Ingemar Björk

On the interaction between polysaccharides and other macromolecules: II. The transport of globular particles through hyaluronic acid solutions☆

Abstract A study of the diffusion behaviour of albumin, α-crystallin, fribrinogen, and turnip yellow mosaic virus in hyaluronic acid media has shown that the diffusion rates of these substances are markedly decreased by the presence of the polysaccharide. The relative decrease in the same as the corresponding effect observed on the sedimentation rates of these substances. Investigation of eleven globular particles with diameters of 47–3650 A established that the relative decrease of the sedimentation rate of a particle in the presence of hyaluronic acid was essentially a function of the diameter of the particle and the concentration of the polysaccharide and that the effect could be expressed by a simple exponential relationship. The results observed have been interpreted as a macromolecular-sieving effect of the polysaccharide.

Studies on γ-crystallin from calf lens: II. Purification and some properties of the main protein components

Four proteins belonging to the γ-crystallin group were purified by chromatography on sulphoethyl-Sephadex and phosphate-cellulose columns. The proteins were homogeneous in gel and immunoelectrophoresis experiments and could be crystallized. Their molecular weights, N-terminal amino acid sequences and antigenic structures were all similar, but their amino acid compositions and the sulphydryl groups contained showed certain dissimilarities. It is probable that the four proteins possess small differences in their primary structure, which are not associated with the antigenic sites and which may have arisen from mutations during evolution.

Chromatographic separation of bovine α-crystallin

α-Crystallin, isolated from bovine lenses by vertical-column zone electrophoresis, has been separated into two fractions by chromatography on deae -cellulose. The two fractions gave single precipitation lines in immunodiffusion and immunoelectrophoresis experiments. They exhibited a marked difference in their free-electrophoretic mobility at pH 8·0 and a small difference in their sedimentation constants, but their amino acid composition, amide group content, intrinsic viscosity, optical rotatory dispersion and molecular weight, as determined by light scattering, were the same. Furthermore, double-diffusion tests revealed an immunological identity between the fractions. These observations suggest either that the two fractions possess slightly different secondary or tertiary structures or that one of the fractions contains a larger amount of an electrically charged compound bound to the protein molecule. The relative proportions of the two fractions in the lens were found to vary considerably with the age of the animal, indicating a transformation of α-crystallin in the lens during the life span of the animal or a slightly different mechanism for the synthesis of α-crystallin in young and old lenses.

Studies on γ-crystallin from calf lens: III. Comparison of the main protein components by peptide mapping

New methods, such as preparative polyacrylamide-gel electrophoresis and isoelectric focusing, have been applied to the purification of the four main γ-crystallin proteins. The purified proteins were studied by peptide mapping, and differences between them were clearly demonstrated. Molecular weight determinations of the fully reduced and carboxymethylated proteins suggested that two or more polypeptide chains held together by disulfide bonds do not exist in the four proteins.

Fractionation of β-crystallin from calf lens by gel filtration

β-Crystallin was isolated from calf lenses by a combination of gel filtration on Sephadex G-75 and vertical-column zone electrophoresis. It was subdivided into four fractions by subsequent gel filtration on dextran gels with low cross-linkage. Each fraction showed only one major peak in the ultracentrifuge, the sedimentation coefficients of which were 13·6, 9·6, 4·9 and 4·2 S for fractions I–IV, respectively. However, all four fractions contained some additional minor components, as revealed by sedimentation and immunoelectrophoretic analyses. The fractionation procedure may be of value as a first step in the isolation of individual proteins of the β-crystallin group.

Comparative studies of α-crystallin from lenses of different mammalian species *

α-Crystallin, prepared by zone electrophoresis from lenses of eight different mammalian species, has been investigated by agarose gel electrophoresis, ultracentrifugation and immunological methods. The molecular weights and electrophoretic mobilities of the α-crystallins differ for most species, but nevertheless all the proteins have similar immunological characteristics. Disc electrophoresis in 7 m urea gave a large number of bands in all cases. These data suggest that all mammalian α-crystallins are built up of subunits, and furthermore that the number of subunits and the relative proportions of different subunits vary from species to species. The subunits may have identical or similar antigenic sites, or alternatively, all mammalian α-crystallins may contain all the antigenically different subunits, but in varying amounts.

Recovery of the native conformations of the variable and constant halves of an immunoglobulin light chain upon renaturation from the linear random coil state.

Abstract The ability of separated constant and variable light chain halves (obtained by proteolytic digestion of the A.J. lambda light chain) and of the intact light chain to refold into their native conformations after complete reduction and unfolding, has been studied. The renatures proteins were analyzed by gel chromatography, quantitative precipitin curves, optical rotary dispersion, circular dichroism and fluorescence measurements. The results indicate that each half regains its native structure upon renaturation, both when it is free and when it is attached to the other half. The implication of this finding is that during light chain biosynthesis each half folds into its three-dimensional structure independently of the oather half. This is in accord with the idea that the genetic information for the light chain is derived from two separate genes. Preliminary studies of the denaturation of intact light chain by heat and by guanidine hydrochloride have been carried out. Stable intermediates might be expected in such experiments since it is highly probable that the two light chain domains, which independent in other respects, also denature independently. Inspection of the transition curves do not reveal the presence of such intermediates. More accurate analyses, however, are needed to study this problem.