M. Rabaey

Electrophoretic and immunoelectrophoretic studies on the soluble proteins in the developing lens of birds.

The embryonic lenses of chick and duck embryos were investigated by means of electrophoresis and immunoelectrophoresis. By this method α-crystallin is not found in embryonic lenses before 6 days of incubation. This is explained by the fact that in the early stages of lens formation α-crystallin is bound to more or less insoluble cellular elements. Much earlier, from the 3-day embryo on, another important soluble protein (called first important soluble crystallin) is found, which predominates in the electrophoretic and immunoelectrophoretic pattern during almost the whole embryonic life. In this respect there is a great resemblance between the pattern obtained in the chick, duck and pigeon embryos, although the distribution of proteins in the adult lens is entirely different. By using an immunoelectrophoretic method, it was possible to trace this important soluble crystallin in the complicated pattern of several birds throughout the order of Aves. The response to the proteolytic enzymes trypsin and chymotrypsin is characteristic. Ultracentrifugal analysis permitted us to classify this protein as a member of the so-called group of β-crystallins.

Gel filtration of the soluble proteins from normal and cataractous human lenses

The soluble proteins of normal and cataractous lenses were separated according to their molecular size on the polysaccharide gel Sephadex G-100. In cataractous as well as in normal lenses 4 different components were obtained. During the evolution of eataract there is a preferential decrease of the low molecular weight proteins of the lens. These disappeared completely in mature and hypermature eataract. On the other hand, the proteins of high molecular weight (e.g., α-crystallin) are very resistant to the pathological process. The electrophoretic pattern of the low molecular weight proteins in the normal lens revealed several fractions distributed over a large mobility area. After immunoelectrophoresis, 3 different precipitin lines were obtained.

Low molecular weight protein (γ-crystallin) in the lens of birds

Abstract A method is described for the isolation of low molecular weight protein (γ-crystallin) from the lens of birds, based on a combination of salting-out and gel filtration. The isolated protein appears as a single component after gel filtration, ultracentrifugation and immuno-electrophoretic analysis. Specific antiserum to γ-crystallin of two birds was prepared and the first results of the immunological cross-reactivity with the γ-crystallin of other vertebrates are reported.

Distribution of protein molecular groups in the normal and cataractous lens

As a continuation of previous investigations, a comparative study of the protein composition in the normal and cataractous lens was made using a thin-layer gel filtration technique (Sephadex G-200). This technique permits a good separation of proteins with high molecular weight. The remarkable persistence of α-crystallin, and the diminution in concentration of some proteins with low molecular weight, are discussed.

Ontogeny and localization of γ-crystallin antigen in the developing pigeon (Columba livia) lens

Ontogeny and localization of the lens γ-crystallin antigen were investigated in the embryonic and post-embryonic pigeon lenses by the indirect immunofluorescence with antiserum from rabbit immunized with isolated pigeon lens γ-crystallin. The results show that γ-crystallin appears for the first time in a 4-day embryonic lens and is distributed uniformly over the entire fibre cells. In a late and also post-embryonic lens γ-crystallin was also detected in the epithelium and annular pad areas and the intensities of immunofluorescent reaction gradually increased from embryonic to post-embryonic lens.

Immunoelectrophoresis of the proteins of the corneal epithelium.

The proteins of the corneal epithelium are separated into a greater number of fractions by high-tension agar electrophoresis than by paper electrophoresis. The distribution of the different fractions is greatly influenced by the ionic strength of the buffer solution. Although in agar electrophoresis a larger number of fractions are found, there is, nevertheless, a great similarity between the pattern obtained after paper electrophoresis and that obtained after agar electrophoresis at relatively high ionic strength. The existence of a greater number of fractions is confirmed by immunoelectrophoresis, which reveals at least eight different precipitation lines corresponding to different tissue proteins. The fraction present in highest concentration, which we have called the principal fraction, is found not only in humans, but also in several other mammals. In view of the fact that, in all cases, the corresponding precipitation line appeared when antiserum to bovine corneal epithelium was used, it would seem that the principal fraction has a very definite organ specificity. This fraction has poor stability in solution. It rapidly decomposes into two or three components, which are separated during electrophoresis in agar, where their mobilities are slightly different.