Some constituents of tissues of the eye

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. One compound is a basic tripeptide, with the probable structure arginyllysylglycine, which is present in lens at a concentration of about 0.02mM. Lysine, arginine and glycine were liberated on hydrolysis, and quantitative analysis of the hydrolysate with ninhydrin gave a ratio of 1 : 2.2 for neutral to basic amino-acids. Dinitrophenylation indicated N-terminal arginine. The complete amino-acid sequence was obtained by digestion of the peptide with trypsin which yielded the three amino-acids. A peptide with the alternative structure would have yielded arginine and the dipeptide glycyllysine. One other arginine tripeptide, leucylarginylleucine, has been isolated from pig pituitary gland.
The other two substances have been only partially characterized. One may be an aliphatic amine with a single unsubstituted amino group and possible between 6 and 12 carbon atoms. No substance of this type has been identified in tissue. Its concentration in lens is about 0.04 mM. This substance travels near the solvent front on chromatography in three solvents; it is unaffected by hydrolysis but appears to be attacked by nitrous acid. It yields a picrate and probably a hydrochloride. The second substance contains phosphorus and does not react with ninhydrin. It has a characteristic blue fluorescence in ultraviolet light after electrophoresis at pH 2.3 but not at pH 4 or pH 9. Guanine is liberated on mild acid hydrolysis but since the hydrolysate contains only traces of inorganic phosphate, it seems unlikely that this substance is guanosine mono- or tri-phosphate, both of which are believed to be present in lens.
Other tissue constituents
In general, little is known about the distribution of low molecular-weight compounds in lens. Many of the compounds known to be present in lens were found in individual fractions of extracts, and a number of other compounds have been identified by electrophoresis and by chromatography. These are mostly present in plasma and are transported through the aqueous humour into the lens. No quantitative studies have been carried out but it seems likely that some at least of the compounds are at a higher concentration in lens than in plasma.
All the common protein amino-acids were found free in calf lens. Previous data (on cow lens) had indicated the presence only of alanine, glycine, glutamine, serine, threonine, valine, leucine, aspartic and glutamic acids. The concentration of cystine is greater than in most other tissues and is probably connected with the high concentration of glutathione in lens. Lens, unlike aqueous humour, contains more free cystine than methionine. The amino-acids were identified with markers and detected with ninhydrin, also with reagents specific for particular groups in the molecule wherever possible.
A number of non-protein amino-acids have been identified in the free state. &alpha-Aminobutyric; acid, which is present bound as ophthalmic acid, is also found free at a higher concentration than many protein amino-acids. Other compounds which have been detected are ornithine, 1-methylhistidine, β-alanine, γ-aminobutyric acid, and ethanolamine. Citrulline and cystathionine are probably present but the evidence for these is not as firm as with the others. Thurine had been detected previously.
Only a few ninhydrin-negative compounds were characterized and one, ergothioneine, was detected in lens for the first time. It was identified with a marker by its electrophoretic and chromatographic behaviour, and was located under ultraviolet light and with Pauly reagent. The apparent concentration of ergothioneine in lens, about 0.1mM, is similar to that in plasma.
Investigation of whole extracts
Preparation and investigation of fractions of lens extracts required comparatively large amounts of tissue and was a lengthy procedure. A considerable amount of material was obtained which permitted identification of some compounds present in only small amounts. Electrophoresis or chromatography in a single direction did not give a sufficiently good separation for analysis of complex fractions. Satisfactory separations were achieved on single papers by low-voltage electrophoresis at pH 1.6 in the first direction followed by chromatography in butanol-acetic acid-water in the second. Since compounds in individual fractions tended to occupy different areas of the paper, the method was extended to analysis of unfractionated extracts. Much information could be gained quite quickly from extracts of single lenses.
A combination of electrophoresis and chromatography has one main advantage over two-dimensional chromatography for the study of tissue extracts. Electrophoresis acts as a desalting process by removing most inorganic ions and a more reproducible pattern is obtained without treatment of the deproteinized extract before analysis. This method does not seem to have been applied previously to analysis of tissue extracts although protein hydrolysates and enzymic digests of proteins have been studied by similar systems. The relative pattern of spots was constant but actual positions varied slightly in the direction of chromatography. Many compounds were identified by comparison with markers. Papers were viewed under ultraviolet light then treated with ninhydrin or with another location reagent. The blue fluorescence of tryptophan, present in unhydrolysed extracts acted as a useful internal marker. A number of different extracts were compared.
Satisfactory results were obtained with half of a calf lens extract or quarter of a cow lens extract on a single paper, that is about 7 mg total solid. About 60 spots were detected with ninhydrin before hydrolysis, of which about half have been identified with some certainty, another 10 have been identified tentatively, and the remainder are unidentified. Most of the unidentified substances are present in only trace amounts and virtually no information has been obtained about them. About 35 spots were detected in hydrolysed extracts, and only a few of these substances are unidentified. Some compounds have also been identified in extracts which were examined with reagents other than ninhydrin. Coloured spots were sometimes given by compounds present in high concentration which do not normally react with the reagents.
Since lens extracts were obtained initially from stored tissue, a comparison was made between trichloroacetic acid extracts of stored and of fresh lens. The extracts were almost the same with the exception that in stored lens ascorbic acid had decomposed, glutamine had cyclized, and some breakdown of peptides seemed to have occurred. The last is probably a consequence of enzyme activity which is known to persist in vegetables stored at low temperature. No significant difference was observed between extracts of fresh calf lens prepared by removal of protein with phenol or with trichloroacetic acid. Cow and calf lenses gave almost identical patterns and the concentrations of many compounds were similar in both extracts. S-(α,β-dicarboxyethyl)-cysteine, S-(α,β-dicarboxyethyl)-glutathione, and the new basic peptide were all detected in fresh cow and calf lenses. The unidentified ninhydrin-positive spots may be peptides, but their concentration is low relative to that of the amino-acids, and most of the amino-acid spots are not noticeably more intense after hydrolysis.