Capillary electrophoresis for the investigation of prostate-specific antigen heterogeneity

Prostate-specific antigen (PSA) is a single-chain glycoprotein that is used as a biomarker for prostate-related diseases. PSA has one known posttranslational modification, a sialylated diantennary N-linked oligosaccharide attached to the asparagine residue N45. In this study capillary electrophoresis (CE) was employed to separate the isoforms of seven commercially available free PSA samples, two of which were specialized: enzymatically active PSA and noncomplexing PSA. The free PSA samples examined migrated as four to nine distinct, highly resolved peaks, indicating the presence of several isoforms differing in their oligosaccharide compositions. Overall, the use of CE provides a rapid, reproducible method for separation of PSA into its individual isoforms.     

Analysis of the heterogeneity of human collagens by two-dimensional polyacrylamide-gel electrophoresis.

The heterogeneity of the CNBr-cleavage peptides of human types I, II, III and V collagens were studied by using two-dimensional electrophoresis combining non-equilibrium pH-gradient-gel electrophoresis and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Specific 'maps' were produced by the peptides obtained from the chains of each type of collagen, and most peptides had at least three charged forms of the same molecular weight. Specific 'maps' were also produced by the peptides of types I, III and V collagens from insoluble dermis and the peptides of types I and V collagens from decalcified bone. The alpha 1(I) CB7 and alpha 1(I) CB8 and the alpha 2 CB4 peptides obtained from the type I collagens of these tissues contained the same number of charged components, but there was a relative increase in the more basic components in bone. Some aspects of the involvement of the alpha 1(I) CB6 and the alpha 1(III) CB9 peptides in cross-linkages were also studied. The recovery of the alpha 1(I) CB6 peptide from bone and dermis was decreased and the alpha 1(III) CB9 peptide was not detected in dermis. Additional peptides, which were probably cross-linked peptides involving the alpha 1(I) CB6 peptide, were also observed.     

Fractionation of human neutrophils into subpopulations by countercurrent distribution: surface charge and functional heterogeneity.

Isolated blood neutrophils from normal healthy subjects were separated into fractions by sequential countercurrent distribution (CCD) in a charge-sensitive dextran/polyethylene glycol aqueous phase system. The neutrophils separated as a broad profile, and in a charged phase procedure the separation was based upon differences in cell surface electrokinetic properties, as confirmed by electrophoretic mobility measurements of fractions across the profile using analytical cytopherometry. The CCD cell fractions were generally pooled as three or four major subfractions for analysis of functional and metabolic differences. These included measurements of chemotaxis, phagocytosis, and respiratory burst. An inverse relationship was found between the electrophoretic mobility (EPM) of the subfraction pools and their functional competence, with the less electronegative cell fraction pools often as much as 2 to 3-fold more active than the more electronegative pools. This demonstration of electrokinetic and functional heterogeneity in 'resting' neutrophil subpopulations separated by CCD may reflect changes during their sojourn in the circulation that determine selective margination and recruitment of cells to inflammatory foci and sites of infection.     

Demonstration of protease isoenzymes by two-dimensional polyacrylamide electrophoresis

A two-dimensional polyacrylamide electrophoresis system is described for the detection of protease isoenzymes using 4-methoxy-2-naphthylamine derivates as substrates. With this technique is possible to detect isoenzymes differing in molecular weight by separation according to size and shape. This is not possible by the use of isoelectric focusing alone.     

Peptide bond synthesis catalyzed by alpha-chymotrypsin.

alpha-Chymotrypsin [EC 3.4.21.1] catalyzed the syntheses of peptide bonds with various N-acylated amino acids or peptides having aromatic or hydrophobic amino acid residues at the C-terminal position as carboxyl components, and amino acid derivatives, peptides or their derivatives as amine components. A neutral pH was most efficient and quite high concentrations of alpha-chymotrypsin and starting materials were required for synthesis. Four amine components, hydrophobic or bulky amino acid residues were useful at the N-terminal position. Stereospecificity was also observed at the N-terminal position of amine components. Peptide synthesis was not usually seen when the products were soluble in the reaction mixture. This could be partly overcome by increasing the concentration of either the carboxyl or the amine component to more than ten times that of the other.     

Alpha 1-antitrypsin: apparent molecular weight heterogeneity shown by two-dimensional electrophoresis.

Two-dimensional (2-D) gel electrophoresis was used to examine charge and molecular weight variability of alpha 1-antitrypsin. Two-D electrophoresis resolved distinctive differences among individual phenotypes. Microheterogeneity of charge was seen for the different alleles that corresponded to the charge variability observed on isoelectric focusing gels. The molecular weights of the major components of each allele appeared to differ from each other by approximately 1,000, suggesting, that in addition to sialic acid, there may be differences in neutral sugar composition between the individual components. In comparison to the M allele components, the corresponding S and Z components had higher molecular weights. The MZ and MS phenotypes showed characteristic patterns of protein spot doublets. Computerized quantitation was used to separate and estimate the contribution of each component to the overall allele composition. The Z allele components contained about 15% of the total MZ quantity. The 2-D electrophoresis technique may offer a new approach for molecular structural studies of alpha 1-antitrypsin variants and similar glycoproteins.