The interaction of Cibacron Blue F3GA with troponin and its subunits.
Binding of troponin to Cibacron Blue F3GA-agarose column and its selective release from the gel in the presence of 0.5 M KCl provides the basis for a new purification method. The two-step procedure consists of isoelectric precipitation of tropomyosin and chromatography of the resultant crude troponin supernatant on Affi-Gel Blue column. Adsorption of troponin to the immobilized dye appears to occur through the troponin-T subunit. Troponin-I and troponin-C do not bind to the blue agarose column, whereas troponin-T binds to it very tightly. Binding of the dye to troponin-T prevents formation of troponin-T-troponin-C complex, but does not interfere with direct interaction of troponin-T with troponin-I. The activity of troponin in conferring calcium sensitivity on actomyosin ATPase is not affected by Cibacron Blue. Circular dichroism and difference absorption measurements of complexes of the blue dye with troponin and its subunits reveal the presence of a tight binding site on whole troponin and on troponin-T (KA greater than or equal to 10(6) M). The existence of weak binding sites for the dye on troponin and all of its subunits is deduced from difference absorption studies. Cibacron Blue appears to be a sensitive probe for subunit interactions in troponin.
D000199:Actins; D000818:Animals; D000873:Anthracenes; D001665:Binding Sites; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D046911:Macromolecular Substances; D009124:Muscle Proteins; D009218:Myosins; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D012685:Sepharose; D013053:Spectrophotometry; D014227:Triazines; D014335:Tropomyosin; D014336:Troponin
D000199:Actins; D000873:Anthracenes; D046911:Macromolecular Substances; D009124:Muscle Proteins; D014227:Triazines; D014335:Tropomyosin; D014336:Troponin; C012842:Cibacron Blue F 3GA; D012685:Sepharose; D009218:Myosins