D. Cavallini

The copper catalyzed oxidation of cysteine to cystine

Abstract The addition of cysteine to an alkaline solution of CuII produces the immediate appearance of a yellow color. The spectrum of this color is characterized by a broad peak at 330 mμ and a shoulder at 400 mμ. By chemical analysis and EPR spectrometry, it has been demonstrated that this yellow compound is to be identified with a cysteine-copperII complex, in which the cysteine to copper ratio is 2:1. This complex accounts for nearly all the copper present in solution; remains unchanged during the cysteine oxidation by molecular oxygen, and quickly disappears at the end of the reaction. At this time no more cysteine is present in solution in its reduced form and all the copper may be recovered as CuI if immediately trapped by neocuproine. The yellow cysteine-CuII complex is also immediately produced in the absence of oxygen, however, under this condition, it disappears slowly, CuII being reduced by excess of cysteine. Some kinetic approaches indicate that both the formation of the yellow complex, and its anaerobic reduction, are very complex reactions. The results obtained indicate that the cysteine-CuII complex represents the real intermediate catalyst in the copper-catalyzed oxidation of cysteine.

Quantitative determination of keto-acids by paper partition chromatography

Abstract A simplified method for the quantitative determination of keto-acids by means of paper chromatography of their 2,4-dinitrophenylhydrazones is reported. 1. 1. The identification of these compounds on the filter paper is discussed, especially of those compounds which give more than one spot. 2. 2. The method is described in detail, espicially with regard to the pyruvic and α-ketoglutaric acid content in biological material. The recovery of these two keto-acids, starting from known aqueous solutions, averages 94%; on the basis of these results a standard curve can be drawn. According to this curve, recovery from deproteinized extracts of liver and blood averages 91.6% for pyruvic acid and 96.7% for α-ketoglutaric acid. 3. 3. Using this method the pyruvic and ketoglutaric acid content of rat blood, muscle, liver and brain is estimated.

Novel findings on the copper catalysed oxidation of cysteine

SummaryThe oxidation of cysteine (RSH) has been studied by using O2, ferricytochrome c (Cyt c) and nitro blue tetrazolium (NBT) as electron acceptors. The addition of 200μM CuII to a solution of 2mM cysteine, pH 7.4, produces an absorbance with a peak at 260 nm and a shoulder at 300 nm. Generation of a cuprous bis-cysteine complex (RS-CuI-SR) is responsible for this absorbance. In the absence of O2 the absorbance is stable for long time while in the presence of air it vanishes slowly only when the cysteine excess is consumed. The neocuproine assay and the EPR analysis show that the metal remains reduced in the course of the oxidation of cysteine returning to the oxidised form at the end of reaction when all RSH has been oxidised to RSSR. Addition of CuII enhances the reduction rate of Cyt c and of NBT by cysteine also under anaerobiosis indicating the occurrence of a direct reduction of the acceptor by the complex. It is concluded that the cuprous bis-cysteine complex (RS-CuI-SR) is the catalytic species involved in the oxidation of cysteine. The novel finding of the stability of the complex together with the metal remaining in the reduced form during the oxidation suggest sulfur as the electron donor in the place of the metal ion.

A possible role for rhodanese: The formation of ‘labile’ sulfur from thiosulfate

Rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) is a well-known mitochondrial protein [ 1,2] . In spite of its widespread occurrence and abundance, its physiological role is very uncertain. In vitro it catalyzes the transport of sulfur from thiosulfate to a nucleophilic acceptor (cyanide, reduced lipoate) [3,4] by a double displacement reaction with the formation of an intermediate sulfur-enzyme complex [5,6] . Although some cyanide may be formed in vivo, this seems insufficient to explain the ubiquity and abundance of rhodanese. The possible significance of its action on reduced lipoate is also unclear. In the present paper a new role for rhodanese is outlined, namely the possibility that the enzyme may contribute to the formation of ‘labile sulfur’ in non-heme iron proteins from thiosulfate.

Luminol chemiluminescence studies of the oxidation of cysteine and other thiols to disulfides.

Abstract The luminol chemiluminescence excited by autooxidizing thiols has been investigated. When the copper-catalyzed thiol oxidation in alkaline medium is allowed to take place in the presence of luminol, a sharp light flash is observed at the end of the reaction. It has been demonstrated that the luminol chemiluminescence is due to the sudden degradation of the H2O2 accumulated during the reaction. By a chemical method, H2O2 production during thiol oxidation has been demonstrated. H2O2 is accumulated during the reaction and is immediately destroyed when all the thiol is oxidized to the disulfide. A comparison of the degradation of H2O2 and the excitement of luminol chemiluminescence shows that both reactions are strongly dependent upon pH. At high pH values H2O2 is suddenly destroyed by cupric ions, and chemiluminescence appears. At neutral pH values it is more stable and cannot excite luminol chemiluminescence. It has been shown that, under the experimental conditions described, the degradation of H2O2 requires the presence of a free metal, and only in these conditions may it excite the chemiluminescence of luminol. The results obtained indicate that during thiol oxidation the metal is strongly complexed by the thiol itself. At the end of the reaction, when thiol is no longer present in solution, the metal is again free and immediately decomposes the accumulated H2O2, thereby exciting the luminol chemiluminescence.

Transamination of L-cystathionine and related compounds by a bovine liver enzyme. Possible identification with glutamine transaminase

A transaminase which catalyses the monodeamination of L-cystathionine was purified 1100-fold with a yield of 15% from bovine liver. The monoketoderivative of cystathionine spontaneously produces the cyclic ketimine. Other sulfur-containing amino acids related to cystathionine such as cystine, lanthionine and aminoethylcysteine were also substrates for the enzyme. The relative molecular mass of the enzyme was determined to be 94 000 with a probable dimeric structure formed of identical subunits. The isoelectric point of the enzyme was at pH 5.0 and the maximal enzymatic activity was found at pH 9.0--9.2. Kinetic parameters for cystathionine and for the other sulfur amino acids as well as for some alpha-keto acids were also determined. Among the natural amino acids tested, glutamine, methionine and histidine were the best amino donors. The enzyme exhibited maximal activity toward phenylpyruvate and alpha-keto-gamma-methiolbutyrate as amino acceptors. The broad specificity of the enzyme leads us to infer that the cystathionine transaminase is very similar or identical to glutamine transaminase.

Cleavage of cystine by a pyridoxal model.

In the presence of catalytic amounts of pyridoxal and copper ions at pH 8.5 and 38 °, cyatine is oxidized and extensively degraded. Pyruvate, ammonia, CO2, free sulfur, thiosulfate, cysteinesulfinic acid, and alanine thiosulfonate are the main products of the reaction. The addition of sulfinates to the reacting mixture leads to the production of large amounts of the corresponding thiosulfonate instead of free sulfur. Evidence is obtained that thiocysteine (alanine hydrogen disulfide) is a possible intermediate in a cyclic process complicated by many side reactions.

Transamination of l-cystathionine and related compounds by bovine brain glutamine transaminase

Glutamine transaminase (EC 2.6.1.15) has been purified 113 fold from bovine brain. The product is free of aspariate amino transferase (EC 2.6.1.1.) and other common transaminases. The enzyme shows a wide specificity similar to that reported from the same transaminase purified from bovine kidney and liver as regards both the amino donor and the amino acceptor. Of interest is the transamination and cyclization of l-cystathionine, l-lanthionine, l-cystine and S-aminoethylcysteine. The latter result indicates that the deamination and the cyclization of the sulfur containing diamino acids described for bovine liver and kidney enzyme is feasible also in the brain and suggests the possible endogenous origin of cyclothionine and thiomorpholine dicarboxylate recently detected in bovine brain.

Detection of 2H-1,4-thiazine-5,6-dihydro-3,5-dicarboxylic acid (lanthionine ketimine) in the bovine brain by a fluorometric assay

A new sulfur imino acid, 2H-1,4-thiazine-5,6-dihydro-3,5-dicarboxylic acid (lanthionine ketimine), has been detected in the bovine brain by means of fluorometric and HPLC procedures. The fluorometric assay is based on the fluorescent property of the copper-ketimine interaction product at pH 11.5. Other ketimines do not fluoresce in these conditions. The fluorophore exhibits an excitation maximum at 353 nm and an emission at 462 nm and is stable for at least 24 h. In the test conditions the fluorescence is proportional to the ketimine concentration from 1 to 200 microM. Detection of endogenous lanthionine ketimine has been performed after a simple enrichment procedure (brain deproteinization and extraction with diethyl ether) which minimizes degradative by-reactions of the unstable ketimine. The concentration of this new sulfur imino acid in the brain ranges from 0.5 to 1 nmol/g in three different samples. Identification and quantitations were confirmed by an HPLC procedure which takes advantage of the selective absorption at 380 nm of the phenylisothiocyanate-ketimine adduct. The identification of lanthionine ketimine in nervous tissues may have important metabolic and physiological implications.

Studies of the metabolism of thiazolidine carboxylic acid by rat liver homogenate.

Abstract The metabolism of thiazolidine carboxylic acid in a rat liver extract has been investigated. Evidence is presented for the occurrence of an oxidative reaction leading to the desaturation of the thiazolidine ring. Diformylcystine has been identified among the final metabolic products. The existence of other final metabolites, not yet identified, has been indicated by chromatography and spectrophotometry. Diformylcystine does not appear to be an intermediate in the formation of the final products.