Problems Related to the Assay of Activities Following

8 PROBLEMS RELATED TO THE ASSAY OF ACTIVITIES FOLLOWING THEIR PURIFICATION BY HPLC 

While most of the problems in the assay of an activity purified by HPLC are expected and typical of chromatographic work with enzymes, the introduction of this technique into the purification scheme may lead to problems if the fractions obtained from the HPLC purification step are to be measured for enzymatic activity. For example, the salt in each fraction may inhibit any enzymatic activities it contains. Moreover, when ion-exchange HPLC is used the salt concentration will vary in the fractions. Thus it is prudent to study the effects of salt, at the concentration used for elution, on enzyme activity before the chromatography. If the salt is found to be detrimental, it will have to be eliminated or at least reduced in concentration before the chromatography. Removing the salt by dialysis may not be the appropriate way to proceed, however, since the inactivation of enzyme activities is not always reversible. 

it is emphasized that because salt solutions often act to precipitate detergents, as in the precipitation of sodium dodecyl sulfate by potassium, it is necessary to check the solubility of the protein solutions in the detergent against the salt solution at the concentration that will be present at the conclusion of the gradient. 

Two other problems often arise following the use of HPLC for purification. The first has to do with the volume of the sample used for assaying activity. Upon successful completion of the ion-exchange step, it is necessary to determine enzymatic activity. These determinations are performed on samples taken from a series collected during the course of the purification. With HPLC purification, however, the volume of each sample collected will probably be no more than a few hundred microliters, and often less. Further, the number of samples is usually small. This situation in HPLC is in contrast to that found in traditional chromatography, where the volume of each sample can be in the milliliter range and the total number of samples or fractions collected can be in the hundreds. 

Since to locate the enzyme, it is necessary to assay each of the fractions for activity, the concentration of salts will not be the same in each fraction when gradients are used. And since salts often inhibit activity, false data may be obtained on the distribution of activity across the column if salts remain in the sample. 

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