Cystine titration

The -ATPase gene sequence indicates the presence of eight cysteine residues in the molecule [37,38]. In order to ascertain the chemical state of these cysteine residues, direct chemical studies with established cysteine and cystine reagents were carried out [44]. Titrations with the cysteine reagent, dithiobisnitrobenzoate, and the cystine reagent, nitrothiosulfobenzoate, indicated the presence of six free cy-... 

Nur wenigc davon kdnncn im intaktcn Protein quantitativ erfaBt wer-den. So z.B, Tyiosin nnd Tiyptoplian durch Messung der Ultraviolett-Absorption, Trj ptophan durch spezifische koloriinetrische Methoden, Cystin und Cystein durch Titration oder durch photometrischc Methoden (siehc z.B. Block (9), Light und Smith 69)). 

Neuberg and Popowsky, as also Abderhalden and Kempe, have introduced a few alterations in the procedure, such as evaporation in vacuo, and Levene and Rouiller suggested in 1906 that the tryptophane, on account of its proneness to decompose on evaporation of its solution with consequent loss, be estimated colorimetrically the mercury sulphate precipitate is decomposed, and the solution, freed from hydrogen sulphide, is titrated with bromine water in presence of amyl alcohol. Both cystine and tyrosine react with bromine water the latter can, however, be removed, but for the former a correction has to be made. Up to the present no values concerning the amount of tryptophane in various proteins have appeared, and it will be of interest to see if the values so obtained are very much higher than those obtained by crystallisation of the tryptophane. 

The molecular weight of 320,000 obtained for the muscle enzyme from sedimentation-diffusion data at 2-6 mg/ml and v = 0.75 (132) is to be compared with 270,000 obtained by Wolfenden et al. from s20,w = 11.1 S and D2 ,w = 3.75 X 10 7 cm2 sec1, and v = 0.731 calculated from the amino acid content (92). The rabbit muscle enzyme has a normal amino acid content, that is, no unusually low or large amount of a particular amino acid was found. Of the 32 cysteine/half-cystine residues per mole based on a molecular weight of 270,000, 6.2 were rapidly titrated with p-mercuribenzoate (92). Typical protein absorption spectra were reported for elasmobranch fish (126), carp (125), rat (127), and rabbit muscle enzyme (68). An E m at 280 nm = 9.13 has been reported for the rabbit muscle enzyme (133). The atypical absorption spectrum with a maximum at 275-276 nm observed by Lee (132) is indicative of contaminating bound nucleotides. 

When ferredoxin was obtained in crystalline form, one of the basic questions was whether the unstable form of sulfur was a special constitutent of ferredoxin or whether it arose from the half-cystine residues. This question has not been answered unequivocally, but available evidence supports the presence of two types of sulfur in ferredoxin half-cystine sulfur and inorganic sulfide. This conclusion is based mainly on the specificity of the method used for determination of inorganic sulfide, on amino acid and elemental analyses, and on mercurial titration data. 

Lovenberg, Buchanan, and Rabinowitz (65) tested the response of ferredoxin to mercury compounds. Two mercurial reagents used, p-mer-curibenzoate (PCMB) and o-((3-hydroxymercuri-2-methoxypropyl)car-bamyl)phenoxyacetate (sodium mersalyl) reacted rapidly with ferredoxin and caused a bleaching of the visible spectrum and a concomitant loss of biological activity. C. pasteurianum ferredoxin was titrated with PCMB as described by Boyer (24) and the results showed that 20 moles of PCMB reacted with 1 mole of ferredoxin. In another determination, 2 moles of PCMB reacted with 1 mole of sodium sulfide. Since ferredoxin contained 7 moles of inorganic sulfide and 8 moles of half-cystine residues, 22 (7 x 2 = 14 14 + 8 = 22) moles of PCMB would be expected to react with 1 mole of ferredoxin. These data, summarized in Table 8, are consistent with the existence of two types of sulfur in ferredoxin. This conclusion was supported by the presence of half-cystine residues in ferredoxin after inorganic sulfide had been removed by acid hydrolysis, as well as results of sulfur analyses, which showed an amount of sulfur greater than could be attributed to half-cystine residues. 

Lovenberg, Buchanan, and Rabinowitz found that treatment of ferredoxin with iodoacetate or N-ethylmaleimide in either the presence or absence of 8 M urea had no effect on its spectral characteristics. Less than 1 mole of carboxymethyl cysteine was formed per mole of protein when native ferredoxin was treated with iodoacetate-1-C14 (Table 10). Sobel and Lovenberg (96) showed recently that C14-iodoacetate did not react appreciably with reduced ferredoxin. However, Table 10 shows that if ferredoxin was treated with 2-mercaptoethanol in 8 M urea, it was alkylated with iodoacetate. This demonstrated that the half-cystine residues of native ferredoxin were not present as free sulfhydryls, and the mercurial titration data given above showed that they were not present as disulfides. The two observations were consistent, therefore, with a structure in which the half-cystine residues are present as cysteine and are bonded with the iron by a sulfide bridge. 

On treatment with 1,4-dithiothreitol or 2-mercaptoethanol in dodecyl sodium sulfate, components II and III both yielded199,586 subunits of molecular weight 31,000. Poly(acrylamide) gel-electrophoresis, in the presence of 1% of dodecyl sodium sulfate alone, gave586 a component in the range of 60,000. Amino acid analysis (see later) showed two half-cystine residues per subunit. Direct titration of components II and III with 5,5 -dithiobis(2-nitrobenzoic acid), in the absence or presence of 8 M urea or dodecyl sodium sulfate, gave199,588... 

Paramyosin has no cystine residues and a low number of cysteine residues (11, 46, 47), whereas LMM Fr. I (35) has half-cystines and shows no trace of free sulfhydryl groups by the p-chloromercuribenzoate titration. Tropomyosin also has at least one disulfide bond (11) which may link the two chains of the coiled coil (65). 

P-Amylases.— Five half-cystine residues in soybean p-amylase can be titrated with 4-chloromercuribenzoate after denaturing the enzyme with guanidinium hydrochloride. The reactivities of these groups towards 4-chloromercuri-benzoate differ and are pH-dependent. Differential titrations at different pH values and in the presence of maltose showed that reaction of only one sulphydryl group with 4-chloromercuribenzoate results in loss of enzymic activity and a conformational change. 

A more successful reagent which is used for the analysis of thiols is a standard solution of cupric copper. The titration, performed in ammoniacal solution, is more selective than argenti-metric procedures, and it is possible by this titration (which is dependent on the reduction of divalent copper with cysteine to cuprous copper) to analyse mixtures of cysteine and cystine. 

Procedure. Deoxygenate 25-100 ml. of 0-05 n ammonia in the titration cell and add a sample of 4 X 10 M to 1 X 10 M cystine or cysteine. Make the solution O l m to sodium sulphite and carry out the titration with a 0-01 M solution of divalent copper which is preferably prepared from metallic copper. Measure the current at —0-4 V with a rotating platinum electrode. The titration curve obtained should be of the form given in Fig. 47. 

Cystine has also been determined in wool using a polarometric titration< based on the principle that sulphite reacts with... 

Miscellaneous Procedures. Most of the other procedures for the determination of —SH groups are variants of the processes indicated above. Of particular interest are the several amperometric titration procedures which, in effect, depend on mercaptide formation. Polarographic methods have also been used. An interesting submicro method for cystine by a Cartesian diver technique has recently been developed the method depends on the catalytic effect of —SS— groups on the decomposition of azide ion to nitrogen (78,94). The method cannot be applied to the direct determination of —SH groups. 

---