A new relation of .... modification of enzyme lysozyme

Abstract

The characterisation of a new reaction of o-benzoquinone at the indole moiety of tryptophan and its application in the chemical modification of the enzyme lysozyme constitutes the present thesis. o-Benzoquinone is a highly reactive compound whose reactions with proteins and amino acids have great biological relevance. The chemistry of the o-quinone reactions with proteins and amino acids is complex. Some of the functional groups known to be involved in these reactions are the amino, imino, sulfhydryl, and thioether groups. Preliminary investigations showed that o-benzoquinone undergoes a reaction at acid pH with tryptophan. A detailed study has now been made of this reaction of o-benzoquinone. o-Benzoquinone prepared by the oxidation of catechol was used in this study. The quinone had a characteristic absorption spectrum with absorption maxima in the region of 380 nm (? = 1800). The quinone was found to be stable in solvents like ether, chloroform, hexane, and glacial acetic acid. It was relatively less stable in aqueous solvents. The reactions of the quinone with amino acids and proteins have generally been carried out either in glacial or aqueous acetic acid or in dilute aqueous hydrochloric acid solutions. o-Benzoquinone was found to react with tryptophan, N?acetyl?DL?tryptophan, N?acetyl?DL?tryptophan methyl ester, N?acetyl?DL?tryptophan amide, and other 3?substituted indoles such as 3?methylindole, 3?dimethylaminoindole (grammine), indole?3?butyric acid, and indole?3?acetic acid within a relatively short period of less than three minutes to give products incorporating phenolic chromophores. Tryptophan?containing dipeptides L?tryptophyl?L?glycine and L?tryptophyl?L?tyrosine were also found to give similar condensation products. The reaction of o-benzoquinone with an amino acid mixture showed that methionine and tryptophan are the only amino acids capable of reacting with o-benzoquinone under the said experimental conditions. The reaction products of o-benzoquinone with N?acetyl?DL?tryptophan and other 3?substituted indoles were found to have distinct chromatographic mobility and characteristic absorption spectra. The o-benzoquinone derivatives of N?acetyl?DL?tryptophan and 3?methylindole were prepared and were chromatographically homogeneous. The o-benzoquinone derivative of N?acetyl?DL?tryptophan prepared in the presence of hydrochloric acid had an empirical formula C??H??O?N?Cl, and the o-benzoquinone derivative of 3?methylindole had an empirical formula C??H??O?NCl. A detailed study was made regarding the electrophoretic, spectral, and potentiometric titration characteristics of the o-benzoquinone derivative of N?acetyl?DL?tryptophan. It was found that the derivative prepared in the presence of hydrochloric acid contained one equivalent of chloride per mole of derivative. The derivative was also found to have an excess positive charge at pH 2 as compared to N?acetyl?DL?tryptophan. This was indicative of the possible presence of a quaternary nitrogen atom in this derivative. In acidic solutions (pH 2), the o-benzoquinone derivative of N?acetyl?DL?tryptophan was found to have an absorption spectrum with one absorption maximum at 283 nm, a minimum at 262 nm, and high absorption below 250 nm. At pH values above 6, spectral changes characteristic of phenolic ionisation were observed. These changes were found to be of two kinds: (i) a reversible ionisation below pH 9.9, and (ii) an irreversible ionisation above pH 9.9. The derivative was found to undergo decomposition by acid as well as basic hydrolysis. It did not undergo reduction on catalytic hydrogenation at atmospheric pressure and at five atmospheres. Potentiometric titration data of the o-benzoquinone derivative of N?acetyl?DL?tryptophan showed that the derivative has three ionisable groups with pK values 3.5, 9.7, and 12.05, which could be assigned to a carboxyl, a phenolic hydroxyl with a low pK, and another phenolic hydroxyl, respectively. The molecular weight of the derivative was found to be around 380 from titration studies. The IR spectrum of the derivative clearly showed the presence of a carboxyl group (1715 cm?¹) and the possible existence of a quaternary nitrogen atom. The PMR spectrum of the derivative in D?O showed resonance signals at 2.14?, 3.2?, and 6–7.6?. These signals can be attributed to the methyl protons of the acetyl group, the ??methylene protons, and the aromatic protons, respectively. The signals were also present in the ratio 3:2:8, indicating that the derivative has eight aromatic protons in contrast to the native N?acetyl?DL?tryptophan, which has only five. The extra three aromatic proton signals arise due to the incorporation of the o-benzoquinone moiety in the derivative. Combining the above results, the following structure has been proposed for the o-benzoquinone derivative of N?acetyl?DL?tryptophan.