Arthur Stevens

The effects of isolation buffers on the properties of α-crystallin

This work was undertaken in order to resolve some of the controversy in the literature concerning the properties of α-crystallins isolated in different laboratories. Bovine lens proteins were extracted and isolated by gel chromatography using ‘Hoenders buffer’ (0.02 M Tris- HCl, 1 mM EDTA, 80 mM NaCl, pH 7.3), ‘Tardieu buffer’ (0.04 M phosphate, 1 mM EDTA, 0.2 mM DTT, 0.06 M KCl, pH 6.8) and ‘Thomson/Augusteyn’ buffer (0.05 M Tris-HCl, 2 mM EDTA, 0.2 mM DTT, pH 8.0). The α-crystallin peaks were then divided into 12–16 poolsand subjected to detailed physicochemical characterization. Fractionation by HPLC-GPC and quasi-elastic light scattering indicated that the size of the proteins decreased with increasing elution volume and that they were stable for at least 9 months at 20°C. Molecular masses were found to range from over 2 mDa at the front of the peaks to around 600 kDa at the end. The size distributions, for the three buffers, were indistinguishable. No differences could be detected in the polypeptide distributions by SDS-PAGE. The proteins were also identical in their near- and far-UV circular dichroism spectra, accessibility of their sulphydryl groups to DTNB, tryptophan accessibility to quenching by acrylamide and iodide, and immunoreactivity with two monoclonal antibodies with different specificities. It is concluded that identical α-crystallins are isolated with the three different buffers and that variations in pH (6.9–8.0), ionic strength (60–150 mM) and cation (K, Na, Tris) during the isolation do not affect the properties of the protein. Claims that differing observations on the properties of α-crystallin may be attributed to the buffers used, are untenable.

Specific dissociation of αB subunits from α-crystallin

Abstract Exposure of bovine α-crystallin to 0.1 M glycine at pH 7 decreases the average molar mass of the protein from 700 to 420 kDa. When the pH is lowered to 2.5, in the same buffer, the αB chains specifically dissociate from the aggregates, leaving a particle of 290 kDa containing only αA chains. The decrease in the molar mass corresponds to the mass of the αB chains in the original aggregate. The pH-dependent dissociation is fully reversible. Similar changes were observed with rat and kangaroo α-crystallins but the dogfish protein was not affected. Sedimentation velocity analyses and fluorescence spectroscopy yielded a p K , for the dissociation, of 3.7 for α-crystallin and 4.0 for a homopolymer constructed from purified αB 2 polypeptides. An αA 2 homopolymer was virtually unaffected by the lowering of pH. The products from the dissociation were isolated and their properties studied by sedimentation analysis and acrylamide quenching of tryptophan fluorescence. The αB chains were found to be completely denatured, whereas the structure of the αA chains, in the 290 kDa, particle, were only slightly altered. Comparisons of the sequences of the various proteins examined suggested that decreased ionization of aspartic acid 127 in the αB chain was responsible for the specific dissociation of this polypeptide.

Chaperone activity in the lens

Introduction: α‐crystallin, the major protein of the eye lens, is a molecular chaperone that is able to prevent the precipitation of denatured proteins. This activity is thought to be important for the maintenance of lens transparency. Loss of the activity has been postulated to contribute to the development of cataract. The purpose of this study was to determine how chaperone activity was affected by growth and ageing of the lens.

Evaluation of equilibrium constants from precipitin curves: Interaction of α-crystallin with an elicited monoclonal antibody

A simple procedure, based on the precipitin curve and the antibody:antigen ratios of the precipitates, is described for evaluation of the intrinsic association constant (k) governing the interaction between a multivalent antigen and a bivalent antibody. Its application is illustrated with a study of the interaction between alpha-crystallin and an elicited monoclonal antibody, which is shown to exhibit essentially identical affinities (k = 9 X 10(4) M-1) for the alpha m and alpha c forms of the antigen.

Isolation of α-crystallin and its subunits by affinity chromatography on immobilized monoclonal antibodies

Use has been made of the specific interactions between monoclonal antibodies and the alpha A or alpha B subunits of alpha-crystallin to devise methods for the purification of the intact protein or its subunits. alpha A and alpha B subunits were separated by affinity chromatography on an immobilized monoclonal antibody specific for alpha A chains, using a pH gradient. Use of an antibody which binds both subunits has enabled the isolation of intact alpha-crystallin aggregates. Gel electrophoresis, conformational probing and size analysis showed that the affinity purified proteins were purer but otherwise indistinguishable from the alpha-crystallins isolated by other methods.

Distribution of proteins across the porcine lens

In an attempt to determine the feasibility of using the porcine lens as a model for research into human lens ageing and cataract we have investigated the distribution of proteins across the lens. Analysis of the soluble protein distributions in the porcine lens indicated that the distribution of high, middle and low molecular weight proteins changed in a way similar to that observed for a young human lens. Changes in the amount of water insoluble proteins across the lens, which are used as an indicator of age‐dependent changes and cataract formation, also resembled those observed in the human lens. Coupled to previous observations showing the similarity in metabolism between porcine and human lenses, we suggest that the porcine lens may be more appropriate than the current animal models such as rat, rabbit or bovine, for studies of human lens ageing and cataract. (Clin Exp Optom 1995; 78: 3: 87–92)

Contribution of glucose and its metabolites to diabetic cataracts

Despite extensive research, the mechanisms responsible for diabetic cataract formation are still unknown. While recent data have favoured non‐enzymatic glycation as the most likely mechanism, there is relatively little information on the types of glycolytic metabolites and die way in which they are involved in diabetic cataracts.

A review of current research on the effect of diabetes mellitus on the eye

It is estimated that almost one million Australians will have diabetes by the year 2000. Of those with diabetes a significant proportion will have eye‐related conditions, the most debilitating being diabetic retinopathy.

Considerations in the routine assessment and treatment of anisometropic amblyopia

Anisometropic amblyopia has traditionally been treated by direct occlusion with or without refractive correction of the anisometropia. This treatment has generally been considered so successful that practitioners prescribe occlusion for a time and consider treatment complete when visual acuity is no longer improving. The aetiology of anisometropic amblyopia, addressing the functional deficits of the amblyopic system and patient compliance issues, are important to the success of amblyopia treatment in anisometropia and are briefly reviewed in this paper. We propose that, while each case of anisometropic amblyopia may have slightly different attributes, initially they should all be treated with occlusion and full‐time spectacle correction. Some presentations may be responsive to individual treatment strategies to correct binocular functions such as stereopsis, accommodative response and fusional vergences.