Free Sulphydryl Groups Research Articles

Inhibition of L-type Ca current in guinea pig ventricular myocytes by blocking free sulphydryl groups

Inhibition of L-type Ca current in guinea pig ventricular myocytes by blocking free sulphydryl groups

Anomalies in reduction-mediated technetium-99m labelling of monoclonal antibodies

A reduction-mediated technetium-99m labelling method has been evaluated with a range of tumour-specific monoclonal antibodies. Antibodies reduced with 2-mercaptoethanol (2-ME) had free sulphydryl groups, but their number was much higher than could be accounted for by only limited intra-chain reduction of disulphide bonds. Reduced antibody could be labelled efficiently with 99mTc using an methylene diphosphonate (MDP) bone-scanning kit, although this seemed to depend on the presence of residual 2-ME in the preparations. With a carcinoembryonic antigen (CEA)-specific antibody, immunoreactivity of labelled antibody was confirmed, and after injection into nude with CEA-producing xenografts there was localisation into the tumours. Sephacryl S300 gel filtration showed the radiolabel eluting at a single discrete peak at the expected 150 kDa. However, examination of all of the labelled antibodies by polyacrylamide gel electrophoresis (PAGE) and autoradiography showed the presence of a large number of radiolabelled low molecular weight degradation products of the antibodies. These degradation products seemed to be formed in previously reduced antibodies during processing for PAGE, indicating some fragility of the reduced antibody.

Detecting pathogenic microorganisms in products for human consumption by eliciting enzyme production by the microorganism and detection with a fluorogenic substrate : Berg J D US-117481; 12 September 1990

Glucose oxidase (Gox), transglutaminase (TG), and pentosanase (Pn) were investigated for their effect on bread quality. The changes introduced in wheat protein by the action of these enzymes were analysed to explain dough behaviour. Gox treatment decreased free sulphydryl groups (SHf), increased glutenin macropolymer contents, and modified the electrophoretic pattern of protein fractions. Gox modified mainly albumin, globulin, and glutenin, forming large protein aggregates. These modifications explained the high strength of the dough and the low bread specific volume of samples with Gox. TG treatment modified solubility in SDS of protein and decreased glutenin macropolymer content. However, it formed large protein aggregates. The new cross-linking bonds introduced by this enzyme were different to S–S bonds and, consequently, the dough was less extensible and showed high resistance. Pn treatment increased water soluble pentosan content. Moreover, in these samples a tendency to increase SHf content was observed. In addition, Pn increased protein solubility in isopropanol, which indicates that the reduction of pentosans size decreases steric impediment of insoluble pentosans, thus increasing interaction among protein and making their extraction easier. These changes at the microscopic level allowed explaining the formation of softer dough and the production of higher specific volume in breads with Pn.

Beneficial effects of N-acetylcysteine and cysteine in stunned myocardium in perfused rat heart.

1. The objective of this study was to evaluate the effects of three sulphydryl (SH) compounds, N-acetylcysteine (NAC), cysteine (Cys) and cystamine, on functional recovery and ventricular arrhythmias (VF) in stunned myocardium in the isolated perfused heart of the rat. 2. Hearts (n = 7-8 per group) were perfused by the Langendorff procedure for 20 min to stabilize and then assigned to one of five groups: saline, sham, NAC, Cys and cystamine. After the stabilizing period, the drugs (at 3.6 microM min-1) or their vehicle (saline) were infused into coronary vessels throughout the experimental period. Ten min after administration of drugs, the left anterior descending coronary artery (LAD) was ligatured for 20 min and then untied to reperfuse for 30 min. In the sham group, a ligature was placed around the LAD but not tied. 3. NAC and Cys had a significant effect in attenuating myocardial stunning: the percentage recovery of rate-pressure product measured 30 min after reperfusion as an index of heart function, was improved with the NAC (98.3 +/- 4.5) and Cys groups (104.0 +/- 6.5) compared with the saline (only 73.6 +/- 3.8, P < 0.01) group. Cystamine did not show these beneficial effects. This may be due to the difference in chemical structure between NAC, Cys and cystamine since the latter does not have a free SH group with a disulphide bond formed. This phenomenon suggests that a free SH group is essential for the protective effects of compounds like NAC and Cys in myocardial injury. 4. NAC and Cys prevented the fall in coronary flow during the LAD occlusion and enhanced coronary flow during reperfusion but cystamine did not have such a beneficial effect. 5. The incidence of VF in the saline, cystamine, Cys and NAC groups was 6/8 (75.0%), 4/7 (57.1%), 3/8 (37.5%) and 2/7 (28.6%), respectively, and no significant differences (P > 0.05) were noted between the saline- and drug-treated groups. 6. An in vitro study with electron spin resonance indicated that Cys effectively scavenged the hydroxyl radical (-OH) generated by Fenton's reaction but did not scavenge superoxide generated in an irradiated riboflavin system. NAC and cystamine showed a scavenging effect on -OH to a certain extent but this effect did not reach statistical significance (P > 0.05 vs saline). 7. Our results demonstrate that NAC and Cys treatment before ischaemia and reperfusion can reduce myocardial stunning. This beneficial effect may be mainly due to their ability to preserve and enhance coronary flow during coronary occlusion and reperfusion and in part due to scavenging -OH and/or replenishing intracellular glutathione. The results also indicate that the condition of coronary perfusion can produce a great impact on postischaemic ventricular performance.

Time-dependent polymerization of β-lactoglobulin through disulphide bonds at the oil-water interface in emulsions

Time-dependent intermolecular sulphydryl-disulphide interchange involving β-lactoglobulin adsorbed at the oil—water interface in n-tetradecane-in-water emulsions (10 wt% oil, 0.5 wt% protein, pH 7.0) has been investigated using sodium dodecyl sulphate—polyacrylamide gel electrophoresis (SDS—PAGE). While only monomers are detected in the adsorbed protein immediately after emulsion formation with pure β-lactoglobulin, on storing the emulsion the amount of polymerized β-lactoglobulin and the sizes of the oligomers are found to increase with time. There is no polymerization of adsorbed protein in emulsions made with pure α-lactalbumin after 72 h, or in emulsions made with β-lactoglobulin in the presence of a reagent ( N-ethylmaleimide) for modifying sulphydryl groups. Analysis by two-dimensional SDS—PAGE of adsorbed protein from aged emulsions made with a mixture of α-lactalbumin + β-lactoglobulin shows some linking by disulphide bonds between α-lactalbumin and β-lactoglobulin at the interface. Taken together with earlier time-dependent surface viscosity measurements, the results indicate the important role of free sulphydryl groups in the development of the high surface viscoelasticity of adsorbed globular proteins at the oil—water interface.

Allergenicity and Physicochemical Characterization of House Dust Mite Derived Amylase

The enzyme amylase was shown to be present in extracts prepared from both house dust and spent growth medium used in the culture of the mite Dermatophagoides pteronyssinus. In dust, it was shown to correlate with both mite counts and concentrations of the faecally derived mite allergen, Der p I. Mite amylase was isolated from the culture medium and shown to be a single chain protein with a molecular weight of 56,000. The enzyme contained free sulphydryl groups and had the N-terminal sequence, KYXNPHFIGXRSVITXLME. It was found to be an allergen using sera from adults (46% positive) and children (25%) who were mite allergic. The expression of allergenicity was dependent on the integrity of intra-chain disulphide bonds.

Evidence for the Importance of a Carboxyl Group in the Binding of Ligands to the D2 Dopamine Receptor

A series of group specific modifying reagents were tested for their effects on [3H]spiperone binding to brain D2 dopamine receptors to identify amino acid residues at the binding site of the D2 dopamine receptor that are critical for ligand binding. The dependence of ligand binding to the receptor on the pH of the incubation medium was also examined. N-Acetylimidazole, 5,5'-dithiobis(2-nitrobenzoic acid), 1,2-cyclohexanedione, and acetic anhydride had no specific effect on [3H]spiperone binding, indicating the lack of participation of tyrosine, free sulphydryl, arginine, or primary amino groups in ligand binding to the receptor. N,N'-Dicyclohexylcarbodiimide (DCCD) potently reduced the number of [3H]spiperone binding sites, indicating that a carboxyl group is involved in ligand binding to the receptor. The effects of DCCD could be prevented by prior incubation of the receptor with D2 dopamine receptor selective compounds. The pH-binding profile for [3H]spiperone binding indicated the importance of an ionising group of pKa 5.2 for ligand binding which may be the same carboxyl group. Diethyl pyrocarbonate, the histidine modifying reagent, also inhibited [3H]spiperone binding, reducing the affinity of the receptor for this ligand but the effects were not at the ligand binding site. From the effects of pH changes on ligand binding some evidence was obtained for a second ionising group (pKa 7.0) that specifically affects the binding of substituted benzamide drugs to the receptor. It is concluded that the D2 dopamine receptor binding site contains separate but over-lapping binding regions for antagonists such as spiperone and substituted benzamide drugs. The former region contains an important carboxyl group; the latter region contains another group that may be a second carboxyl group or a histidine.

Thiols in flour and baking quality

Baking quality (LV) was defined as loaf volume per gram of loaf protein. Further attempts to find negative correlation between LV and small thiols bound by disulphide bonds (SS) to gluten failed, but this was almost certainly because the method was flawed. A new approach was devised to measure on the same sample: free sulphydryl groups (SH) in glutenin, diffusible thiols in flour, and thiols bound to protein by SS. Both methods showed the existence of bound thiols; corrected values in the old method had a mean of 1·5 μmol per gram of gluten; in the new they ranged from ∼ 0·9 to 2·3 μmol per gram of flour protein with a mean of 1·6. Altogether three ways were tried of measuring diffusible thiols in flour (possibly the rheologically active ones). The most specific reagent measured 14–16% of accessible thiol as diffusible. A second method gave slightly lower values but this could be explained by side reactions. High values, with a third reagent, were almost certainly due to incomplete reaction. Probably for the first time, it was shown that untreated glutenin molecules in flour carry free SH. The use of phenyl mercuric acetate suggested ∼ 1/8 SH a glutenin chain (of average mol. wt 50 000). Higher results with iodoacetamide ranged from 0·17 to 1·1, but these probably included extra SH, from reaction of glutenin with active thiols before they could be blocked, and/or from attack on labile SS in glutenin. The amount of free SH on glutenin was negatively correlated with LV in the Chorleywood Bread Process (CBP), but not significantly in the 3 h long fermentation process (LFP). The results suggest that an active thiol attacks a glutenin SS when flour is wetted to add both SH and bound thiol; it can also form an SS with an SH on glutenin by oxidation. The hypothesis that the mol. wt of glutenin controls about half the variation in baking quality is compatible with recent findings that baking quality depends on the type of high molecular weight subunit in glutenin. Payne found one-third to two-thirds the variation in baking quality was linked with variation of subunit composition, so both the mol.wt of glutenin, and its high mol.wt subunits, affect baking quality. Further studies of the effect of thiols on the mol.wt of glutenin (and therefore on baking quality) could well be done on wheats of constant subunit composition. With a commercial flour the CBP was less tolerant of added thiols than the LFP. Cysteine is only about a tenth as active as the others in the LFP. Its effect (when large amounts were used) did not seem to involve an enzyme since the d- and l-forms were equally active. In a trial with 24 flours, protease activity was uncorrelated with LV. A further measurement was made of SH in three stored flours.

Effect of a Free Sulphydryl Group on Expression of HLA-B27 Specificity

Sequence studies indicate that the alpha-1 domain of the HLA-B27 molecule has a characteristic unpaired cysteine residue at position 67, adjacent, because of secondary structure, to a lysine at position 70. Simple chemical considerations predict that this cysteine should have an exceptionally reactive sulphydryl group. We have shown by ELISA and flow cytometry that the binding of some monoclonal antibodies to B27 on lymphoid cell lines can be inhibited by reagents which react with sulphydryl groups. However this inhibition is never complete: the evidence suggests 2 forms of B27 molecule, one of which is already blocked. We propose that some HLA molecules with oxidised sulphydryls are recognised as different from the reduced forms. Whether they are also recognised as foreign will depend on an individual's history of thymic learning. Oxidation to 'foreign' HLA in the adult is likely to predispose to inflammatory reactions.

Changes of Oat Globulin During Storage With Sodium Dodecyl Sulfate

Structural changes occurring during storage of oat globulin solutions containing 0 to 37mM sodium dodecyl sulfate (SDS) were examined by SDS polyacrylamide gel electrophoresis (PAGE). Oat globulin solutions stored with SDS (20mM) at room temperature showed dissociation of the oat globulin subunit (mean MW 58,000) into acidic (AP) and basic (BP) polypeptides of mean MW 36,000 and 22,000 respectively. This dissociation of subunits in the absence of a reducing agent (β-mercaptoethanol) was caused by a base-catalyzed sulphydryl-disulfide exchange reaction, since blocking of free sulphydryl groups by iodoacetate or N-ethylmaleimide prevented the dissociation of subunits. The changes emphasize the need to avoid extended storage before SDS-PAGE analysis of oat globulin and other proteins containing disulfide bonds and sulphydryl groups.

The reaction of S-mercuric-N-dansylcysteine with acetylcholinesterase and butyrylcholinesterase.

S-mercuric-N-dansylcysteine was investigated as a potential probe of protein sulphydryl groups using bovine serum albumin, S-carboxymethyl-bovine serum albumin, lysozyme, and partially reduced lysozyme as test proteins. Criteria used to assess covalent binding through mercury-bridged mercaptide linkages include a finite reaction time (minutes to hours), abolition of the characteristic fluorescence spectrum following addition of a reducing agent, and failure to separate probe and protein after chromatography or electrophoresis. By these criteria, both Torpedo californica acetylcholinesterase and human serum cholinesterase (butyrylcholinesterase) contain four free sulphydryl groups per tetrameric enzyme molecule whereas Electrophorus electricus acetylcholinesterase has none. Labeled acetylcholinesterase and butyrylcholinesterase remain active and responsive to the inactivator Zn2+. Zn2+ promotes an increase in the fluorescence of bound S-mercuric-N-dansylcysteine, whereas activators such as Mg2+ or gallamine promote a decrease, suggesting that the label may be a useful probe of ligand-induced conformational changes. With T. californica acetylcholinesterase, but not with human serum cholinesterase, Zn2+ also promotes access to two additional groups that are reactive towards the sulphydryl reagent.

Structure of a major pea seed albumin: Implication of a free sulphydryl group

AbstractRecent studies in a number of laboratories have characterised many of the properties of a major pea albumin (designated PMA or PA2: subunit Mr∼26 000) which is not readily degraded in the cotyledon upon germination. In the present report, some of the physicochemical properties of this protein have been studied in greater detail. A non‐helical structure, stable at high pH, is evident. Of special significance is the presence of a single free sulphydryl group, although studies reported elsewhere show that the protein consists of four copies of a strongly conserved repeated sequence. It is postulated that this free sulphydryl group in PA2 (5–10% of total pea seed protein) causes polymerisation through disulphide interchange and thereby is a likely source of partial insolubility in protein isolates following an initial alkaline extraction.

Purification and biochemical properties of pitton's type 2 beta-lactamase (SHV 1)

A five-step procedure has been developed for purifying Pitton's type 2 plasmid-mediated beta-lactamase (PIT-2, also called SHV-1) from cultures of a hyperproducing variant of an Escherichia coli K12 strain carrying the plasmid p453. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a single protein band with a molecular weight of about 27,500. The amino acid composition of the protein and the amino acid sequence of the NH2-terminal region have been determined. PIT-2 enzyme contains 272 amino acid residues with 2 cysteines. Studies of the S-carboxymethylated protein (previously reduced or unreduced) suggest that these two residues are presumably in the form of free sulphydryl groups in the native protein. Conversely, TEM-2 beta-lactamase contains 2 cysteine residues which are in the form of a disulphide bond. Comparison of PIT-2 with other beta-lactamases was made using the difference index (DI) of Metzger et al. The PIT-2 enzyme appeared more closely related to the TEM-type penicillinases (DI of 6.5).

Chemical studies on protease A of Bitis arietans (puff adder) venom

Protease A of Bitis arietans venom is probably a metalloprotease, since it is inhibited by o-phenanthroline and contains 0.77 moles of zinc per mole protein. The enzyme comprises 213 amino acids, including 9 methionine residues and one free sulphydryl group. It contains one polypeptide chain, which is terminated at the carboxyl end by serine. The amino terminal sequence of protease A is: Arg-Ser-Ser-Asp-Pro-Asn- Lys-Tyr-Phe-Asn-Val-Ile-Val-Val-Val-Asp-Asn-Arg-Met-Val-Asn-Tyr-Tyr-Lys-Gly-Glu-Leu- Asn-Lys-Ile-Thr-. Despite difficulties with ‘insoluble peptide core’ formation, a number of peptides were purified from peptic and tryptic digests of S-derivatized protease A.

Modification of the free sulphydryl groups of bovine serum albumin to probe conformational transitions in the neutral region

The free SH group in bovine serum albumin has been modified by covalent coupling with 2-chloromercuri-4-nitrophenol and 2-chloromercuri-2,4-dinitrophenol. The ionization of the phenolic OH group of the former label when bound to albumin can be followed spectrophotometrically. The pK of this group was influenced by the presence of Ca 2+. This is not a direct effect but proceeds via an effect of Ca 2+ on the protein conformation. Similar results were obtained by following the c.d. signal of this label. This conformational change seems to be different from the one which can be detected by measuring the induced c.d. of a non-covalently bound ligand like diazepam.

Properties of the thiosulphate-oxidizing multi-enzyme system from Thiobacillus versutus

Enzyme A of the thiosulphate-oxidizing multi-enzyme system of Thiobacillus versutus was shown by dialysis equilibrium, using [ 35S]thiosulphate, to bind 1 mole of thiosulphate per mole of enzyme. The formation of this complex is believed to the the first step of the thiosulphate-oxidizing sequence, leading to sulphate. Sulphite was a competitive inhibitor of thiosulphate oxidation by the multi-enzyme system, with an apparent K 1 of 25 μM, and was also an inhibitor of thiosulphate binding to enzyme A, with an apparent K d of 70 μM. Activity of the multi-enzyme system was inhibited by sulphydryl group reagents, indicating the importance of SH groups in the overall oxidation process. Inhibition by N-ethylmaleimide, p-hydroxymercuribenzoate and HgCl 2, but not by iodoacetamide, was reversed by reduced glutathione. SH group reagents did not inhibit the binding of thiosulphate to enzyme A, supporting the indication from sulphite-inhibition of binding that binding was effected through the sulphonate group of thiosulphate and did not directly involve free sulphydryl groups. Dithionate and methane sulphonate did not affect binding by enzyme A or thiosulphate oxidation by the multi-enzyme system. The overall stoichiometry of thiosulphate oxidation resulted in 8 mol cytochrome c being reduced for each thiosulphate ion oxidized, thereby eliminating the possibility that an oxygenase was involved in the system. A hypothetical scheme describing thiosulphate binding and oxidation is presented.

Chemical synthesis of oligonucleotides containing a free sulphydryl group and subsequent attachment of thiol specific probes.

Oligonucleotides containing a free sulphydryl group at their 5'-termini have been synthesised and further derivatised with thiol specific probes. The nucleotide sequence required is prepared using standard solid phase phosphoramidite techniques and an extra round of synthesis is then performed using the S-triphenylmethyl O-methoxymorpholinophosphite derivatives of 2-mercaptoethanol, 3-mercaptopropan (1) ol or 6-mercaptohexan (1) ol. After cleavage from the resin and removal of the phosphate and base protecting groups, this yields an oligonucleotide containing an S-triphenylmethyl group attached to the 5'-phosphate group via a two, three or six carbon chain. The triphenylmethyl group can be readily removed with silver nitrate to give the free thiol. With the three and six carbon chain oligonucleotides, this thiol can be used, at pH 8, for the attachment of thiol specific probes as illustrated by the reaction with fluorescent conjugates of iodoacetates and maleiimides. However, oligonucleotides containing a thiol attached to the 5'-phosphate group via a two carbon chain are unstable at pH 8 decomposing to the free 5'-phosphate and so are unsuitable for further derivatisation.

Myeloperoxidase reduces the opsonizing activity of immunoglobulin G and complement component C3b

The effect of myeloperoxidase, hydrogen peroxide (H 2O 2) and a halide (Cl) on the opsonizing molecules in immunoglobulin G (IgG) and complement factor C3b was assayed. At concentrations of the enzyme (1 μg/ml) that can be found in the extracellular fluid during inflammation, the myeloperoxidase-H 2O 2-Cl system inhibited the opsonizing effect of IgG and C3b measured as phagocytiuptake and superoxide generation. The effect was related to the enzymatic peroxidative activity of the protein. The presence of albumin (10 mg/ml) reduced the effect of myeloperoxidase with 10–20%. Taurine, which in the presence of myeloperoxidase-H 2O 2-Cl forms hydrophilic chloramines, and D-penicillamine, which scavenges HOCl, neutralize the inhibitory effect of myeloperoxidase. This suggests that either hypochlorous acid or lipophilic chloramines may exert its effect by oxidizing free sulphydryl groups exposed on the opsonizing ligands. Since the myeloperoxidase-H 2O 2-halide system also affects chemotactic factors, leukotrienes, proteinases and membrane receptors, the system may in several ways affect the development of the inflammatory response.

Human fibronectin: cell specific alternative mRNA splicing generates polypeptide chains differing in the number of internal repeats.

The nucleotide sequence of five independent cDNA clones, which cover 4843 nucleotides from the poly(A) addition site of human fibronectin (FN) mRNA was determined. The deduced amino acid sequence (1383 residues) covers the COOH-terminal 60% of human FN, spanning the C-terminus, fibrin-, heparin- and cell-binding domains, and shows the exact location of the only two free sulphydryl groups present in each subunit chain. We have recently reported two different FN mRNA species; one of them containing an additional 270 nucleotide insert (ED) that encodes exactly one of the homology type III repeats of the protein. The two mRNAs arise by alternative splicing of a common precursor. S1 nuclease mapping of cDNA/RNA hybrids shows that the expression of the two mRNAs is cell specific. Liver only produces the mRNA without the ED, whereas hepatoma cells, breast tumor cells and normal fibroblasts produce both forms of mRNA. Another area of alternative splicing generating three different FN mRNAs in rat liver has been reported by Schwarzbauer et al (16). We here provide evidence for the existence in human cells of a fourth mRNA species different from the three described in rat liver.

Binding of antigens to tissues: The example of Boophilus microplus and bovine skin

Two antigens from the tick Boophilus microplus, one an esterase, the other an inhibitor of proteolytic enzymes, may have a pronounced tendency to aggregate or bind non-specifically to other proteins. It is suggested that this may be important in enhancing their retention in the tick's feeding lesion on the host. Using [ 125J] labelled proteins it was shown that the tick proteins when injected into bovine skin were retained there significantly longer than were model proteins from non-parasitic sources. It was shown that the tick esterase contained three free sulphydryl groups per molecule and evidence was obtained that these could contribute to binding of the protein in bovine skin.