Cysteine protection assay

Theoretical rate of modification of an introduced cysteine using four different concentrations of MTS (10 xM, 20 ulM, 30 ii.M, and 60 ulM, ). The pseudo first-order rate constants (kfl are 0.1285, 0.2445,0.4849, and 0.7564, respectively. When corrected for the concentration-dependence of MTS modification, the second-order rate constants (k2) are 12,900, 12,275 12,122 and 12,606 M s respectively. Although all k2 values are similar, the early phase of modification (<20 s) is less well described when higher concentrations (>20 xM) of the reagents are applied (see inset). Based on these data, we would use 10 xM for control experiments and protection assays... 

The same authors (G8, G7) also found very substantial decreases in riboflavin (approx. 80%), and niacin (P9) fared little better. When mixtures were irradiated unusual events occurred. Riboflavin and ascorbic acid were each protected by niacin. Addition of cystine or cysteine apparently sensitized the niacin (P10). Since initial rates were not given, and the doses were considerably above the oxygen breakpoint (Sec. IIIA2), no mechanistic interpretation is possible. There also appears to be some doubt about the reliability of the colormetric assay used by these workers. 

The original evidence for activation by modification of cysteine residues came from studies with 2,4-fluorodinitrobenzene (15). Incubation of the crystalline enzyme preparations with 4 equivalents of FDNB led to a marked increase in activity in the neutral pH range, together with a small decrease in the activity assayed at alkaline pH (Fig. 4). The modified enzyme showed two broad and nearly equal activity maxima one at pH 7.7 and the other at pH 9.0. When dinitrophenylation was carried out at pH 7.5, this change in catalytic properties was associated with the modification of only 2 of the 20 cysteine residues in the protein (16). These 2 highly reactive cysteine residues were found to be completely protected against the action of FDNB by addition of FDP. 

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