Thiol-disulphide Interchange Research Articles

The adenovirus protease is activated by a virus-coded disulphide-linked peptide

In common with many other viruses, adenoviruses code for a protease essential for the development of infectivity. Recombinant adenovirus protease was active in crude in vitro complementation assays but was inactive with peptide or purified protein substrates. Activity was reconstituted by a component of adenovirus virions, which was identified as GVQSLKRRRCF, a peptide derived from the virus protein pVI. Synthetic peptides were used to demonstrate that the cysteine is essential and that the disulphide-linked dimer is required for activity. It is proposed that the adenovirus protease is a cysteine protease and that its activation by the peptide involves thiol-disulphide interchange, which serves to expose the active site cysteine. This represents a novel strategy for controlling the activity of a protease that is required for virus maturation.

Gingivain; A Cysteine Proteinase Isolated fromPorphyromonas gingivalis

The extracellular proteinase produced by Porphyromonas gingivalis , which has often been described as ‘trypsin-like’, was isolated by thiol-disulphide interchange covalent chromatography. Evidence from the method of isolation, thiolspecific inhibition reactivation cycle and thiol-specific reactivity probe kinetics, all involving 2-pyridyl disulphides, compels the view that this enzyme, for which the name gingivain is here proposed, is a cysteine proteinase. All of the hydrolytic activity towards α-N-benzoyl-L-arginine-4-nitroanilide(L-BAPNA) and azocasein in the P. gingivalis vesicle/supernatant mixture was shown to be thiol dependent by its complete inhibition by 2,2’-dipyridyl disulphide and complete reactivation by 2-mercaptoethanol. The vesicles and all of the hydrolytic activity were isolated by precipitation in 70 per cent saturated ammonium sulphate, centrifugation for 22 h at 150,000 g and 4°C. The cysteine proteinase was prepared in fully active form by sequential elution covalent chromatography on Sepharose-glutathione 2-pyridyl disulphide gel, which separated the enzyme from the vesicles and from other thiol-containing protein devoid of catalytic activity towards L-BAPNA. The fully active isolated enzyme was: (a) completely inhibited by reaction with 2,2’-dipyridyl disulphide with complete reactivation by 2-mercaptoethanol, and (b) shown to possess a key catalytic site characteristic, typical of many cysteine proteinases. Thus, stopped-flow kinetic analysis of the reaction of its catalytically essential thiol group with 2,2’-dipyridyl disulphide showed the reactivity to be minimum at ca. pH 6, behaviour characteristic of the existence of a catalytic site cysteine-histidine interactive system. Keywords: Gingivain; a cysteine proteinase; P. gingivalis proteinase.

Isolation, purification and characterisation of 2-oxoglutarate reductase from Fusobacterium nucleatum

2-Oxoglutarate reductase from Fusobacterium nucleatum was isolated by thiol-disulphide interchange covalent chromatography. The enzyme was purified approximately 4000-fold and had a molecular mass of 68 kDa. The Michaelis constants for 2-oxoglutarate and NADH were 6.4 x 10(-5) and 0.4 x 10(-5), respectively. The involvement of sulphahydryl groups in catalysis was shown from the inhibition of 2-oxoglutarate reduction in the presence of 2,2'-dipyridyl disulphide and reactivation with 2-mercaptoethanol. Allosteric effectors did not alter the rate of the reaction, or the enzyme stability. With the exception of 2-oxoglutarate, none of the other oxo-acids such as oxaloacetate, pyruvate, 2-oxobutyrate and glyoxylate were reduced. Although 2-oxoglutarate oxidised NADPH to a limited extent (3%), the enzyme was almost entirely specific towards NADH. 2-Oxoglutarate reductase was stable at 45 degrees C for 10 min, while incubation at 60 degrees C abolished all activity.

Isolation, purification and characterisation of 2-oxoglutarate reductase fromFusobacterium nucleatum

2-Oxoglutarate reductase from Fusobacterium nucleatum was isolated by thiol-disulphide interchange covalent chromatography. The enzyme was purified approximately 4000-fold and had a molecular mass of 68 kDa. The Michaelis constants for 2-oxoglutarate and NADH were 6.4 x 10(-5) and 0.4 x 10(-5), respectively. The involvement of sulphahydryl groups in catalysis was shown from the inhibition of 2-oxoglutarate reduction in the presence of 2,2'-dipyridyl disulphide and reactivation with 2-mercaptoethanol. Allosteric effectors did not alter the rate of the reaction, or the enzyme stability. With the exception of 2-oxoglutarate, none of the other oxo-acids such as oxaloacetate, pyruvate, 2-oxobutyrate and glyoxylate were reduced. Although 2-oxoglutarate oxidised NADPH to a limited extent (3%), the enzyme was almost entirely specific towards NADH. 2-Oxoglutarate reductase was stable at 45 degrees C for 10 min, while incubation at 60 degrees C abolished all activity.

Light-dependent de-activation/re-activation of Anabaena variabilis ferredoxin: NADP+ reductase

The activity of ferredoxin: NADP+ reductase (FNR) was found to decline to approximately 20% maximal levels with little or no loss in enzyme levels when cultures of the cyanobacterium Anabaena variabilis were maintained in the stationary phase of growth. Re-activation of enzyme activity occurred when cells were diluted into either fresh or re-utilized media and illuminated. This reversible de-activation/re-activation process was found, in vivo, to be dependent on the intensity of light illuminating the cells. The de-activated form of FNR was purified to homogeneity and exhibited the same molecular mass, isoelectric-focusing pattern and N-terminal amino acid sequence as the native form. Both de-activated and native FNR preparations each exhibited three reactive thiol groups on denaturation in urea; however, the rate of reaction with Ellman's reagent was much faster with the de-activated form than with the native form. Both preparations contain a single disulphide bond. Upon reduction of the disulphide bond in either form of the enzyme, the five reactive thiol groups exhibited identical reactivities in the presence of urea. Steady-state kinetic analysis of the de-activated form showed a marked increase in Km values for NADPH in diaphorase assays and an increase in Km for ferredoxin in the ferredoxin-mediated reduction of cytochrome c. No significant difference in kcat. was observed in comparison of the de-activated with the native form in any of the above assays; however, the de-activated form did exhibit a lower kcat. value in the transhydrogenase assay. The de-activated form of FNR bound ferredoxin with a 16-fold lower affinity than the native enzyme. These data suggest that the de-activation of FNR in vivo in response to low light intensity involves an alteration in protein structure, possibly via an intramolecular thiol disulphide interchange, which influences the interaction of the enzyme with its substrates.

Highly efficient propane-1,3-dithiol mediated thiol–disulphide interchange: a facile and clean methodology for S–S reduction in peptides

Propane-1,3-dithiol, at room temperature and in the absence of any promoter, neatly brings about the S–S → 2SH change, and the latter can be isolated as acrylonitrile adducts; the methodology has been tested with several substrates including c-lysozyme, insulin and oxytocin, and spontaneous imidazole promoted S-deprotection was observed in the case of Z-Cys(S-CH2CH2CN)His-OMe.

The effect of reactive dyes on damage in wool dyeing

This paper demonstrates that reactive dyes exert a significant fibre–protective effect when dyeing wool fabric; this effect may be measured, in a reproducible manner, using the wet–burst strength test. The effect of dyebath pH, dye concentration, dyebath temperature and dyeing time on wool can thus be evaluated. The magnitude of the above protective effect has been compared with that achieved using commercially available fibre–protective agents. It is concluded that reactive dyes are significantly more effective than the latter agents when employed in medium to full depths. It is proposed that the unusual protective effect afforded by reactive dyes is related to the following factors. Reactive dyes readily react with sulphur nucleophiles, thus inhibiting thiol–disulphide interchange reactions and thereby significantly interfering with the level of set produced in a boiling dyebath. Reactive dyes react preferentially with non–keratinous proteins in the intercellular cement and the endocuticle thus reducing their tendency to hydrolyse and to partially dissolve in the hot aqueous dyebath.

Potassium iodate-induced proteolysis in ultra heat treated milk during storage: the role of β-lactoglobulin and plasmin

SummaryThe report that addition of KI03(0·1 mm) to milk before ultra high temperature (UHT) treatment induces extensive proteolysis during subsequent storage at 37 °C was confirmed. None was produced by addition of H202KMn04or K2Cr207. The pH optimum for KI03-induced proteolysis was between 7·0 and 8·0 and the temperature optimum 37—45 °C. β-Casein was particularly susceptible and the proteolysis pattern was similar to that caused by indigenous alkaline milk proteinase (MPA, plasmin). Addition of plasmin to milk before UHT treatment (140 °C/10 s) caused slight proteolysis during subsequent storage but addition of 0·1 mm-KI03and plasmin caused extensive proteolysis which was prevented by addition of soyabean trypsin inhibitor, indicating the probable involvement of plasmin in KI03-induced proteolysis in UHT-treated milk. Equally extensive proteolysis occurred in serum protein-free casein micelle systems (SPFCM), with or without KI03, during storage at 37 °C following UHT treatment, indicating a role for whey proteins in KI03-induced proteolysis. Addition of β-lactoglobulin (β-lg) to a SPFCM system inhibited proteolysis, but extensive proteolysis occurred in a SPFCM system containing both β-lg and KI03. MPA-free Na caseinate (prepared by heating at 140 °C for 7 min) underwent extensive proteolysis when treated with plasmin before UHT treatment; proteolysis was inhibited by addition of °-lg to this system and KI03reversed the inhibitory effect of β-lg. Plasmin proteolysis of isolated αs1-casein was inhibited by denatured β-lg (90 °C/30 min) at a level of 4 mg/ml but not by native β-lg. When denatured in the presence of KI03, β-lg had a lower free SH content than the control and was less inhibitory for plasmin in proteolysis of isolated αsl-casein. The results show that denatured β-lg inhibits plasmin proteolysis of caseins in UHT milk and that inhibition is prevented by KI03. This inhibition may occur via thiol–disulphide interchange, which is prevented if the SH group of ²-lg is oxidized by KI03, thus permitting the stimulatory effect of KI03on proteolysis in UHT-treated milk.

Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives.

Chymopapain A was isolated from the dried latex of papaya (Carica papaya) by ion-exchange chromatography followed by covalent chromatography by thiol-disulphide interchange. The latter procedure was used to produce fully active enzyme containing one essential thiol group per molecule of protein, to establish that the chymopapain A molecule contains, in addition, one non-essential thiol group per molecule and to recalculate the literature value of epsilon 280 for the enzyme as 36 000 M-1 X cm -1. The Michaelis parameters for the hydrolysis of L-benzoylarginine p-nitroanilide and of benzyloxy-carbonyl-lysine nitrophenyl ester at 25 degrees C, and I 0.1 at several pH values catalysed by chymopapain A, papaya proteinase omega, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were determined. Towards these substrates chymopapain A has kcat./km values similar to those of actinidin and of papaya proteinase omega and significantly lower than those of papain or ficin. The environment of the catalytic site of chymopapain A is markedly different from those of other cysteine proteinases studied to date, as evidenced by the pH-dependence of the second-order rate constant (k) for the reaction of the catalytic-site thiol group with 2,2'-dipyridyl disulphide. The striking bell-shaped component that is a characteristic feature of the reactions of S-/ImH+ (thiolate/imidazolium) ion-pair components of many cysteine-proteinase catalytic sites with the 2,2'-dipyridyl disulphide univalent cation is not present in the pH-k profile for the chymopapain A reaction. The result is consistent with the presence of an additional positive charge in, or near, the catalytic site that repels the cationic form of the probe reagent. Resonance Raman spectra were collected at pH values 2.5, 6.0 and 8.0 for each of the following dithioacyl derivatives of chymopapain A: N-benzoylglycine-, N-(Beta-phenylpropionl)glycine- and N-methoxycarbonylphenylalanylglycine-. The main conclusion of the spectral study is that in each case the acyl group binds as a single population known as conformer B in which the glycinic N atom is in close contact with the thiol S atom of the catalytic-site cysteine residue, as is the case also for papain and other cysteine proteinases studied. Thus the abnormal catalytic-site environment of chymopapain A detected by the reactivity-probe studies, which may have consequences for the acylation step of the catalytic act, does not perturb the conformation of the bound acyl group at the acyl-enzyme-intermediate stage of catalysis.

Cysteamine depletes cystinotic leucocyte granular fractions of cystine by the mechanism of disulphide interchange.

Cystinotic lysosome-rich leucocyte granular fractions, loaded with [35S]cystine, were exposed to different cystine-depleting agents. During a 30 min incubation at 37 degrees C, untreated cystinotic granular fractions lost negligible [35S]cystine when corrected for lysosome rupture. Granular fractions exposed to 0.1 mM-cysteamine lost 64% of their initial cystine, and hexosaminidase activity was decreased by 10%. This was accompanied by the formation of high concentrations of [35S]cysteine-cysteamine mixed disulphide within the granular-fraction pellet, and, in the presence of N-ethylmaleimide, increasing amounts of [35S]cysteine-N-ethylmaleimide adduct outside the granular fraction. In separate experiments, [35S]cystine exited cystinotic leucocyte lysosomes at a negligible rate (half-times 199 and 293 min), but [35S]cysteine-cysteamine mixed disulphide exhibited substantial egress (half-times 66 and 88 min) and was recovered intact outside the granular-fraction pellet. We conclude that cysteamine depletes lysosomes of cystine by participating in a thiol-disulphide interchange reaction to produce cysteine and cysteine-cysteamine mixed disulphide, both of which traverse the cystinotic leucocyte lysosomal membrane.

In situ interaction of cartilage proteoglycans with matrix proteins

The interaction of proteoglycans with other matrix proteins via thiol-disulphide interchange was explored. Chick sternal cartilage was extracted with 4 M guanidine hydrochloride in the presence and absence of N-ethylmaleimide and the proteoglycans from the centrifugation A2 fractions were isolated. Those from extracts without N-ethylmaleimide were linked with reducible bonds with 10–15 proteins-glycoproteins including the link proteins, the 148 kDa and 36 kDa proteins. The same was observed with extracts of pig laryngeal and sheep nasal cartilage. The linked proteoglycans from sheep amounted to 2–3% of the extractable uronic acid and belonged to two populations. The major fraction was included by Sepharose 6B ( M r 110 000) had twice as long chondroitin sulphate chains, higher 4-sulphated residues and a high content of aspartic acid and leucine-rich protein. The larger proteoglycans had a size and composition similar to those of aggregating proteoglycans.

Preparation of cathepsins B and H by covalent chromatography and characterization of their catalytic sites by reaction with a thiol-specific two-protonic-state reactivity probe. Kinetic study of cathepsins B and H extending into alkaline media and a rapid spectroscopic titration of cathepsin H at pH 3-4.

A procedure for the isolation of cathepsin B (EC 3.4.22.1) and of cathepsin H from bovine spleen involving covalent chromatography by thiol-disulphide interchange and ion-exchange chromatography was devised. The stabilities of both cathepsins in alkaline media are markedly temperature-dependent, and reliable kinetic data can be obtained at pH values up to 8 by working at 25 degrees C with a continuous spectrophotometric assay. Both enzyme preparations contain only one type of thiol group as judged by reactivity characteristics towards 2,2'-dipyridyl disulphide at pH values up to 8; in each case this thiol group is essential for catalytic activity. Cathepsin H was characterized by kinetic analysis of the reactions of its thiol group with 2,2'-dipyridyl disulphide in the pH range approx. 2-8 and the analogous study on cathepsin B [Willenbrock & Brocklehurst (1984) Biochem. J. 222, 805-814] was extended to include reaction at pH values up to approx. 8. Cathepsin H, like the other cysteine proteinases, was shown to contain an interactive catalytic-site system in which the nucleophilic character of the sulphur atom is maintained in acidic media. The considerable differences in catalytic site characteristics detected by this two-protonic-state reactivity probe between cathepsin B, cathepsin H, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) are discussed. Reaction with 2,2'-dipyridyl disulphide in acidic media, which is known to provide a rapid spectrophotometric active centre titration for many cysteine proteinases, is applicable to cathepsin H. This is useful because other active-centre titrations have proved unsuitable in view of the relatively low reactivity of the thiol group in cathepsin H.

The thiol groups of the Folch-Pi protein from bovine white matter. Exposure, reactivity and significance.

The number and the reactivity of accessible thiol groups of the Folch-Pi apoprotein and proteolipid (50% of myelin proteins) were studied, by using a specific thiol-disulphide interchange reaction, in connection with the known solubility of this protein in organic and aqueous solvents. The high reactivity of 2,2'-dipyridyl disulphide towards thiol groups leads to the titration of 4.8 mol of SH groups/mol of protein (Mr 30000) in alkaline and acidic chloroform/methanol (2:1, v/v). Unlike previous findings, this value was consistently found from batch to batch and remained stable with time. In the proteolipid 1 mol of SH groups/mol was not accessible as compared with the apoprotein. In aqueous solvents, a similar number of 4.4 mol of SH groups/mol was also found. For the first time, kinetic studies carried out in chloroform/methanol discriminated between two classes of thiol groups. The reaction of 2 mol of SH groups/mol was characterized by apparent second-order rate constants whose values were 5-10-fold higher than those of the other class. Kinetic studies and cyanylation experiments in aqueous solvents also indicated the high reactivity of these thiol groups with Ellman's reagent. Together with kinetic results, studies on the stoichiometry of the interchange reaction of equimolar solutions of protein and disulphide indicate that these highly reactive thiol groups are near to each other in the amino acid sequence. The location of the thiol groups at the boundary between hydrophilic and hydrophobic domains of the Folch-Pi protein is suggested in connection with their possible structural and biological significance.

Drug protein conjugates—VI. Role of glutathione in the metabolism of captopril and captopril plasma protein conjugates

Previous metabolic studies of captopril suggest that the rapid dissociation of captopril-plasma protein conjugates in vivo is dependent upon endogenous thiols such as glutathione and cysteine. Consistent with this hypothesis, we have found that cysteine (0.06–3 mM) and glutathione (0.02–1 mM) cleave 14C-captopril-plasma protein conjugates in vitro. Dissociation of the drug-protein conjugate was accompanied by formation of the corresponcling mixed disulphide which indicates that the reaction proceeds via a spontaneous thiol-disulphide interchange. Administration of high doses (50–300 mg/kg) of CP produced a time-dependent and dose-dependent decrease in hepatic glutathione concentrations in the mouse and the rat. The depletion of glutathione observed was similar to that produced by equimolar doses of D-penicillamine and paracetamol. Acute and chronic (7 days) administration of captopril (100 mg/kg) produces the same (11–12%) depletion of hepatic glutathione. However, changes in liver function as determined by elevation of serum glutamic-pyruvic transaminase was only observed at doses of 200 and 300 mg/kg. Thus, although thiol-disuiphide interactions between captopril and plasma proteins may contribute to the perturbation of hepatic glutathione concentrations, it is unlikely that this process will be of toxicological significance during therapeutic administration of captopril.

Role of cytoplasmic thioltransferase in cellular regulation by thiol-disulphide interchange.

Cytoplasmic thioltransferase purified from rat liver [Axelsson, Eriksson & Mannervik (1978) Biochemistry 17, 2978--2984] catalyses the formation and decomposition of mixed disulphides of proteins and glutathione. The enzyme was found to catalyse the reversible thiol-disulphide interchange between glutathione disulphide and a crude thiol-containing protein fraction from rat liver. This finding indicates a role of the thioltransferase in the regulation of the 'glutathione status' of the cell. Specifically, it was found that thioltransferase catalyses the reactivation of pyruvate kinase from rat liver that had previously been inactivated by glutathione disulphide. It is suggested that thioltransferase may have a general role in regulatory processes involving thiol-disulphide interchange.

Use of thiol-disulphide interchange reactions in preparative work on plasma proteins

Use of thiol-disulphide interchange reactions in preparative work on plasma proteins

Immobilised lipoamide dehydrogenase. 2. Properties of the enzyme immobilised to agarose through spacer molecules of various lengths.

1. Pig heart lipoamide dehydrogenase (NADH: lipoamide oxidoreductase, EC 1.6.4.3) has been immobilised to Sepharose by thiol-disulphide interchange via a series of thiolated spacer molecules of increasing length. A number of properties of the immobilised enzyme have been investigated in order to ascertain the effects of proximity to the matrix backbone. 2. Proximity to the matrix backbone reduced the specific activity for lipoamide as substrate but enhanced by 3-8-fold the diaphorase activity with 2,6-dichloroindophenol. These observations are explained in part by an increase in the apparent Km for lipoamide when the enzyme is covalently attached to Sepharose via a short spacer molecule. 3. Both the thermal stability at 90 degrees C and the stability in 30% (v/v) dioxane are enhanced by up to 200% when the enzyme resides close to the matrix but approach those of the native enzyme as the length of the spacer molecule is increased. 4. These data have been correlated with measures of the accessibility of the enzyme as the nominal length of the spacer arm was increased. Thus, as the chain length increased, the rate of cleavage of the disulphide linkage between the enzyme and spacer increased and the enzyme became more susceptible to proteolysis by thermolysin. In contrast, increasing the chain length of the spacer made the enzyme less amenable to inhibition by a specific antibody. 5. These data are discussed in terms of the effect of the matrix on the conformation of the bound enzyme.

The Function of Thioredoxin and Glutathione in Deoxyribonucleic Acid Synthesis

Ribonucleotide reductases (EC 1 .I 7.4.1 and EC 1.17.4.2) catalyse the reduction of ribonucleoside 5’-diphosphates or triphosphates to the corresponding 2’-deoxyribonucleotides and are key enzymes for the onset of DNA replication during the cell cycle (Reichard, 1968; Follmann, 1974). The reduction of the 2’-hydroxyl group of the ribose moiety in a ribonucleotide is dependent on NADPH, but this coenzyme will not function directly with ribonucleotide reductase. In vitro, purified ribonucleotide reductase requires certain dithiols like lipoic acid or dithiothreitol for activity (Reichard, 1968). The thioredoxin system of Escherichia coli was discovered in 1964 by Reichard and coworkers (Laurent et al., 1964) as a physiological NADPH-dependent hydrogen-donor systemforribonucleotidereductase. Thioredoxin is asmall acidic dithiol protein (mol.wt. 12000) which can occur in an oxidized (thioredoxin-S,) or a reduced [thioredoxin-(SH),] form. Thioredoxin reductase is a flavoprotein (mol.wt. 66000) which will reduce thioredoxin-S, by NADPH (Reichard, 1968). Ribonucleotide reduction as studied in vitro thus appears as a complex multienzyme system. Further, evidence exists that the organization in vivo of the ribonucleotide reductase system may involve interactions between the proteins to form a highly effective multienzyme complex of unknown properties. This is supported by the low activity of ribonucleotide reductase obtained by reconstitution in vitro as compared with the activity in cells made permeable to nucleotides by diethyl ether treatment or the activity in ‘high-speed’ supernatants from gently lysed E. coli cells (Eriksson, 1975). Steady-state kinetics of ribonucleotide reductase indicated that the enzyme acted by a Ping Pong mechanism, in the presence of thioredoxin-(SH)z, that is, it akernated between two stable forms during catalysis (Thelander, 1974). The electron acceptor in ribonucleotide reductase was suggested to be oxidation-reduction-active disulphides of protein B1 (Thelander, 1974). The Ping Pong mechanism might explain the difliculties in observing any binding between Escherichia coli thioredoxin and ribonucleotide reductase in vitro (A. Holmgren, unpublished work). From an X-ray-crystallographic study of E. coli thioredoxin-S, to 0.28 nm (2.8A) resolution, the oxidation-reductionactive disulphide bond has been located in a protrusion of the molecule (Holmgren et al., 1975). Reduction of thioredoxin-Sz is suggested to be accompanied by a local conformational change within this active-centre protrusion (Holmgren et al., 1975). From such a model the simultaneous binding of thioredoxin to thioredoxin reductase and ribonucleotide reductase seems unlikely. Both these enzymes have oxidationreduction disulphide bonds at their active sites (Thelander, 1974) that should interact with the S-S bond protrusion in thioredoxin for transfer of electrons via thiol-disulphide interchange. The structural data for thioredoxin thus also support Ping Pong mechanisms for the interaction of thioredoxin with both ribonucleotide and thioredoxin reductase. A completely new and unexpected role for thioredoxin in DNA synthesis was discovered in the work of Richardson and co-workers (Mark & Richardson, 1976). E. coli thioredoxin was found to be a subunit of a phage TFinduced DNA polymerase, detected after virus infection, which is essential for the phage life cycle. Phage T7 DNA polymerase is composed of one 84000-dalton phage-coded subunit and 1 molecule of thioredoxin (Mark & Richardson, 1976). The specific function of thioredoxin in the phage T7 DNA polymerase is presently unknown. However, since the enzyme can be reconstituted in vitro from purified gene-5 protein of phage T7 by addition of E. coli thioredoxin, the function of the thioredoxin subunit can be studied in some detail. The discovery of the new function for E. coli thioredoxin as subunit of phage T7 DNA polymerase was based on the isolation of E. coli mutants (tsnC) which could not

A reversibly refolding non-covalent complex of three peptides from Staphylococcus aureus penicillinase [proceedings

Reduced and ‘scrambled’derivatives of NO2-ribnuclease appear to be less stable than the corresponding derivatives of unmodified ribonuclease. In our hands, reduced ribonuclease is stable in solutions of pH4 for 4-5 days at 4”C, whereas reduced NO,-ribonuclease is reoxidized with a half-time of several hours. Likewise, the background enzymic activity of ‘scrambled‘ NO,-ribonuclease increases during storage in solution at pH8.0, 4“C, whereas scrambled ribonuclease is comparatively stable. Although this instability may be a limitation, the nitrotyrosine residue in these derivatives is a useful probe of the conformational changes accompanying disulphide-formation and interchange. The conversion from ‘scrambled‘ to ‘native’ NO,-ribonuclease corresponds to a vertical transition between the titration curves of Fig. 1. As a result of the shift in PIC., if this conversion is carried out between pH6.0 and 7.4, it should be accompanied by a significant change in A428. We find that this conversion is catalysed by protein disulphide isomerase (EC 5.3.4.1), an enzyme catalysing thiol-disulphide interchange, and hence disulphide-bond isomerization in proteins (Freedman & Hawkins, 1977). Incubation of ‘scrambled‘ NO,-ribonuclease with partially purified protein disulphide isomerase (Hawkins & Freedman, 1976) in the standard conditions for assay of this enzyme (Ibbetson & Freedman, 1976) leads to the appearance of increased ribonuclease activity and an increase in A42s. The rate of increase in ribonuclease activity with ‘scrambled’ NO,-ribonuclease is 2-10 % that found with scrambled unmodified r i b nuclease. With ‘scrambled’ NO,-ribonuclease in the range O.l-O.Smg/ml, rates of increase in absorbance were 0.0035-0.0125 AA428 units/min per mg of enzyme. So nitrated ribonculease derivatives may provide useful probes for the kinetic characterization of disulphide-bond formation and isomerization processes, including those catalysed by protein disulphide isomerase.

A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe.

1. A convenient method of preparation of jack-bean urease (EC3.5.1.5) involving covalent chromatography by thiol-disulphide interchange is described. 2. Urease thus prepared has specific activity comparable with the highest value yet reported (44.5 +/- 1.47 kat/kg, Km = 3.32 +/- 0.05 mM; kcat. = 2.15 X 10(4) +/- 0.05 X 10(4)s-1 at pH7.0 and 38 degrees C). 3. Titration of the urease thiol groups with 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) and application of the method of Tsou Chen-Lu [(1962) Sci. Sin. 11, 1535-1558] suggests that the urease molecule (assumed to have mol.wt. 483000 and epsilon280 = 2.84 X 10(5) litre-mol-1-cm-1) contains 24 inessential thiol groups of relatively high reactivity (class-I), six 'essential' thiol groups of low reactivity (class-II) and 54 buried thiol groups (class-III) which are exposed in 6M-guanidinium chloride. 4. The reaction of the class-I thiol groups with 2-Py-S-S-2-Py was studied in the pH range 6-11 at 25 degrees C(I = 0.1 mol/l) by stopped-flow spectrophotometry, and the analogous reaction of the class-II thiol groups by conventional spectrophotometry. 5. The class-I thiol groups consist of at least two sub-classes whose reactions with 2-Py-S-S-2-Py are characterized by (a) pKa = 9.1, k = 1.56 X 10(4)M-1-s-1 and (b) pKa = 8.1, k = 8.05 X 10(2)M-1-s-1 respectively. The reaction of the class-II thiol groups is characterized by pKa = 9.15 and k = 1.60 X 10(2)M-1-s-1. 6. At pH values 7-8 the class-I thiol groups consist of approx. 50% class-Ia groups and 50% class-Ib groups. The ratio class Ia/class Ib decreases an or equal to approx. 9.5, and at high pH the class-I thiol groups consist of at most 25% class-Ia groups and at least 75% class-Ib groups. 7. The reactivity of the class-II thiol groups towards 2-Py-S-S-2-Py is insensitive to the nature of the group used to block the class-I thiols. 8. All the 'essential' thiol groups in urease appear to be eeactive only as uncomplicated thiolate ions. The implications of this for the active-centre chemistry of urease relative to that of the thiol proteinases are discussed.