Free Thiol Form Research Articles

Crystallization of Porcine Liver Ribonuclease Inhibitor a Member of the Family of Proteins Containing Leucine-rich Repeats

Single crystals of the RNase inhibitor from porcine liver have been obtained from 30 to 34% saturated ammonium sulphate solutions at pH 6·0 to 7·2, containing 20 mM dithiothreitol, at room temperature over a period of two to three weeks. Because the inhibitor contains 30 1/2-cystinyl residues, all of which occur in the free thiol form, crystallization experiments were carried out in a desiccator under a nitrogen atmosphere. The crystals belong to the tetragonal space group I4, with cell dimensions a = b = 134·76 Å and c = 83·65 Å. The asymmetric part of the unit cell contains two molecules with a molecular mass of 49,093 Da, as could be shown with a self-rotation function calculated in the resolution range 10·0 to 3·2 Å. The crystals diffract to at least 3·2 Å resolution and are suitable for an X-ray structure determination.

Concentration-Dependent Protection against X-Ray-Induced Chromosome Aberrations in Human Lymphocytes by the Aminothiol WR-1065

WR-1065, the free-thiol form of WR-2721, has radioprotective effects in various biological systems. We measured the efficiency of WR-1065 in modifying the induction of chromosome aberrations by X rays in human lymphocytes. G0 lymphocytes were incubated for 30 min in medium containing 1-12 mM WR-1065, exposed to 0 or 3.1 Gy 220-kV X rays, washed, and cultured for evaluations of chromosome aberrations and micronuclei (MN). Neither proliferation kinetics nor baseline frequencies of aberrations or MN were affected in nonirradiated cultures incubated in WR-1065 for up to 45 min. Radiation-induced chromosome aberrations and MN varied inversely as a logarithmic function of thiol concentration. At extracellular concentrations of 8-12 mM, WR-1065 protected against > 85% of X-ray-induced chromosome damage as measured by either cytogenetic end point. WR-1065 is more efficient in modulating X-ray-induced chromosome aberrations than dimethyl sulfoxide, which provides protection by scavenging OH radicals. Our data suggest that mechanisms in addition to OH radical scavenging are involved in radioprotection by WR-1065.

The Effect of the Radioprotector WR-2721 and WR-1065 on Mitochondrial Lipid Peroxidation

The radioprotective agent WR-2721 is dephosphorylated to the free thiol form WR-1065 in vivo. The effects of WR-2721, WR-1065 and reduced glutathione on a mitochondrial lipid peroxidation system were compared. WR-2721 had no effect on mitochondrial lipid peroxidation in vitro, and could not prevent the inactivation of mitochondrial enzymes. Both WR-1065 and glutathione were effective inhibitors of mitochondrial lipid peroxidation induced by ADP/Fe/NADPH or by ADP/Fe/ascorbate. Both thiols correspondingly delayed the free radical-mediated inactivation of succinate dehydrogenase and isocitrate dehydrogenase. WR-1065 was able to reduce cumene hydroperoxide non-enzymatically, and proved to be weak substrate for glutathione peroxidase. The disulfide formed from WR-1065 could be reduced by glutathione without the participation of glutathione reductase. A redox cycle is proposed between WR-1065, glutathione and glutathione reductase to explain the inhibitory effect of WR-1065 on lipid peroxidation.

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.

Local interactions favor the native 8-residue disulfide loop in the oxidation of a fragment corresponding to the sequence Ser-50-Met-79 derived from bovine pancreatic ribonuclease A.

A 30-residue peptide was obtained from ribonuclease A by chemical cleavage with cyanogen bromide, subsequent sulfitolysis with concomitant S-sulfonation, and finally enzymatic cleavage with Staphylococcus aureus protease. The peptide was converted to the free thiol form by reductive cleavage of the S-sulfo-protecting groups with D,L-dithiothreitol. This peptide consisted of residues 50-79 of the native sequence of ribonuclease A, with the exception that methionine-79 had been converted to homoserine. Included in this sequence are residues cysteine-65 and cysteine-72, which form a disulfide bond in the native enzyme, as well as cysteine-58. This molecule may form one of three possible intramolecular disulfide bonds upon thiol oxidation, viz. one loop of 15 and 2 of 8 residues each. These isomeric peptides were prepared by oxidation with cystamine, 2-aminoethanethiolation of residual thiols, and fractionation by reverse-phase high-performance liquid chromatography. Disulfide pairings were established by mapping the tryptic fragments and confirming their composition by amino acid analysis. After protracted incubation under oxidizing conditions at 25.0 degrees C and pH 8.0, the 26-member ring incorporating the native disulfide bond between residues 65 and 72 is the dominant product. Assuming that equilibrium is established, we infer that local interactions in the sequence of ribonuclease A significantly stabilize the native 8-residue disulfide loop with respect to the non-native 8-residue loop (delta G degree = -1.1 +/- 0.1 kcal mole-1). The implications of this observation for the oxidative folding of the intact protein are discussed.

Factors Influencing the Oxidation of the Radioprotector WR-1065

N-(2-Mercaptoethyl)-1,3-diaminopropane (WR-1065) is the free thiol form of the radio- and chemoprotector S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). Interest currently exists in the clinical use of WR-2721 and WR-1065 as radio- and chemoprotectors of normal tissues. However, measurement of plasma levels of WR-1065 has proven difficult, due to rapid drug oxidation. Therefore, we studied factors influencing the oxidation of WR-1065, in Hepes-buffered saline as well as in tissue culture media containing 10% fetal bovine serum. The rate of oxygen consumption by WR-1065, as determined using the Clark oxygen electrode system, was faster in medium plus serum than in Hepes-buffered saline. That this effect is largely due to the presence of trace metal ions in tissue culture media and serum was indicated by the observation that addition of Cu2+ or Fe3+ to buffer stimulated oxygen consumption. Addition of KCN inhibited the reaction of WR-1065 with oxygen, and this effect was dependent on KCN concentration. That KCN blocked WR-1065 oxidation to the disulfide was verified using Ellman's reagent to quantitate the free thiol form. The rate of oxygen consumption was shown to be affected by temperature as well as concentration of WR-1065. Catalase reduced the rate of oxygen consumption of WR-1065, indicating that peroxide is formed in this system. Superoxide dismutase had a stimulatory effect. WR-1065 was found to stimulate the hexose monophosphate shunt in A549 cells. Since this stimulation was prevented by the presence of catalase, it appeared to be due to the response of the cells to peroxide, formed as a result of WR-1065 autooxidation.

Role of the essential thiol group in the thiol-activated cytolysin from Clostridium perfringens.

A hemolysin, 0-toxin, produced by Clostridium perfringens has one cysteinyl residue in the free thiol form which is essential for its hemolytic activity. The cysteinyl residue was shown to be located at a position about 5 kDa from the C terminus of the molecule by the method of cysteine-specific chemical cleavage. Modification of the residue with a thiol-blocking agent, 5,5'-dithiobis(2-nitrobenzoic acid), reduced the binding affinity of the toxin to sheep erythrocytes to 1/100 that of intact toxin, resulting in a failure of binding at low cell concentrations (0.5%). Thus the failure of hemolysis at low cell concentrations is primarily ascribed to a decreased affinity of the toxin for erythrocytes. Effects of the modification on the lytic processes were examined using high cell concentrations where considerable amounts of modified toxin bound to the cells. The modified toxin hemolyzes erythrocytes once it binds to them; however, the efficiency of hemolysis is reduced by the modification. These, and additional results indicating that modification alters the sensitivity of toxin molecules to protease digestion, show that thiol-modification inactivates the toxin by affecting both binding and the subsequent lytic processes, probably through a conformational change introduced in the toxin molecules.

A method for the combined measurement of ethiofos and WR-1065 in plasma: Application to pharmacokinetic experiments with ethiofos and its metabolites

An analytical method for the combined measurement of ethiofos (WR-2721) and a major metabolite (WR-1065) in plasma is described. Plasma samples were subjected to conditions which quantitatively converted both ethiofos and bound WR-1065 to free WR-1065 which was subsequently separated by HPLC and detected electrochemically using established procedures. Although bound WR-1065 in plasma is thought to exist mainly in the form of mixed disulfides, the symmetrical disulfide, WR-33278, also was quantitatively converted to the free thiol form. Standard curves were linear over the range 0.10 to 25 μg/mL (0.75 to 186 μmol/L). Mean precision over the range was 5.4% (coefficient of variation, CV) and recoveries of various mixtures of ethiofos, WR-1065 and WR-33278 averaged 102% (CV = 6.6%). This analytical procedure and others specific for ethiofos, free WR-1065 and WR-33278 were applied to dosing experiments in which the parent drug and its major metabolites were variously administered to beagle dogs and rhesus monkeys. Following i.v. administration of ethiofos (120–150 mg per kg body weight) to monkeys, plasma concentrations of unchanged drug ranged from 477 μg/mL (2.23 mM) down to the minimum detectable limit of the analytical procedure (0.05 μg/mL, 0.23 μM) 2–3 hours postinfusion. Clearances averaged 43.5 ± 13.4 (SD) mL min −1 kg −1 and half-lives observed in the 20–60 minute postinfusion period were 8–15 min.

Dissociation of bovine cytochrome c1 subcomplex and the status of cysteine residues in the subunits.

Purified bovine heart two-band cytochrome c1 subcomplex was dissociated by treatment with p-chloromercuribenzoic acid (pCMB) into its heme subunit and a colorless subunit called hinge protein, which is essential for the formation of cytochrome c1-c complex. The subcomplex was found by titration to react with 4 mol of pCMB per mol of cytochrome c1. The contents of mercury of the dissociated heme subunit and the hinge protein were 3 and 1 mol per mol of polypeptide, respectively. These results, together with the sequence analysis, indicated that the three cysteine residues in cytochrome c1 heme subunit not involved in heme-binding existed in free thiol form. One of the five cysteine residues in the hinge protein was in free form and four in two disulfide bonds. The dissociated hinge protein was digested with staphylococcal protease and the cysteine-containing peptides were separated by reversed-phase high-performance liquid chromatography (HPLC). The content of mercury and the result of performic acid oxidation of cystine peptides revealed that Cys-30 existed in free thiol form and two disulfide bridges were formed between Cys-24 and Cys-68 and between Cys-40 and Cys-54. The conformation of the hinge protein was predicted to be composed largely of either two-alpha-helical or four-alpha-helical conformation with the amino (N)-terminal 20 residues being in a random structure.

ChemInform Abstract: ON THE INTRAMOLECULAR CYCLIZATION OF A THIAZOLIUM SALT

AbstractDas Thiazoliumchlorid (I) liefert bei der basischen Hydrolyse anstelle des erwarteten freien Thiols den Bicyclus (II).

Characterization of hydrogen sulphide reaction with rat‐tail tendon Type I collagen in vitro

Type I acid‐soluble rat‐tail tendon collagen was reacted up to four days with hydrogen sulphide (H2S) concentration equivalent to that produced by putrefying saliva in in vitro systems. After reaction, the head‐space was assayed for H2S by gas chromatography (GC), and the reaction mixture was separated into salt‐soluble supernatant fraction and sediment which was solubilized in 0.5 M acetic acid. Both fractions were analyzed by SDS‐polyacrylamide gel disc electrophoresis (SDS‐PAGE) and assayed for free aldehyde, free thiol, total available thiol, and hydroxyproline content. The GC analysis of head‐space samples showed that more than 85% of the added H2S was absorbed within the initial 24 h by the reaction medium and totally removed from the head‐space in four days. Although no discernible qualitative or quantitative changes were visually detected in H2S‐treated systems by SDS‐PAGE, increased levels of hydroxyproline in the supernatant fraction indicated production of a neutral salt‐soluble product. Chemical analysis showed that the reaction resulted in the incorporation of H2S in free thiol and disulphide forms in both fractions. [35S]‐H2S/paper chromatography methods confirmed that H2S was incorporated into collagen. An increase in free aldehyde groups of both fractions inferred exposure of aldehyde groups presumably through cleavage of intramolecular cross‐linkages.The reversion of acid‐soluble collagen to a more soluble product, which is considered more susceptible to enzymatic degradation, suggests one mechanism how the volatile thiol compounds may contribute to the etiology of periodontal disease.

On the intramolecular cyclization of a thiazolium salt

3-Acetonyl-4-methyl-1,3-thiazolium chloride is resistant to basic hydrolysis to its free-thiol form and undergoes instead a unique intramolecular cyclization to yield a stable bicyclic form.