Effect of Visible Light on Amino Acids—ii. Histidine

Abstract

FOR U N D E R S T A~I N G the role of histidine in the biological activity of enzymic and hormonal proteins, photooxidation technique has been frequently employed.(1-6) The main interest in these experiments has rested in the quantitative aspect of the disappearance of histidine in the protein molecule. Weil(?* e, has shown that photooxidation of free histidine results in destruction of the imidazole group. Lukton and colleagues(s) and Weilo measured the rate constants for the destruction of this amino acid by visible light at varying pH and temperature. Information in regard to the chemical nature of the resultant products is not available.The experiments presented in this report show that when an aqueous solution of histidine containing methylene blue (MB), is irradiated with visible light, four chemically distinct products are formed which can be detected by paper chromatography. It was found, using labelled histidine, that these products retain carbon-2 of the imidazole ring. None of them were formed when a solution of histidine was subjected to the action of either hydrogen peroxide or u.v.-light. Experimental details have been described earlier. (lo) MB was removed by adsorption on active carbon; contrary to the observation made with tryptophan,(lO) histidine and its irradiation products were not adsorbed on active carbon. Irradiation at pH 7 or 9 in the presence of MB resulted in a decrease in the u.v.-absorption at all wave lengths between 200-230 mp (Figs. 1 and 2). It was shown by Saidel and associates(ll) that the imidazole group has characteristic maximum at 210-212 mp in acid but not in neutral media. An irradiated solution of histidine did not show this maximum in an acid medium ( Fig. l), suggesting that the observed decrease in the intensity of absorption at 210-212 mp on irradiation is due to the disappearance of the imidazole group. Irradiation of histidine in the absence of MB resulted in a slight increase in the absorption in the above mentioned range, which could be due to slight evaporation during the irradiation period.Separation of the products obtained by irradiation of a solution of histidine in the presence of MB was attempted using paper chromatography in two different solvents.

Photooxygenations of nitrogen heterocycles

“…29 Sensitized photooxygenations of different fe/T-butylpyrroles have been studied in detail.30,31 It has been shown, for example, that both 2,5-di-te/T-butylpyrrole (66) and 2,3,5-tri-fe/T-butylpyrrole (68) are converted to the corresponding 2-hydroperoxides 67 and 69, respectively (Scheme XI).30 In contrast, the photooxygenations of mono-fe/T-butylpyrroles follow a different pathway.31 Thus, 1-ferf-butylpyrrole (70) on photooxygenation in methanol and using Rose Bengal gives a mixture of products consisting of 5-methoxy-1-ferT-butyl-A3-pyrroline-2-one (71), 5-hydroxy-1-ferf-butyl-Á3-pyrrolin-2-one (72), and N-tertbutylmaleimide (73). The Methylene Blue sensitized photooxygenations of 70 in acetone, however, gives a mixture of 72 and /3-(A/-fe/T-butylformamido)acrolein (74) (Scheme XII). Similarly, the photooxygenation of 2-ferf-butylpyrrole (75) in methanol yields a mixture of the 5-methoxylactam 76, the 5-hydroxylactam 77, and pivalamide (78), whereas the photooxygenation of 75 in acetone gives a mixture of 77,78, the keto amide 79, and the hydroxy ketone derivative 80 (Scheme XII).…”

A Study of the Methylene Blue‐sensitized Oxidation of Amino Acids

“…Gurnani et a1. [18] and Weil el al. [4,5] have shown that the sensitized oxidation of tryptophan in air results primarily in the oxidation of the ring system, and that substitution or removal of the carboxyl and a-amino groups has no effect upon the oxygen uptake by the indole moiety.…”

“…Histidine. Histidine is considered to be the most sensitive amino acid to sensitized oxidation [4,5,7,19] and Weil found it to be nearly as effective as tryptophan in reducing methylene blue at pH 8. However, under our conditions, it slightly inhibits the fading of the dye.…”

Studies on Lysozyme: Effect of Visible Light on the Conformation of the Enzyme in the Presence of Methylene Blue

“…The proteolytic enzymes were then inactivated by adding 0-1 ml of 1 N HCI and heating at 90" for 15 min, and the mixture was dialysed against water. Water was removed from the solution outside the dialysis bag in a vacuum desiccator and the residue taken up in a small volume of water; this solution was used for paper chromatography and for the detection and estimation of tryptophan and its modified products [23,24].…”

“…However, the tryptophan spot from the photooxidized enzyme contained, on an average, 30 per cent less amino acid than the tryptophan spot from the native enzyme. Further, a new spot reacting with paradimethylaminobenzaldehyde was obtained on the chromatogram of the hydrolysate of the photooxidized enzyme; The Rf value of this spot in various solvents [23] suggested that it might be identical with the major product of the degradation of tryptophan exposed to visible light in the presence of methylene blue (component IV in [23]). Lysine was the only N-terminal amino acid in both native and photooxidized lysozyme, which shows that no peptide bonds were broken during the exposure of the enzyme to light in the presence of MB.…”