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The development of the eye lens in the prenatal phase is accompanied by a gradual change in the polypeptide composition of α-crystallin. As the lens matures from that of a 6-week-old embryo to that of a new-born calf, there is a successive appearance of different polypeptides although two obviously principal polypeptides, A2 and B2, are present at every stage of development. The youngest embryonic lenses studied contain four polypeptides. Two of them differ from the well-known polypeptides as found in cortical bovine α-crystallin.

To investigate the possible role of glycation in the aging of lens proteins, we used bovine lenses as a model. We studied crystallins isolated from prenatal bovine lenses, calf lenses and lenses from mature animals (up to 20 years old). The experiments show an increase in glycation levels with age in all crystallin fractions. Regarding the lysine content of the different crystallins, gamma-crystallin showed relatively high levels of early glycation products. The results also revealed high levels of early glycation products for the HM material (containing mainly alpha-crystallin). In alpha-crystallin, alpha A-subunits were glycated to a higher extent compared with the alpha B-subunits. There is an age-related increase in advanced glycation products, measured as specific fluorescence (excitation/emission wavelengths 370/440 nm), mainly present in the HM and alpha-crystallin fraction.

To investigate the possible role of glycation in the onset of diabetic cataract we used calf lens crystallins as a model. After incubation with reducing sugars, the proteins were investigated by high-pressure gel permeation chromatography, SDS-PAGE and analytical ultracentrifugation. Glucose-6-phosphate incubation resulted in an increase in mean molecular weight of all crystallin fractions and the occurrence of high-molecular weight material, partly formed by disulphide bonds. The glycated crystallins showed a decrease of tryptophan fluorescence and an increase of a specific non-tryptophan fluorescence. This fluorescence was, however, not exclusively associated with the high molecular weight protein, but was present in all protein fractions.

Water-soluble crystallins were obtained from clear human lenses of different age (4-81-year-olds) and lenses of individuals showing senile or diabetic cataracts. Levels of early glycation products were high in the high molecular weight material (HM) and the alpha-crystallin fractions, compared with beta- and gamma-crystallins. This difference becomes more prominent upon aging. The content of total early glycation products in HM and alpha-crystallin increases clearly with age, whereas levels remain relatively constant in the beta- and gamma-crystallins. There is an elevation of early products in cataractous lenses from diabetic individuals compared with those suffering from senile cataract. Specific non-tryptophan fluorescence (excitation/emission wavelengths 370/440 nm), used as an indicator for late glycation products, increased dramatically with age and was 2-fold higher in the diabetic subjects. Levels of fluorescence decreased in the order HM > alpha- > beta- > gamma-crystallins. The results suggest an increase in glycation rate in alpha-crystallin as a result of aging and diabetes, while the rate of glycation of beta- and gamma-crystallins remains almost constant.

Glycation is a non-enzymatic modification of proteins by sugars, probably responsible for the initiation of complications in diabetes patients and aging individuals. Our in vitro experiments show an inhibition of sugar attachment in the presence of Diclofenac. The levels of advanced glycation products, measured as specific fluorescent groups, were also lowered due to Diclofenac. These results were compared with inhibition by Aspirin (acetylsalicylic acid), a known inhibitor of the glycation process. The protection by Diclofenac is based on a non-covalent interaction of the drug with serum albumin. There is evidence that Diclofenac specifically blocks at least one of the major glycation sites of human serum albumin.

The structural bovine eye lens protein alpha-crystallin was dissociated in 7 M urea and its four subunits, A1, A2, B1, and B2, were separated by means of ion-exchange chromatography. Homopolymeric reaggregates of these subunits were prepared by removal of the denaturant via dialysis. It was found that subunits were exchanged upon incubation of mixtures of two homopolymers under native conditions. New hybrid species were formed within 24 h as demonstrated by isoelectric focusing. Moreover, native alpha-crystallin molecules also exchanged subunits when incubated with homopolymeric aggregates of B2 subunits. Subunit exchange between native alpha-crystallin molecules is postulated, and a "dynamic quaternary structure" is presented that allows the polydisperse protein to adapt to changes in cytoplasmic conditions upon aging of the lens tissue.

Calf lens nuclear α-crystallin was separated into the three known molecular weight populations on Ultrogel AcA22. Electron microscopy illustrates that the two higher molecular weight classes, which are formed upon aging of the lens, are chain-like oligomers and polymers of the spherical monomeric α-crystallin molecules of 15 nm average diameter and 1·1 × 106 average molecular weight which form the lowest molecular weight class. Our combined sedimentation and electron microscopy data make the alleged non-equilibrium state within these populations questionable. From the polypeptide chain composition, determined by isoelectric focusing, it can be inferred that the total amount of C-terminal shortened chains increases progressively from 33 to 53% from monomeric to polymeric α-crystallin. Reassociation of different types of isolated shortened A-chains in various mixtures indicates a tendency for formation of higher molecular weight reaggregates with increasing relative amount of degraded chains. The involvement of degraded chains in polymerization is postulated although it is not likely to be the only factor causing aggregation.

The stability of the native quaternary structure of bovine alpha-crystallin was studied, by sedimentation analysis and electron microscopy, as a function of pH (7--11), ionic strength (0.01--0.5), temperature (6--60 degrees C) and calcium ion concentration (0 and 10 mM). Three successive transitions are distinguished at 20 degrees C. Firstly, a slow transconformation step, which is independent of pH, ionic strength or calcium ions. Secondly, an irreversible primary dissociation step, favoured by increasing pH above 8 and/or a lower ionic strength, with formation of 'alkali-modified alpha-crystallin', which is spherically shaped like the native protein but has a smaller average diameter, sedimentation coefficient and molecular weight. Thirdly, with further increase of pH above 9, a rapidly reversible dissociation of alkali-modified alpha-crystallin characterized by a single reaction boundary in sedimentation velocity analysis. In the presence of calcium ions the quaternary structure is stabilized to the extent that no dissociation is observed up to at least pH 10.3. Upon increase of temperature, at pH 7.3, a slow irreversible dissociation and swelling run parallel until a limit is reached around 37 degrees C with formation of 'temperature-modified alpha-crystallin', which is indistinguishable from the native protein by electron microscopy, but has a higher relative viscosity and lower sedimentation coefficient and molecular weight. Calcium ions have little or no effect on this transition. Above 37 degrees C a reversal of this transition or aggregation is indicated. These findings, together with previous structural data on microheterogeneity, reassociation from urea, and aging of alpha-crystallin in vivo, are incorporated into a hypothetical scheme of transitions, based on a three-layer model for the quaternary structure.

Racemization of aspartyl residues in proteins is a post-translational process, related to ageing. A method is presented for the detection of aspartic acid enantiomers in protein hydrolysates, based on chiral capillary gas chromatography. It is fast, easy and preferable to the usual diastereomeric dipeptide technique. We present evidence that traces of metals that are extracted from the glassware during acidic hydrolysis are the main cause for high background racemization, which often troubles accurate measurements. Effective ways to reduce this background and its standard deviation to acceptable levels are discussed, and a mathematical approach to correct for background racemization is given. Hydrolysates of aged human eye lens proteins were used to demonstrate the enantiomeric separation.

Evaluation of sulfhydryl data derived from the nucleus and cortex of individual human lenses, ranging in age from 40 to 90 years, revealed the following: (a) no significant correlation between the age and the sulfhydryl content of normal and nuclear-cataractous lenses; (b) a significant relationship between the distribution of non-protein-bound sulfhydryl compounds among the lens parts and both age and cataractous state; (c) no significant difference in sulfhydryl data from the cortex between groups of cataractous lenses with increasing nuclear color; and (d) a significant difference in nuclear protein sulfhydryl content between the groups of lenses, varying in cataractous state. If lens parts are separated on the basis of cataract localization, sulfhydryl oxidation in nuclear cataractogenesis seems to be confined to the colored nuclear region. It is suggested that nuclear cataract develops from the inner nucleus towards the cortex of the lens.

Electron micrographs of high (40-S) and very-high-molecular-weight (240S) alpha-crystallin preparations show large aggregates with a random arrangement of individual low-molecular-weight alpha-crystallin molecules. The electron microscopic data are completed with viscosimetric and light-scattering experiments. All data obtained with higher-molecular-weight alpha-crystallins point to aggregates with non-spherical structures.

The concentration of the free aromatic amino acids tryptophan and tyrosine, the antioxidant uric acid and some unidentified compounds have been determined in the water-soluble extracts of parts from clear and nuclear-cataractous human lenses. With age (29-90 years) and cataract formation, associated with increasing nuclear pigmentation, no significant changes were observed in content of tryptophan, tyrosine and uric acid. Furthermore, these compounds did not show variation in distribution within the lens. An unidentified fluorophore, excitation and emission maxima of 345 and 425 nm, respectively, increases significantly in content with nuclear cataract formation, especially in the nucleus. Another unidentified, presumably aromatic compound shows a striking age-related decrease.

Yellow protein derived from European and Pakistani human nuclear cataractous lenses was digested. Anthranilic acid was found in 50 times greater amount in Pakistani than in European material. Treating

Oxidative processes are suggested to be a main cause of the covalent cross-linking of lenticular proteins. For a major part these cross-links are disulfides (cystine). With progression of nuclear cataract, cystine may be oxidized further to cysteic acid. Another oxidative degradation product of cystine is the symmetric thioether lanthionine. In this study, we clearly demonstrate that lanthionine is a protein cross-link of cataractous human lenses. Possible mechanisms of its formation are discussed.

The very acidic polypeptide chains found in aging human eye lenses are products of post-synthetic alterations known to occur in mammalian eye lens proteins. Isoelectric focusing in the presence of ure

In α-crystallin from the nuclear part of the lens, polypeptide chains were found, which were not present in the cortex of the lens. These additional subunits were fractionated by means of chromatography on DEAE-cellulose (DE-52) or CM-cellulose (CM-52). Characterization of two of them showed that they were identical to shorter αA-chains, and hence they were designated as αA21–151and αA11–151. By studying α-crystallin as function of fiber cell development it turned out that the subunit structure of α-crystallin is not constant. The polypeptide chains in the α-crystallin aggregates are subject to specific and limited degradation processes, causing an ever changing polypeptide chain composition.

A highly sensitive method was used to determine d - and l -aspartyl residues in protein fractions from normal and cataractous human lenses. A linear relationship with age was found in all fractions from normal lenses. However, no correlation with cataract could be established. It is concluded that racemization of aspartyl residues is a continuous process in eye-lens proteins but plays no role in cataract formation.

A recently developed chiral gas chromatographic method and a diastereomeric high-performance liquid chromatographic method for the analysis of aspartic acid enantiomers in protein hydrolyzates have been evaluated. Although both techniques are fast and convenient, the latter is preferred because of its higher reproducibility and shorter analysis time. Furthermore, this method offers the possibility of on-line derivatization and analysis.

Limited proteolysis of α-crystallin with 10 different endopeptidases in vitro shows that only 5–20 residues of the carboxy termini of A and B chains are accessible to these enzymes, without disruption of the quaternary structure. The A chains are more susceptible to proteolysis in vitro than B chains, which corresponds with the degradation in vivo upon aging of α-crystallin in the lens. Six proteases specifically cleave off the carboxyl-terminal pentnpcptide of all B chains, which indicates that the B (Thr170 -Ala171) bond, the only observed degradation site of B chains in vivo, is surface-exposed and proteolytically very labile. None of the endopeptidases split the A (Asp151 -Ala152) bond, the main degradation site of A chains in vivo, but a nearby segment A156–163 iS very accessible to several enzymes. We postulate that this A151–152 bond is close to the surface of α-crystallin and, although proteolytically stable in the native protein, it could become exposed as a result of age-related changes in secondary and/or tertiary structure. Two minor tryptic cleavage sites in A chains of native α-crystallin become more accessible in reassociated a-crystallin, and particularly in pure A-chain reaggregates, which indicates a change in secondary/tertiary structure upon dissociation-reassociation. Although the accessibility of B chains suggest that they are in equivalent positions in the α-crystallin quaternary structure, the A chains exhibit various rates of proteolysis and therefore occur in more than one orientation. The anomalously slow migration of B chain in dodecylsulfate gel electrophoresis is abolished after removal of its carboxyl-terminal pentapeptide, Ala-Ala-Pro-Lys-Lys.

Intumescent cataractous lenses were investigated as to their water content and protein composition. Nuclei were separated from cortices. Water-soluble, water-insoluble, urea-soluble and urea-insoluble portions were quantified and the subunit composition of the water-soluble crystallin fraction was looked at on SDS-gel electrophoresis. Compared to normal lenses it is observed that the water content in intumescent cataract is increased, water-soluble and urea-soluble fractions are diminished, whereas the urea-insoluble portions are augmented. No major changes are noted in the subunit composition of the soluble protein fractions. Particularly, the relative amount of gamma-crystallins is diminished in intumescent cataractous lenses.

Total extracts of both water-soluble and water-insoluble portions are obtained from fetal and adult human lenses. In the water-soluble extracts of lenses older than 14 years of age little difference is seen between the cortical and nuclear preparations. In lenses older than 14 years a polypeptide chain of approximately 10 k appears. In fetal and newborn lenses a 43 k polypeptide is detected which becomes more diffuse upon aging. Concomitant with a decrease of a 29·5 k polypeptide, there is a relative increase of a 29 k chain, suggesting a situation comparable with the transition from calf βB1a to βB1b. In young lenses a major β-polypeptide co-migrating with calf βBp is seen. As for the water-insoluble portions, no difference between cortical and nuclear preparations is detectable after 14 years of age. In fetal lens there is a high amount of the 29·5 k polypeptide, which decreases with aging and finally disappears. Till 6 months four bands with molecular weights of 200 k, 100 k, 57 k and 43 k are detected, co-migrating with calf myosin, α-actinin, vimentin and actin, respectively.

Contrary to the elution pattern of bovine eye lens proteins chromatographed on a TSK GEL G5000 PW type column, an additional peak, representing 4.10 6 dalton aggregates, was observed on a combined G5000 PW–G4000 SW type system. This extra peak, found between the G5000 PW total exclusion fraction and α-crystallin (M r  1.1 · 10 6 ), appeared to correspond to the G4000 SW void volume. Simulation calculations substantiated that this peak is the result of coupling the two columns and does not originate from a distinct sub-population of molecules. Such artificial peaks may be expected whenever a broad molecular weight distribution extends beyond the fractionation limits of at least one of the constituents of a combined gel permeation system. It is inferred that the hypothetical exclusion limit for compact, globular, symmetrical proteins on TSK GEL G5000 PW is near 10 8 dalton.

Cortial alpha-crystallin was size-fractionated by gel filtration on Ultrogel AcA22 and charge-fractionated by anion-exchange chromatography on DE-52 DEAE-cellulose using gradient elution. Electron microscopy demonstrates that both native and reassociated alpha-crystallin are heterogeneous populations of spherical or slightly ellipsoidal molecules with diameters of 13.5--16.0 nm (maximum at 14.0--15.0 nm) for native alpha-crystallin and 8.5--12.5 nm (maximum at 10.0--10.5 nm) for reassociated alpha-crystallin. An enormous charge heterogeneity of native alpha-crystallin was detected, which is shown to arise from variations in the stoichiometry of the 5 main types of subunits. The molar ratio of acidic chains (A2, A1 and A1/2-151) to basic chains (B2 and B1) varies from 70/30--80/20 (averaging about 3/1) and the amount of deamidated chains (A1 and B1) varies from 7--37%. Recombination of the subunits, after dissociation in 6 M urea, leads to a charge heterogeneity of reassociated alpha-crystallin very similar to that of native alpha-crystallin. Therefore, specific formation of pure A or B chain aggregates is not preferred. Instead, random combination of subunits is theoretically shown to be sufficient to describe the observed charge microheterogeneity of both reassociated and native alpha-crystallin. No obvious relationship exists between size and charge heterogeneity. Within these ranges of molecular weight and subunit composition there are more than 1000 different combinations of A2, A1, A1/2-151, B2 and B1 conceivable.

Knowledge of the quaternary structure of α-crystallin is required to understand the age-dependent superaggregation processes, eventually leading to lens opacification. Different approaches, e.g. sulfhydryl modification, chemical cross-linking, limited proteolysis and dissociation studies revealed new information, providing a basis for further studies of aging and higher-order structures. A model for the architecture of native α-crystallin from calf lens cortex, featuring subunit arrangement and surface exposure, will be presented.

The molecular weight of bovine alpha-crystallin, isolated at 37 degrees C, was studied and found to be about 800,000. This contrasts with the results of Thomson and Augusteyn (Thomson, J. A., and Augusteyn, R. C. (1983) Exp. Eye Res. 37, 367-377) who isolated a species of about half this molecular weight. We show here that this form of alpha-crystallin can only be isolated under unphysiological conditions with regard to buffer pH and ionic strength.

Extracts from epithelial cells, seven successive concentric layers of the cortex, and the nucleus of calf lenses were submitted to gel chromatography on agarose Bio-Gel A-5m. In the mentioned order, corresponding to increasing age of the relevant lens cells, the content of α-crystallin in these extracts decreased significantly whereas the content of a very high-molecular weight fraction, resembling an aggregate composed of the polypeptide chains of α- and β-crystallin, increased markedly. The sedimentation coefficients of the α-crystallin preparations at concentrations of 0·8 mg/ml ranged from 17 to 21 S and passed through a minimum. Gel electrophoresis in polyacrylamide containing 6 m urea revealed significant changes in polypeptide composition. Nevertheless, no clear relation between sedimentation coefficients and polypeptide composition was found. Evidence has been obtained for the interaction of α-crystallin with constituents of β-crystallin which may lead to the formation of very high-molecular, finally insoluble aggregates.

KNOWLEDGE of the intracellular processes of ageing and breakdown of proteins is still very limited1,2, but the extremely slow turnover of lens proteins in the eye provides a unique opportunity to analyse such processes. Throughout life fibre cells are formed from epithelial cells at the periphery of the lens, being successively covered by younger cells. Protein synthesis ceases soon after the differentiation into fibre cells is completed3. As a result the nucleus of the lens of an adult still contains the proteins formed during foetal life. Here we demonstrate that differences in polypeptide structure observed between the cortex and nucleus4–6 of the lens are due to an ageing process. Our data provide the first example of progressive degradation and deamidation of a protein, probably caused by non-enzymatic mechanisms.

Applicability of the isotachophoretic principle to isolation of closely related subunits of α-crystallin has been studied. The conditions for an isotachophoretic separation were searched for on an analytical scale, using polyacrylamide gels. It was found that gel isotachophoresis is not as appropriate as gel isoelectric focusing for analyzing the polypeptide chain composition of α-crystallin. For large-scale separation of the subunits on a polyacrylamide gel, isotachophoresis turned out to be unsuited.

1. 1. Lenses of bovine embryos and fetuses, ranging in age from 6 weeks to 9 months, were investigated. 2. 2. Weight, shape, protein content and protein composition as well as leucine aminopeptidase activity were determined as a function of age. 3. 3. Alpha-crystallin turned out to be the most prominent embryonic crystallin. 4. 4. The complexity of the protein composition of the lens increased in maturity up to birth. 5. 5. The polypeptide composition of the α-crystallins was analyzed by means of isoelectric focusing in polyacrylamide gels. Two polypeptides, specifiic for the embryonic lenses, were found. 6. 6. One of these specific polypeptides appeared to be sensitive to u.v. light.

Cataractous state-related alterations were studied by Curie-point pyrolysis low voltage mass spectrometry performed on the embryonic nuclei from adult normal and nuclear-cataractous human lenses. It was shown that the relative intensities of ion signals assigned to sulfur dioxide and tentatively, to other sulfur oxidation products correlate with the increase in lens nuclear color. Since these ion signals may represent pyrolysis fragments from methionine sulfoxide, methionine sulfone, cysteic acid and disulfide compounds present in the parent material, it is concluded that progressive oxidation of sulfur compounds appears to take place during nuclear cataractogenesis. Automated pyrolysis mass spectrometry coupled with multivariate analysis of the spectral data by computer turned out to be a rapid method of characterizing sub-milligram samples of lens material. The results were supported by data obtained by conventional determination of sulfhydryl and tryptophan groups.

Water-soluble proteins from prenatal (8–16 and 16–30 weeks of gestation) and postnatal (newborn, 14, 40, and 76 years) human lenses were isolated and fractionated. The relative distributions and S20,w values were determined for each crystallin class. NH2-terminal amino acids of the low molecular weight fractions were identified. Isoelectric focusing and SDS-PAGE patterns are shown. Several interesting changes in the subunit composition, particularly in γ-crystallin, during development and aging are noted.

Citraconic anhydride reacts readily with alpha-crystallin's lysine residues at pH 7.4. Upon addition of 2 equivalents of citraconic anhydride per equivalent lysine, 24% of the lysine residues were modified without disrupting the native quaternary structure. Further citraconylation led to dissociation into 10 S aggregates. Complete dissociation into subunits (1.4 S) occurred after adding 100 equivalents of citraconic anhydride, resulting in 98% modification. Decitraconylation did not lead to reaggregates identical with the native ones. The unmodified and the once and twice citraconylated alpha-crystallin subunits were discerned by isoelectric focusing according to their theoretical isoelectric points. In the native alpha-crystallin aggregates, nearly all B chains and approx. 60% of the A chains were found to possess at least one surface-exposed lysine residue. No differences between the susceptibilities to citraconylation of the in vivo deamidated (A1 and B1) and the de novo synthesized (A2 and B2) subunits were found. These results support the three-layer spherical assembly model for the alpha-crystallin quaternary structure.

Protein carboxyl methyltransferase (PCM, EC 2.1.1.77) is an ubiquitous enzyme capable of methylating a great variety of proteins. The reaction that it catalyzes takes place in a sub-stoichiometric way. This puzzling fact was explained when Clarke and Aswad and their co-workers reported that PCM only recognizes D-aspartic and L-isoaspartic residues (for review see reference 1).

The subunits AA2 and N2, present in α-crystallin from the nucleus of young and old bovine lenses, were isolated and characterized. It was found that AA2 is identical to a shorter A-chain and hence it was designated as A1–101. The subunit N2 turned out to be identical with a shorter B-chain and-was designated as B1/2−170. Characterization of the subunit N1, present only in α-crystallin from the cortex of old bovine lenses, suggested that this subunit is a modified B-chain, probably a deamidation product; it was designated as Bo. Comparison of the appearance of degraded and deamidated chains in relation to the age of Uhc fiber cells in old bovine lenses with that in calf lenses revealed that the observed specific limited degradation of the subunits of α-crystallin increased with older age of the tissue. The deamidation process was found not to be related to the aging of the tissue. Eventually, a clear picture concerning the heterogeneity and fickleness of theα-crystallin subunit structure was obtained.

Lenses of 3-month-old calves were divided into nine concentric layers. The protein extracts of these fractions were submitted to gel chromatography on agarose A-5 m. From the epithelium to the nucleus, the content of α-crystallin decreased significantly, whereas the content of a high molecular weight fraction, resembling α-crystallin in u.v. spectrum as well as polypeptide composition, increased markedly. An analogous phenomenon was observed in the group of the β-crystallins. The proportion of the lower molecular weight fraction decreased and that of the higher molecular weight fraction increased from epithelium to nucleus, i.e. according to the age of the fibre cells. The highest content of γ-crystallin was found in the nucleus. The activity of lens leucine aminopeptidase was found to be highest in the outer cortex fractions, i.e. in the synthetically active area of the lens.

In order to obtain deeper insights into the mechanisms that are responsible for the age-related changes in the eye lens, the water-soluble proteins of 6-day-old to over 3-year-old rat lenses were analyzed by high-performance gel-permeation chromatography. Using this technique eleven crystallin fractions could be discerned: HM-, alpha-, three beta H-, two beta L-, beta S- and three gamma-crystallins. The concentrations of the higher molecular weight crystallins (HM-, alpha- and beta H-crystallin) seem to increase with age while those of the lower molecular weight (beta L- and gamma-crystallin) decrease. Taking into account the gradual increase of water-insoluble protein with aging, the relative amount of alpha-crystallin decreases from an age of 0.5 year after an initial increase. Additionally, an age-dependent increase in its molecular weight was found: from 7 . 10(5) to over one million. It appears that the gamma-crystallins are directly involved in the insolubilization process, while alpha- and beta L-crystallin first take part in aggregation processes leading to HM- and beta H-crystallin aggregates. These aggregation and insolubilization processes proceed gradually with increasing age. A steep decrease in gamma-crystallin concentration in the early phase of life, which also causes the relative increase in alpha-crystallin content in this period, may originate from a decrease in biosynthesis of certain gamma-crystallins.

The aggregation and dissociation behavior of bovine alpha-crystallin as well as the folding and unfolding of its subunits were investigated by equilibrium studies using tryptophan fluorescence measurements and two isoelectric focusing techniques, viz. isoelectric focusing across a urea gradient and isoelectric focusing in two dimensions with different concentrations of urea. It was found that the alpha B chains lose their ability to aggregate and start unfolding at a lower concentration of urea than the alpha A chains. Equilibrium intermediates were found upon unfolding or refolding of alpha A subunits, which can be explained by a two-domain organization of these molecules.

Reaction of calf alpha-crystallin, which consists of about 30 A and 10 B subunits, with various bisimido esters at pH 8.0-8.5 and room temperature leads to inter-subunit crosslinking. Little difference is observed in the efficiency of crosslinking by reagents of a homologous series with different lengths (from C6 to C12). At any stage of the reaction an exponential decrease is found in the amounts of crosslinked dimer, trimer, tetramer etc., with no preference for the formation of any particular oligomer, suggesting that all subunits on the surface of the spherical alpha-crystallin molecule are in quasi-equivalent positions. Molecular weight analysis by docdecylsulfate gel electrophoresis and reversible crosslinking show that both A and B subunits occur in an apparently constant ratio in the crosslinked oligomers, from which we infer that both types of subunits are randomly arranged on the surface of the alpha-crystallin molecule. Not all of the subunits can form inter-chain crosslinks, as a limiting value of about 60% of the subunits is found in oligomeric form. A simple explanation could be a core of subunits which is inaccessible to certain chemical reagents, although other alternatives are also discussed.

1 High-molecular-weight fractions were isolated from extracts of the cortex and the nucleus of calf lenses. By chromatography of this material on large-pore agarose gels a separation into two populations, with s20, w-values ranging from 100 to 190 S and from 30 to 55 S was obtained. 2 It is shown that the high-molecular-weight fraction is identical to α-crystallin in polypeptide chain composition. 3 For the α-crystallin fractions from the nucleus a subunit structure different from that of cortical α-crystallin fractions is found. 4 The quaternary structure of the α-crystallin fractions is not constant during the life span of the animal. The variety is subunit structure and state of aggregation of the α-crystallin molecule is supposed to be due to aging processes.

A more informative method for the visualization of proteins on thin-layer gels, based on combining the images of the gel at different stages of destaining, is presented. It is useful whenever important information seems to be lost after prolonged destaining. The method, which makes it unnecessary to run different loads in different channels, has been developed utilizing isoelectric focusing on polyester film-bound agarose gels. The strongly destained gel is superimposed on a negative image of the same gel made at an earlier phase of destaining, thus showing white spots on a gray background for minor components and dark bands in a white field surrounded by the grey background for the abundant ones. In general, the method may be applied to gel images obtained by different staining procedures.

The polypeptide chains of α-crystallin from 60- to 80-year-old human eye lenses were studied to determine whether they are subject to similar post-synthetic alterations as known from bovine α-crystallin. Isoelectric focusing in the presence of 6 M urea revealed that old human α-crystallin consists of five major polypeptide chains, designated as Ax, A2, Bx, By and B2. By dodecyl sulphate gel electrophoresis mainly chains with apparent molecular weights of 22 000 and 20 000 were observed. Ion-exchange chromatography in the presence of urea was applied to obtain enriched preparations of individual polypeptides. Additional data concerning the primary structure of some chains were gathered by tryptic peptide mapping in combination with amino acid analysis. The presence of two cysteine residues in the A-chains was demonstrated. The Ax component turned out to be composed of two chains: deamidated A2 of normal length (172 residues) and, presumably, a shortened A2-chain: A21–151. The Bx- and By-chain with isoelectric points slightly lower and higher, respectively, than that of bovine B1, appeared to be B21–170 and deamidated B2 of normal length (175 residues). A group of polypeptides with very acidic isoelectric points, present in low proportion, showed a heterogeneous molecular weight distribution below 18 000 and a high degree of similarity with the A-chains as judged by tryptic peptide mapping. In spite of several differences in subunit composition, it can be concluded that in human α-crystallin, as in bovine α-crystallin, deamidation and degradation processes occur.

Antiserum against the αB chain of bovine α-crystallin has been prepared. Several antigens of the bovine lens are compared using this antiserum.