ABSTRACT
A method for estimation of amino acid composition and collagen-specific amino acids was developed. The assay was based on the precolumn reaction of amino acids with phenylisothiocyanate. The phenylthiocarbamyl derivatives were analyzed by means of reverse-phase chromatography on octadecyl sorbents, which involved gradient elution with ethanol and sodium or ammonium acetate. The method was used for effective separation of collagen-specific and main amino acids in cartilage hydrolysate from healthy persons and of patients with funnel chest and Ehlers-Danlos syndrome and also for determination of amino acid composition in creatine phosphokinase B- and M-subunits.
Subject(s)
Amino Acids/analysis , Cartilage/analysis , Creatine Kinase/analysis , Cartilage/enzymology , Chromatography, High Pressure Liquid , Humans , Indicators and Reagents , Isoenzymes , Phenylthiourea , Spectrophotometry, UltravioletABSTRACT
Heterodimers of porcine creatine phosphokinase were obtained from dissociated MM and BB isoenzymes and investigated in the enzyme-linked immunosorbent assay. It has been shown that peroxidase conjugates of rabbit polyclonal antibodies against porcine enzymes have low affinity in the sandwich reaction.
Subject(s)
Binding Sites, Antibody , Creatine Kinase/isolation & purification , Animals , Antibody Affinity , Chromatography, Affinity , Chromatography, DEAE-Cellulose , Creatine Kinase/immunology , Creatine Kinase/metabolism , Enzyme-Linked Immunosorbent Assay , Immunization , Immunodiffusion , Isoenzymes , Rabbits , SwineABSTRACT
Amino-acid composition of porcine M and B creatine phosphokinase subunits and a sequence of 22 N-terminal amino acids have been determined. A comparative analysis with the structure of creatine phosphokinase from other sources made it possible to discover a sequence with 8 to 15 amino-acid residues, which is the most suitable for the preparation of polyclonal antibodies.
