Question

Regarding the LDH purification lab, what characteristics of a protein/enzyme allow us to quantify total protein versus enzyme activity?

Answer 1

Protein structure , reaction that enzyme catalyze, cofeactors associated with enzyme are the characterstic that allow to quantify protein vs enzyme activity.

For example LDH( lactate dehydrogenase), an important enzyme involve in anerobic metabolism of glucose. Perform a last step of anaerobic glycolysis. It catalyze interconversion of pyruvate to lactate. This reaction involve conversion of NAD to NADH. LDH is a tetramer of 35KD subunits. There are 2 type of sunit H which predominate in heart and M type which predominate in liver and muscle. These subunits associated to form 5 types of tertramers all with LDH activity but different substrate affinities and different response to regulators. So , this type of enzyme with similiar function but different structure called isozyme.

To determine enzyme activity , we measure conversion of NAD to NADH spectrophometrically at 340 nm wavelength. This method is applicable bacause LDH uses pyridine nucleotide as cofactor to transfer reducing equivalents. So, we can measure enzyme activity and purification using affinity chromatography.

Protein can be purify on the basis of charge, solubility, size and binding affinity. So, after purification we can quantify total enzyme activity.