A note on the molar absorptivity of reduced Ellman's reagent, 3-carboxylato-4-nitrothiophenolate

Collier, H. B.

doi:10.1016/0003-2697(73)90196-6

Cited by 89 publications

(31 citation statements)

References 2 publications

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“…After mixing (25°C for 30 min), the agarose was removed, HCl was added to 20 mM, and the acidified solution was stored at Ϫ20°C. Thiol concentrations were determined by titration with 5,5Ј-dithio-bis(2-nitrobenzoic acid) (26) by using an extinction coefficient of 14.14 mM Ϫ1 ⅐cm Ϫ1 at 412 nm for the thionitrobenzoate anion (27).…”

Section: Methodsmentioning

confidence: 99%

A trypanothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major

Vickers

Greig

Fairlamb

2004

Proc. Natl. Acad. Sci. U.S.A.

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Glyoxalase I forms part of the glyoxalase pathway that detoxifies reactive aldehydes such as methylglyoxal, using the spontaneously formed glutathione hemithioacetal as substrate. All known eukaryotic enzymes contain zinc as their metal cofactor, whereas the Escherichia coli glyoxalase I contains nickel. Database mining and sequence analysis identified putative glyoxalase I genes in the eukaryotic human parasites Leishmania major, Leishmania infantum, and Trypanosoma cruzi, with highest similarity to the cyanobacterial enzymes. Characterization of recombinant L. major glyoxalase I showed it to be unique among the eukaryotic enzymes in sharing the dependence of the E. coli enzyme on nickel. The parasite enzyme showed little activity with glutathione hemithioacetal substrates but was 200-fold more active with hemithioacetals formed from the unique trypanosomatid thiol trypanothione. L. major glyoxalase I also was insensitive to glutathione derivatives that are potent inhibitors of all other characterized glyoxalase I enzymes. This substrate specificity is distinct from that of the human enzyme and is reflected in the modification in the L. major sequence of a region of the human protein that interacts with the glycyl-carboxyl moiety of glutathione, a group that is conjugated to spermidine in trypanothione. This trypanothione-dependent glyoxalase I is therefore an attractive focus for additional biochemical and genetic investigation as a possible target for rational drug design.methylglyoxal ͉ D-lactate ͉ glutathione ͉ nickel

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Section: Methodsmentioning

confidence: 99%

A trypanothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major

Vickers

Greig

Fairlamb

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…i, pH 7.6) and the reaction with SH groups at 412 nm was followed periodically (Merola et al, 1989); the number of SH groups that had reacted was determined by using a molar absorbance coefficient of 14,000 M-' cm" for the nitrothiophenylate anion (Collier, 1973). When activity measurements were made, samples were removed immediately after recording the A412nm and assayed as described above.…”

Section: Urea and Guanidine Denaturationmentioning

confidence: 99%

Catalytic ability and stability of two recombinant mutants of D‐amino acid transaminase involved in coenzyme binding

et al. 1995

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Of the major amino acid side chains that anchor pyridoxal 5"phosphate at the coenzyme binding site of bacterial D-amino acid transaminase, two have been substituted using site-directed mutagenesis. Thus, Ser-180 was changed to an Ala (S180A) with little effect on enzyme activity, but replacement of Tyr-31 by Gln (Y31Q) led to 99% loss of activity. Titration of SH groups of the native Y31Q enzyme with DTNB proceeded much faster and to a greater extent than the corresponding titration for the native wild-type and S180A mutant enzymes. The stability of each mutant to denaturing agents such as urea or guanidine was similar, Le., in their PLP forms, S180A and Y31Q lost 50% of their activities at a 5-15% lower concentration of urea or guanidine than did the wild-type enzyme. Upon removal of denaturing agent, significant activity was restored in the absence of added pyridoxal 5'-phosphate, but addition of thiols was required. In spite of its low activity, Y31Q was able to form the PMP form of the enzyme just as readily as the wild-type and the S180A enzymes in the presence of normal D-amino acid substrates.However, 0-chloro-D-alanine was a much better substrate and inactivator of the Y31Q enzyme than it was for the wild-type or S180A enzymes, most likely because the Y31Q mutant formed the pyridoxamine 5-phosphate form more rapidly than the other two enzymes. The stereochemical fidelity of the Y31Q recombinant mutant enzyme was much less than that of the S180A and wild-type enzymes because racemase activity, i.e., conversion of L-alanine to D-alanine, was higher than for the wild-type or S180A mutant enzymes, perhaps because the coenzyme has more flexibility in this mutant enzyme.

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“…2). Using 14.1 mM-'cm-' as the &412nm of 3-carboxylato-4-nitrothiophenolate ion [18], the number of thiol residues was calculated as 0.8 per enzyme molecule. This value is in close agreement with those previously reported for the yeast enzyme [2,19].…”

Section: Rate Of Reaction Between Phosphoglycerate Kinase and Nbszmentioning

confidence: 99%

Anion Binding to Yeast Phosphoglycerate Kinase

Wrobel

Stinson

1978

European Journal of Biochemistry

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The single thiol of yeast phosphoglycerate kinase was labelled with the chromophoric sulfhydryl reagent, 2-chloromercuri-4-nitrophenol. Sequential additions of individual anions to this modified enzyme brought about a decrease in absorbance at 410 nm that reflected the degree of saturation of the enzyme with anion. The binding curves were analyzed to determine the dissociation constants of a number of anions with charges varying from -1 to -4.1. A linear relationship was found between the charge of the anion and the negative logarithm of the dissociation constant for the labelled enzyme-anion complex. The highly charged anions, such as ATP, bound more tightly than did anions with less charge, such as C1-. The average number of binding sites for those anions for which accurate results could be obtained was 1.06 mol per 47000 g of enzyme. Several lines of evidence suggested that titration of the active center was not being monitored.Anions bound to phosphoglycerate kinase decreased the rate of reaction between the enzyme thiol and 5,5'-dithiobis(2-nitrobenzoic acid). The relationship between the degree of saturation of the anion binding site and the reaction rate constant was used to calculate the dissociation constant between anion and enzyme. Dissociation constants determined in this manner were in good agreement with those determined by titration of the enzyme-mercurial complex.Kinetic studies of phosphoglycerate kinase isolated from a variety of sources suggest that the substrates 3-phosphoglycerate and ATP and 1,, bind to more than one site on the enzyme. Catalytic activity of the yeast enzyme has also been shown to increase in the presence of ATP [6], or a variety of other anions [7,8] (and Stinson, R. A., unpublished results). We have reported previously that 3-phosphoglycerate and phosphate decrease the rate of reaction between yeast phosphoglycerate kinase and the sulfhydryl-binding reagent 2-chloromercuri-

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A note on the molar absorptivity of reduced Ellman's reagent, 3-carboxylato-4-nitrothiophenolate

Cited by 89 publications

References 2 publications

A trypanothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major

A trypanothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major

Catalytic ability and stability of two recombinant mutants of D‐amino acid transaminase involved in coenzyme binding

Anion Binding to Yeast Phosphoglycerate Kinase

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