Hydroxylamine, incubation

Additional support for the mechanism came from a second approach involving chemical modification experiments with hydroxylamine." Incubation of L-DEX with hydroxylamine in the presence of substrate led to the alkylation of Asp-10 and the concomitant loss of catalytic activity. The absence of substrate protected the enzyme from modification and inactivation by hydroxylamine. These findings are consistent with hydroxylamine attack on the carbonyl carbon of the ester intermediate, which results in the formation of catalytically inactive adducts at the site of the aspartate nucleophile. ... 

Stop the reaction by adding 100 mL of freshly prepared 1.5 M hydroxylamine incubate for 30 min. 

Anhydrides, such as acetic anhydride (Sections 4.2 and 5.1, this chapter), may react with carbohydrate hydroxyls even in aqueous environments to form acyl derivatives. The reaction, however, is reversible by incubation with hydroxylamine at pH 10-11. 

Given that hydroxylamine reacts rapidly with heme proteins and other oxidants to produce NO [53], the hydrolysis of hydroxyurea to hydroxylamine also provides an alternative mechanism of NO formation from hydroxyurea, potentially compatible with the observed clinical increases in NO metabolites during hydroxyurea therapy. Incubation of hydroxyurea with human blood in the presence of urease results in the formation of HbNO [122]. This reaction also produces metHb and the NO metabolites nitrite and nitrate and time course studies show that the HbNO forms quickly and reaches a peak after 15 min [122]. Consistent with earlier reports, the incubation ofhy-droxyurea (10 mM) and blood in the absence of urease or with heat-denatured urease fails to produce HbNO over 2 h and suggests that HbNO formation occurs through the reactions of hemoglobin and hydroxylamine, formed by the urease-mediated hydrolysis of hydroxyurea [122]. Significantly, these results confirm that the kinetics of HbNO formation from the direct reactions of hydroxyurea with any blood component occur too slowly to account for the observed in vivo increase in HbNO and focus future work on the hydrolytic metabolism of hydroxyurea. 

The mechanism of zinc deprivation by 3-nitrosobenzamide was elucidated most recently. When the reconstituted nucleocapsid protein p7 of HIV-1 (15 i-M) was incubated with 3-nitrosobenzamide (300 iM) at pH 7.5, three disulfide bonds per protein molecule were formed while 3-nitrosobenzamide was reduced to the hydroxylamine. Molecular masses of p7 adducts augmented by one or two 3-nitrosobenzamide residues were observed by electrospray ionization MS, consistent with covalent bond formation between cysteine sulfur and the nitroso nitrogen atom127. 

The first suggestion of a practical form of antidotal therapy came in 1949 from Hestrin, who found that acetylcholinesterase (AChE) catalyzed the formation of acetohydroxamlc acid when incubated with sodium acetate and hydroxylamine. Critical in vitro studies in the next decade led to the development of a practical approach to therapy. The crucial concept in these studies was the recognition that the compound formed when AChE reacted with a phosphorus ester was a covalent phosphoryl-enzyme Intermediate similar to that formed in the hydrolysis of acetylcholine. 3 Wilson and colleagues, beginning in 1951, demonstrated that AChE inhibited by alkyl phosphate esters (tetraethyl pyrophosphate, TEPP) could be reactivated by water, but that free enzyme formed much more rapidly in the presence of hydroxylamine. 0 21 Similar results... 

Secondary alicyclic amines, such as pyrrolidine and piperidine, have many properties typical of the corresponding aliphatic amines. Metabolic oxidation at secondary alicyclic nitrogens results in the formation of hydroxylamines, which may then undergo enzymic or non-enzymic conversion to nitrones, and in some cases to nitroxide radicals. For example, the 2-substituted piperidino derivative (- )-anabasine (1), a tobacco alkaloid, is metabolized initially to a hydroxylamine (2) and then to the nitrone (3), when incubated with liver and... 

However, with certain secondary hydroxylamines, P-450, too, can catalyze transformation to nitrones. Thus, incubation of N,N-( 1 -phcnylcyclobutyljbenzyl hydroxylamine with P-450 2B1 in the presence of NADPH-cytochrome P-450 reductase and NADPH has been demonstrated to trigger nitrone 36 formation with Km = 48 pM by direct oxidation of the N-hydroxy compound exogenous catalase did not change the amount of product generated55. 

Ciccoli and coworkers have shown that incubation of rat erythrocytes with hydroxy lated metabolites of aniline, dapsone (39) and the corresponding hydroxylamines brings about enhanced release of iron and methemoglobin formation. This did not occur with parent compounds. That xenobiotics are effective only after biotransformation to metabolites in vivo is supported by acute intoxication of rats with aniline or 39 and marked increase in the erythrocyte content of free iron and of methemoglobin144. The potent toxicity of aniline-derived aminophenylnorharman in the liver of the gpt delta transgenic mouse has been demonstrated145,146. 

Twenty micro litre of the above solutions are added to microcentrifuge tubes followed by the addition of 20 pi concentrated hydrochloric acid, 20 pi hydroxylamine hydrochloride solution, 200 pi ammonium acetate buffer, 80 pi 1,10-phenanthroline solution and 860 pi deionized water. The samples are mixed by vortexing and incubated for 20 min at room temperature. 

Degradation of L-cysteine by cysteine desulfhydrase or other PLP enzymes present in the cells was successfully prevented by addition of hydroxylamine or semi-carbazide to the incubation mixture. A mutant strain of Ps. thiazolinophilum lacking cysteine desulfhydrase was isolated and used to produce L-cysteine from d,l-ATC in a molar yield of 95% and at a product concentration of 31.4 g L 1[12S. Pseudomonas desmolytica A] 3872, one of the L-cysteine producers isolated was found to lack the ability to convert d-ATC into L-cysteine it is an ATC racemase-deficient strain 129l. However, little is known about the enzymological properties and function of the racemase. 

The evidence supporting the double-displacement mechanism for phosphoglycerate kinase was less extensive than that advanced for the acetate kinase reaction. The strongest evidence was the formation of a [ P]phosphoenzyme by reaction of the purified enzyme with either [y- PlATP or 1,3,[l- P]diphosphoglycerate. The P was removed from the enzyme by incubation with 3-phosphoglycerate or ADP. Reaction of the P-labeled phosphoenzyme with hydroxylamine also... 

Because the redox state of the complex ZQB controls fluorescence yield, Z must be inactivated in order to probe QB interactions. A pretreatment of chloroplasts with high concentrations of hydroxylamine will completely destroy the oxidizing side of PS II (Z) and produce some reduction of B (13). The experimental protocol was the following first, a chloroplast suspension was treated in the dark with 10 mM hydroxylamine and was measured immediately. After a 5 min dark incubation period, the chloroplasts received a strong actinic illumination that gave rise to the QB form. After complete relaxation (15 min), DCMU-type inhibitors were added producing Q B and difference between the two values was evaluated in relation to the inhibitor concentrations and a half-effect determined. 

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