Methylene blue oxidations of thiols

Kinetic Analysis of the Methylene Blue Oxidations of Thiols... 

The observed rate laws for the methylene blue oxidations of both mercaptoethanol and dithioerythritol at the different pH s are consistent with the mechanisms proposed for these oxidations. The most significant effect of the pH on the reaction is that of establishing the relative amounts of thiol and sulfide ion. Initiation of the chain sequence by the reaction of a sulfide ion with MB+ would be expected to be more rapid, and therefore the overall oxidation. faster, if the pH of the medium is increased. However, at the higher pH s where the relative amount of undissociated thiol is small, the reaction rate diminishes indicating not only that the thiol is likely a reactant in the overall reaction but that it is involved in a rate limiting step of the reaction at higher pH s. While not as supportive as the... 

Reagents reactive solely toward the methionyl side-chain in native proteins have not been described to date. Selective conversion of the thioether to the sulfoxide derivative by photosensitized oxidation has been reported in a protein devoid of free thiol groups. The selective photosensitized oxidation employed methylene blue or hema-toporphyrin as sensitizers, and aqueous acetic acid (30-90% v/v), or acidic buffers at pH 2-6.5, as solvents (Scoffone et al. 1970). Anaerobic photo-oxidation in 4 M aqueous acetone has been reported to lead to the same result (Gennari and Jori 1970). Methionine can be regenerated from the sulfoxide by incubation with thiols (5 % aqueous j9-mercapto-ethanol at pH 8.0, for 24 hr under Nj Jori et al. 1968). 

Walker and Nicholas (338) have reported the isolation and 600-fold purification of an enzyme from P. aeruginosa, which reduces nitrite to nitric oxide. The preparation contained 1.5 nmoles of FAD per mg protein, a c-type cytochrome and an absorption band at 630-635 nm, suggestive of copper. As electron donors, reduced FMN, FAD, riboflavin, pyo-cyanine, and methylene blue were effective, but not NADH, NADPH, or reduced cytochrome c. The preparation required phosphate or sulfate for maximal activity. The cytochrome and the 630-635-nm band were reduced under anaerobic conditions with a suitable electron donor and readily oxidized by nitrite. The Km for NaNOj is reported to be 3.1 X 10" M. The presence of an active thiol in the enzyme is indicated by p-mercu-ribenzoate inhibition and glutathione reactivation. 

The observed rate laws for the oxidation of mercaptoethanol by methylene blue under different reaction conditions are consistent with the steady-state rate laws derived from the proposed mechanism. If step 2 of the reaction sequence, namely formation of the disulfide radical anion from a thiyl radical and a sulfide ion, is the rate limiting step of the chain sequence, and therefore only termination by 6 (coupling of thiyl radicals) occurs, the derived rate law for the reaction is Eq. 11. This rate law, which takes.into account the distribution of the thiol and sulfide as determined by the of the thiol and the acidity of the medium, predicts that the observed rate law would be half order in MB and three halves order in mercaptoethanol. This is the rate law expected, however, only if the concentration of MB " is sufficiently high so that the second term in the denominator is neglible. At pH s below the pH maximum, and at a sufficiently... 

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