Preparation of an Active Soluble Lactate Dehydrogenase-Nicotinamide Adenine Dinucleotide Complex Using Glutaraldehyde

The cofactor analogue N⁶-((6-aminohexyl)-carbamoylmethyl)-NAD was bound to beef heart lactate dehydrogenase by the glutaraldehyde coupling method and the enzyme-coenzyme complex subsequently separated from excess of non-coupled cofactor by gel filtration chromatography. The number of cofactor molecules bound per enzyme molecule could be varied between 0 and 8 by altering the coupling time. The enzyme-coenzyme preparation could catalyze the reaction lactate to pyrovate in the absence of externally added cofactor although with low efficiency. The enzyme-coenzyme preparation was also immobilized on Sepharose in such a way that possible contact between complexes was minimized (low substitution of the gel) and the activity of the immobilised complex was foound to be partly retained, indicating that a cofactor molecule can inteact with the enzyme molecule to which it is bound.