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Title: Some constituents of tissues of the eye
Author: Calam, Derek Harold
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1962
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Introduction The lens of the eye is a unique theme in several respects. It is derived entirely from one layer of epithelial cells and, since no cells are shed during growth, it contains cells at all stages of development. It has no blood supply and is dependent upon the aqueous humour for supply of nutrients and removal of waste. Calf lens contains a family of γ-glutamyl peptides which is unique among tissues. Those peptides are related to glutathione, γ-glutamylcysteinylglycine, by replacement of the central amino-acid with alanine in norophthalmic acid, with α-aminobutyric acid in ophthalmic acid, and with S-sulphocysteinein S-sulphoglutathione. Cliffe and Waley have shown that the enzyme system by which these peptides are synthesized will also incorporate other amino-acids into the central position, but the corresponding peptides have not been detected in lens. The function of this family of compounds is unknown and only one other member has been identified in nature: S-(β-carboxypropyl)-glutathione, which is present in onion. At least one other acidic tripeptide was known to be present in calf lens and the main object of this work was to isolate and identify it. Isolation involved fractionation of lens extracts, and the fractions obtained were subsequently analyzed for (mostly) ninhydrin-positive substances. Approximately 60 compounds were detected, some of which are known tissue constituents that have not been identified before in lens, and some of which may be previously unrecognized tissue constituents. A method has been developed for analysis of most of the low molecular-weight substances in small amounts of tissue by a combination of electrophoresis and chromatography on a single paper. New acidic compounds Fractionation of trichloroacetic acid extracts of lens was performed by two methods. The first, which has not been applied previously to tissue extracts, was by continuous-flow paper electrophoresis; the second was by anion-exchange chromatography. A better gross fractionation was obtained by continuous electrophoresis at pH 4 than by chromatography since basic and neutral substances were not adsorbed on the resin and were not separated. Acidic compounds were fractionated more satisfactorily by chromatography and the material obtained by the first method, which was homogenous on electrophoresis at pH 2.3 and pH 4, was split into two peaks by the ion-exchange column. These peaks were given by two different compounds which have been identified, by degradation and by comparison with synthetic material, as the peptide S-(α,β-dicarboxyethyl)-glutathione and the related amino-acid S-(α,β-dicarboxyethyl)-cysteine. Contaminations, present in the concentrated solutions after fractionation, prevented isolation of neutral material as pure solids. The peptide, on hydrolysis, yielded equimolar amounts of glycine and glutamic acid, together with a small amount of cysteine and an unidentified amino-acid. This amino-acid gave an atypical mauve colour with ninhydrin-cobalt chloride and contained sulphur. Dinitrophenylation indicated that glutamic acid was N-terminal in the peptide. Both the peptide and the amino-acid were oxidized with performic acid to what appeared to be sulphones. The oxidation products were separated from glutathione sulphonic acid by chromatography but not by electrophoresis at pH 4, nor was the amino-acid sulphone separated from it at pH 2.3. The sulphur atom was assumed to be present as a thioether in the new compounds since neither yielded a sulphonic acid on oxidation. Satisfactory separation of the amino-acid, the peptide, their oxidation products, cysteic acid, and glutathione sulphonic acid, was achieved on a single paper by a combination of electrophoresis at pH 4 and chromatography in butanol-acetic acid-water. The presence of a modified cysteine residue in the amino-acid was indicated by desulphurization of the peptide with Raney nickel to norophthalmic acid. The compounds S-(α,β-dicarboxyethyl)-cysteine and S-(α,β-dicarboxyethyl)-glutathione were known and had been prepared by addition of thiol to the double bond of maleic acid. The amino-acid had also been obtained by addition of cysteine to fumaric acid. These syntheses were repeated under salt-free conditions which facilitated isolation of the products, and the peptide was also obtained by addition of glutathione to fumaric acid. Corresponding products synthesized from each of the unsaturated acids appear to be identical and are probably mixtures of diastereoisomers. The amino-acid was conveniently isolated as a crystalline di-dicyclohexylamine salt. Identity between the natural and the synthetic compounds was established in several ways. The amino-acids were cluted at the same position from a cation-exchange column. They gave identical mauve colours with ninhydrin-cobalt chloride and could not be separated by electrophoresis at two pH values nor by chromatography in two solvents. Products of oxidation with performic acid were indistinguishable on electrophoresis. Both natural and synthetic S-(α,β-dicarboxyethyl)-cysteine yielded approximately 10% of cystine on hydrolysis, and largely cyclized on heating in aqueous solution to form more acidic substances, indistinguishable by chromatography, which gave strong reactions for organic acids but only weak colours with ninhydrin. Identical patterns of products were obtained on electrophoresis and on chromatography of peptide hydrolysates. Both natural and synthetic S-(α,β-dicarboxyethyl)-glutathione were oxidized to the same substance which was separated from glutathione sulphonic acid by electrophoresis at pH 2.3 but not at pH 4. Thiols react with N-ethylmaleimide much more rapidly than with either maleate or fumarate. An extract of fresh calf lens was prepared in the presence of N-ethylmaleimide in order to establish whether or not the new acidic compounds are artifacts. This extract contained both S-(α,β-dicarboxyethyl)-cysteine and S-(α,β-dicarboxyethyl)-glutathione and it seems unlikely that either compound was formed during the extraction procedure. Both compounds are, therefore, probably genuine constituents of calf lens and the concentrations in lens are about 0.05mM for the amino-acid and 0.2mM for the peptide, compared with 7-10mM for glutathione. Lens homogenates do not appear to catalyse the addition of either glutathione or cysteine to fumarate. The amount of free glutathione seemed to decrease in the lens mixture relative to a control solution contains no lens homogenate. Added glutathione may become bound to protein in some way, possibly by hydrogen bonding. The amount of diffusible glutathione is known to increase in the presence of urea or guanidine which rupture hydrogen bonds. Other sulphur compounds The lens extract prepared in the presence of N-ethylmaleimide also contained GSSG, cystine, and S-(N-ethylsuccinimido)-cysteine. The concentration of GSSG, about 3-5% of total glutathione, is in agreement with other values for lens and brain obtained by different methods. Free cysteine has previously been assumed to be absent from lens and is present only in traces in other tissues. The total concentration of cysteine/cystine indicated by this experiment is about the same as that of GSSG, 3-5% of glutathione, which is not sufficient to alter appreciably the amount of glutathione determined by various techniques. New tissue constituents Analysis of individual fractions of lens extracts by electrophoresis and by chromatography before and after hydrolysis indicated the presence of a number of unidentified substances. Three of these have been studied and are believed to be new constituents of tissue.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available