Amino acid-specific ADP-ribosylation: structural characterization and chemical differentiation of ADP-ribose-cysteine adducts formed nonenzymatically and in a pertussis toxin-catalyzed reaction.

ADP-ribosylation is a posttranslational modification of proteins by amino acid-specific ADP-ribosyltransferases. Both pertussis toxin and eukaryotic enzymes ADP-ribosylate cysteine residues in proteins and also, it has been suggested, free cysteine. Analysis of the reaction mechanisms of cysteine-specific ADP-ribosyltransferases revealed that free ADP-ribose combined nonenzymatically with cysteine. L- and D-cysteine, L-cysteine methyl ester, and cysteamine reacted with ADP-ribose, but alanine, serine, lysine, arginine, N-acetyl-L-cysteine, 2-mercaptoethanol, dithiothreitol, and glutathione did not. The 1H NMR spectrum of the product, along with the requirement for both free sulfhydryl and amino groups of cysteine, suggested that the reaction produced a thiazolidine linkage. ADP-ribosylthiazolidine was labile to hydroxylamine and mercuric ion, unlike the ADP-ribosylcysteine formed by pertussis toxin and NAD in guanine nucleotide-binding (G-) proteins, which is labile to mercuric ion but stable in hydroxylamine. In the absence of G-proteins but in the presence of NAD and cysteine, pertussis toxin generated a hydroxylamine-sensitive product, suggesting that a free ADP-ribose intermediate, expected to be formed by the NADase activity of the toxin, reacted with cysteine. Chemical analysis, or the use of alternative thiol acceptors lacking a free amine, is necessary to distinguish the enzymatic formation of ADP-ribosylcysteine from nonenzymatic formation of ADP-ribosylthiazolidine, thereby differentiating putative NAD:cysteine ADP-ribosyltransferases from NAD glycohydrolases.

The photochemistry of p-aminobenzoic acid.

We have studied the photoreactions occurring when p-aminobenzoic acid (PABA), a component of some sunscreens, is irradiated in aqueous solution. These studies were carried out in the presence and absence of oxygen, using light of lambda = 254 nm as well as light of wavelengths greater than 290 nm. In deoxygenated solution between pH 7.5 and 11.0, we found two photoproducts that were identified as 4-(4'-aminophenyl)aminobenzoic acid (I) and 4-(2'-amino-5'-carboxyphenyl)aminobenzoic acid (V); we used 1H and 13C NMR, electron impact mass spectrometry and synthesis by an independent route to identify each of these compounds. Rapid discoloration of the photolyzed sample was observed when PABA was irradiated in aerated solution. Although a number of products were detected under these conditions, the three most abundant stable compounds have been isolated and identified as 4-amino-3-hydroxybenzoic acid, 4-aminophenol and 4-(4'-hydroxyphenyl)aminobenzoic acid (IV). The latter compound was shown to result from rapid photo-induced oxidation of I in the presence of oxygen. Even in the presence of trace amounts of oxygen, the yield of I was significantly reduced in favor of IV. Studies of the thermal oxidation of I, coupled with evidence gathered from studies of the photochemistry of incompletely deoxygenated PABA solutions, indicate that 4-(2,5-cyclohexadien-4-one)iminobenzoic acid (III) is an intermediate on the pathway between I and IV. Qualitatively, we found that the photochemical reactions resulting from irradiation of PABA solutions with lambda = 254 nm light and light with lambda greater than 290 nm were the same. The quantum yields for formation of I and V are highly pH dependent, both being less than 10(-4) at pH 7 and rising steadily to values greater than 10(-3) at pH 11. The detailed pH dependence suggests that the deprotonated PABA radical cation may be an important intermediate entering into the reactions forming I and IV.

Photoaddition of p-aminobenzoic acid to thymine and thymidine.

Several studies in the literature have shown that DNA is damaged after UV irradiation in the presence of the sunscreen agent p-aminobenzoic acid (PABA), both in vivo and in vitro. One type of damage has been shown to be the result of increased yields of pyrimidine cyclobutane dimer formation. However, it has been suggested that other types of lesions are produced as well. We have studied the photochemistry of the thymine-PABA and thymidine-PABA systems and report here the isolation and characterization of thymine-PABA and thymidine-PABA photoadducts. These products have been identified, respectively, as 5-(2-amino-5-carboxyphenyl)-5,6-dihydrothymine and isomeric forms of 5-(2-amino-5-carboxyphenyl)-5,6-dihydrothymine. The quantum yields for the formation of these adducts in deaerated aqueous solutions at pH 7.0 have been determined to be 9.5 x 10(-4) and 4.3 x 10(-3) for the thymine and thymidine based adducts respectively. A pH profile for the thymine-PABA system indicated a maximum quantum yield for adduct formation at pH 6.5, although it could be detected over the whole pH range studied (pH 3.5-11.0).