Another example of the application of capillary electrophoresis for revealing posttranslationally modified proteins is the separation of collagen polypeptide chains after modification by glucose. Deyl and MikSik (1995) performed separations using 4% Polyacrylamide gel-filled capillaries. As shown in Figure 8.7, SDS electrophoresis in capillaries containing linear Polyacrylamide offers good separations of collagen's polypeptide chains, as well as any polymers, separations that are at least comparable with those resulting from slab gel electrophoresis. Moreover, CZE separations offer the possibility of quantitation, by measuring the area of individual peaks, a difficult procedure with slab gels. Also, CE in non-cross-linked gels offers the possibility of separating polymers, (i.e., of components having molecular mass ^300,000). This capability is of considerable importance, because no method offering sufficient selectivity is available for analyzing such polymers. Even in diluted Polyacrylamide slab gels, such high relative molecular mass fractions either stick to the starting line or move as a smear between the y fraction (the collagen a-chain trimer) and the start. Other methods, such as gel permeation low pressure column chromatography, do not provide sufficient selectivity.
Incubation of a collagen type I sample with glucose produces the results seen in Figure 8.8. Such profiles exhibit a twofold difference from untreated collagen electropherograms: the a peaks are split into two each, and the peaks of y-chain polymers and higher are increased. Both these effects can be ascribed to the nonenzymatic reaction of the e amino groups of lysine residues with glucose. Reiser (1991) reported that such reactions are nonspecific, affecting several lysine residues in the collagen molecule and, upon prolonged incubation, leading to progressive insolubilization of the collagen
Figure 8,4 Comparison of capillary electrophoretic (ELPHO) profiles (pH 9.2) of hair keratin samples obtained from control rats (A) and alcohol-treated rats (B). The dilution of extracts was 50 mL to 5 mL. Position of the peak present in alcohol-loaded animals but absent in controls is indicated by an arrow. Assignment of "high-sulfur" and "low-sulfur" proteins is based on the results shown in Figure 8.5. (From Jelinkovi et al., 1995, with permission.)
proteins. Based on these observations, it is reasonable to assume that splitting of the two a-chain peaks into four, following incubation with glucose, may reflect nonspecific binding of glucose to several sites of collagen a chains, resulting in a decrease of positive charges of these polypeptide chains. Because these separations are performed with excess SDS, splitting of the a peaks can hardly be attributed to the change in effective charge of individual polypeptide chains. It is more probable that glycation alters the hydrophobic properties of collagen a chains and modifies the amount of SDS bound to the protein molecule. Decreased binding of SDS to proteins, which possess a high proportion of sugars (though O-glycosidically bonded), has been well documented. The occurrence of polymers with molecular mass higher than y-collagen molecules, as well as the increased proportion of y-chain polymers in the overall profile, can be ascribed to further polymerization of constituting a chains, which is known to lead, under in vitro conditions, to complete insolubilization of collagen proteins.
Was this article helpful?