control of Photorespiration: Steady-State Kinetic and Conformational Changes of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Obtained with O-p-Nitrophenylhydroxylamine

Spinach RuBP carboxylase-oxygenase (Rubisco) purified from the commercially available enzyme, underwent kinetic changes when micromolar concentrations of O-p-nitrophenylhy- droxvlamine (NPHA) were applied on fully activated enzyme. Under steady-state conditions, a decrease of Km CO2 was observed without changes in Vmax. Double reciprocal plots showed a competitive stimulation of carboxylase function. The substrate specificity factor k = Vc Ko/Vo Kc was increased from approximatively 15-20% upon treatment with NPHA, using both parallel and simultaneous determinations of RuBP-carboxylase and oxygenase activities. Such a limited change in partition between Rubisco functions actually resulted into an increase in CO2 fixation of intact soybeans leaves treated with NPHA. arising from a substantial alleviation of O2-inhibition of photosynthesis. From spectrophotometric and sulfhydryl group titration data, evidence was accumulated for a conformational change of the native enzyme, induced by NPHA. bringing about an increased ratio of carboxylase to oxygenase activities. Direct interaction of NPHA with a cysteine residue of Rubisco near the catalytic site, appears as a candidate for such chemically-induced conformational modification.

Characterization of purified cytochrome c1 from Rhodobacter sphaeroides R-26

Cytochrome c 1 from a photosynthetic bacterium Rhodobacter sphaeroides R-26 has been purified to homogeneity. The purified protein contains 30 nmol heme per mg protein, has an isoelectric point of 5.7, and is soluble in aqueous solution in the absence of detergents. The apparent molecular weight of this protein is about 150 000, determined by Bio Gel A-0.5 m column chromatography; a minimum molecular weight of 30 000 is obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The absorption spectrum of this cytochrome is similar to that of mammalian cytochrome c 1, but the amino acid composition and circular dichroism spectral characteristics are different. The heme moiety of cytochrome c 1 is more exposed than is that of mammalian cytochrome c 1, but less exposed than that of cytochrome c 2. Ferricytochrome c 1 undergoes photoreduction upon illumination with light under anaerobic conditions. Such photoreduction is completely abolished when p-chloromercuriphenylsulfonate is added to ferricytochrome c 1, suggesting that the sulfhydryl groups of cytochrome c 1 are the electron donors for photoreduction. Purified cytochrome c 1 contains 3 ± 0.1 mol of the p-chloromercuriphenylsulfonate titratable sulfhydryl groups per mol of protein. In contrast to mammalian cytochrome c 1, the bacterial protein does not form a stable complex with cytochrome c 2 or with mammalian cytochrome c at low ionic strength. Electron transfer between bacterial ferrocytochrome c 1 and bacterial ferricytochrome c 2, and between bacterial ferrocytochrome c 1 and mammalian ferricytochrome c proceeds rapidly with equilibrium constants of 49 and 3.5, respectively. The midpoint potential of purified cytochrome c 1 is calculated to be 228 mV, which is identical to that of mammalian cytochrome c 1.

Heparin modulates conformational states of plasma fibronectin: An electron spin resonance spin label approach

We have examined the interaction between heparin and human plasma fibronectin using electron spin resonance (ESR) spin label methods. The titratable sulfhydryl groups of plasma fibronectin were modified with a maleimide spin label [ Lai and Tooney (1984) Arch. Biochem. Biophys., 228, 465–473 ]. Addition of heparin resulted in a decrease in the maximum splitting value of the ESR spectrum of spin-labeled fibronectin from 66.8 to 64.3 G, suggesting that heparin induces a conformational alteration of plasma fibronectin. This heparin effect was noticeable at a heparin-to-fibronectin ratio of 20 to 1 and reached a plateau at about 100 to 1. Other sulfated carbohydrates were tested; dextran sulfate was found to be as effective as heparin but chondroitin sulfates were ineffective. The results presented suggest that the binding of heparin changes the molecular conformation of plasma fibronectin to a more relaxed or flexible state.

Synthesis, spectral properties, and use of Thiodan, a new thiol-specific fluorescent reagent

A new fluorescent reagent, Thiodan, containing the -SO2-S- function, is described. It can be utilized in protein chemistry as a reversible fluorescent labeling agent of cysteine residues. Thiodan is also shown to be useful for titration of sulfhydryl groups, due to the intensely fluorescent sulfinate anion released during the reaction.

Analytical ultracentrifugation studies on chloroplastic fructose bisphosphatase

Analytical ultracentrifugation studies performed on spinach chloroplast fructose bisphosphatase show that the tetrameric oxidized (inactive) or reduced (active) enzyme dissociates into inactive dimers and monomers at alkaline pH. The dissociation process is, at least, partially reversible if the enzyme is dimeric. Moreover, the oxidized inactive tetrameric enzyme is less prone to dissociation into dimers and monomers than the reduced active tetramer. The irreversibility of the dissociation process may be explained by a sulfhydryl-disulfide interchange. Together with the findings from previously published sulfhydryl group titration experiments (J. Pradel et al., Eur. J. Biochem., 113 (1981) 507), the above results suggest that the activation of the oxidized tetramer involves the reduction of two inter-protomeric disulfide bonds.

A novel low-activity form of carbonic anhydrase I in erythrocytes of patients with primary aldosteronism. Evidence for the presence of a mixed disulfide with glutathione.

A low-activity form of erythrocyte carbonic anhydrase isozyme I was found in patients with primary aldosteronism. The specific activity was very low, but the activity was restored by drug treatment as well as adrenalectomy. The enzyme was purified to homogeneity from one of the patients. With respect to its antigenicity and zinc content, the low-activity form was not distinguishable from the normal enzyme. On the other hand, the inhibition constant and binding affinity to acetazolamide of the low-activity enzyme were lower than those of normal enzyme. Sulfhydryl group titration, amino acid analysis, and peptide-mapping analysis suggested that the low-activity enzyme contains a mixed disulfide with glutathione which is closely associated with its low activity.