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

Abstract

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 noncoupled cofactor by gel filtration chromatography. The number of cofactor molecules bound/enzyme molecule could be varied between 0-8 by altering the coupling time. The enzyme-coenzyme preparation could catalyze the reaction lactate .fwdarw. pyruvate in the absence of externally added free 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). The activity of the immobilized complex was partly retained, indicating that a cofactor molecule can interact with the enzyme molecule to which it is bound.