Cysteine titration

From the bond energies of RS-H, RS-Au, H-H bond which are 87 kcal/mole, 40 kcal/mole and 104kcal/mole respectively, the net enthalpy of binding of thiol to Au is -5 kcal/mole [16], The total heat liberated in the cysteine titration is —966 kcal per mole of Au nanorods. The average number of cysteine molecules attached to Au nanorods thus works out to be around 193. 

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-... 

In one set of experiments a titration of compound is performed to assess its potency in vivo. HeLa cells are maintained in DMEM supplemented with 10% fetal bovine serum (FBS) at 37° in 5% C02. One day prior to labeling, the cells are seeded in 24-well plates at approximately 60,000 cells per well. The next day, cells are washed with warm (37°) PBS and the medium replaced with 250 41 of methionine-free DMEM containing 10% dialyzed serum (Invitrogen). After a 15-min incubation at 37°, different concentrations of compound are added to the cells (which can range from 1 nM to 50 fiM) and the incubation continued for another 45 min. Anisomycin is used as a positive control at a final concentration of 50 /iM. Fifty-five microcuries of 35S-methionine/cysteine [35S-methionine/cysteine express protein labeling mix (1175 Ci/mmol) (Per-kin-Elmer)] is added to each well (220 /(Ci/ml) and the incubation continued for another 15 min. 

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)). 

As deduced from the DNA sequence of the gene, AMDase contains four cysteine residues. Since a-halocarboxylic acids are generally active alkylating agents there is a possibility that a-bromophenylacetic acid reacts with several cysteine residues of the enzyme. Therefore, we tried to clarify how many cysteine residues react with this inhibitor. It is well established that when p-chloromercuri-benzoate (PCMB) binds to a cysteine residue, the absorbance at 255 nm increases due to the formation of an aryl-Hg-S bond. Thus it is possible to estimate the number of free S-H residues of the enzyme by titration with PCMB solution (Fig. 6). When the native enzyme had reacted with PCMB, the absorbance at 255 nm increased by 0.025. On the other hand, when PCMB solution was added to the enzyme solution after the enzyme was incubated with a-bromophenyl-... 

Site-directed mutagenesis is one of the most powerful methods of studying mechanisms of enzyme-catalyzed reactions. Since this technique makes it possible to replace a specific amino acid residue of an enzyme by an arbitrary one, it is particularly useful to specify the amino acid residue(s) which is responsible for the activity [20 - 22]. In the case of AMDase, one of four cysteine residues was presumed to be involved in the catalytic site by the titration experiments. To determine which Cys is located at the active site, preparation of four mutant enzymes, in each of which one of the cysteines is replaced another amino acid, and kinetic studies on them, are expected to be most informative. Which amino acid should be introduced in place of cysteine To decide on the best candidate. 

Fluorescence determinations are important to analyze cysteine, guanidine, proteins, (LSD), steroids, a number of enzymes and coenzymes, and some vitamins, as well as several hundred more substances. A fluorometer can be used to verify conformational changes in multipartite operator recognition by. -repressor as explained in a journal article by Deb et al. (2000). Upon titration with single operators site, the tryptophan fluorescence quenches to different degrees, suggesting different conformations of the DNA-protein complexes. 

Acid-base titration gave the following three pKa values for cysteine 1.70,8.36, and 10.53. Spectro-photometric data allowed H. B. F. Dixon and K. F Tipton (1973, Biochem. 133,837-842) to estimate the following ratio of tautomeric species at pH 9.4 [ -OOC - C(NH3+)- CH2S] / [OOC - C(NH2) -CH2SH] = 2.12, as is also shown in Fig. 6-5. Verify and assign the four microscopic pfCa values. 

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. 

The two preparations from A. oryzae reportedly differ in amino acid and carbohydrate composition. The enzyme prepared by Minato contained 25% carbohydrate no cysteine was detected either by titration with p-mercuribenzoate in 6M urea or by cysteic acid analysis after performic acid oxidation (179). In contrast, Wolfenden et al. (92) reported 14 cysteine residues per mole of enzyme which reacted instantaneously with p-mercuribenzoate in the absence of urea. No explanation is available for this apparent discrepancy. 

The formation of mixed enzyme-CoA or enzyme-acyl carrier protein disulfides was supported by the fact that the activation by these compounds was readily reversed by treatment with glutathione or cysteine (49). Preliminary experiments (50) with radioactive CoA or by titration of sulfhydryl groups in the protein suggest that maximum activation is associated with the incorporation of 4 equivalents of CoA per mole of protein (one per subunit). 

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. 

Fig. 1. (a) Raw calorimetric data obtained by the titration of 0.57 mM cysteine with 1.59 nM gold nanorod solution and (b) binding isotherm plot obtained by integrating each peak in raw data and normalizing with cysteine concentration. 

Fig. 2. UV-visible spectra of gold nanorods (a) before and after calorimetric titration with cysteine and (b) taken at different stages of the calorimetric titration with MPA as indicated in Fig. 4b.

Research aimed at identifying the ligands comprising the flattened tetrahedral blue copper center has been particularly intense in the case of plastocyanin. Direct evidence for a sulfur ligand has come from x-ray photoelectron spectral (XPS) experiments on bean plastocyanin, where a large shift of the S2p core energy of the single cysteine (Cys-85) residue in the protein upon metal incorporation (164.5, apo 169.8, native 168.8 eV, Co(II) derivative) was observed (15). The two histidines in spinach plastocyanin exhibit pK values below 5 in NMR titration experiments,... 

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