Quantitative analysis of protein: immobilized dye interaction.
The interaction of rabbit muscle lactate dehydrogenase with reactive blue 2 immobilized on Sepharose CL-6B was quantitatively evaluated by analytical procedures using both frontal and zonal chromatography and also static equilibrium methodology. All three analytical procedures give a common result, namely (i) that each enzyme molecule is retained by a single immobilized dye molecule; (ii) that the immobilized dye binds to the protein at an NADH binding site with an affinity identical to that of the free dye; and (iii) that less than 2% of the immobilized dye is accessible to the enzyme. The very small fractional accessibility is not due to steric exclusion but likely is a result of adsorption of the immobilized dye to the matrix surface.
D000818:Animals; D004396:Coloring Agents; D004800:Enzymes, Immobilized / Q000378:metabolism*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D011817:Rabbits; D014227:Triazines
D004396:Coloring Agents; D004800:Enzymes, Immobilized; D014227:Triazines; D009243:NAD; C012842:Cibacron Blue F 3GA; D007770:L-Lactate Dehydrogenase