High-performance liquid chromatography in enzymatic analysis. Opening lecture.
During the previous two decades, high-performance liquid chromatography (HPLC) has proven to be an extremely useful technique with which to study the activity of enzymes and this paper will explore some of these uses. The success of the method can be seen not only from the increase in the number of papers utilizing this technique but also from the insights gained from its use on cellular phenomena. Given this success, it is no wonder that HPLC has become the technique of choice for many biologists seeking a more quantitative understanding of biological processes. Based on past experience, there is every reason to expect that the application of HPLC to the assaying of enzymatic activities will usher in another era of fundamental discoveries in the biological sciences. HPLC is particularly well suited to the assay of one activity in the presence of other activities obviating the need for extensive and tedious purification of biological samples. This advantage makes this technique particularly well suited to those who wish to use enzymes as markers for cellular processes, as indicators of metabolic activity and as evidence of gene function. To date, well over 100 activities have been assayed by this method. The method is particularly suited to problem-solving especially in such cases as when the presence of competing reactions prevents the recovery of the expected reaction products. Of the many applications, examples will be given on the use of HPLC for (1) monitoring the activity of an enzyme in a cell-free system, (2) monitoring the flow of metabolites through a multienzyme system and (3) the detection and study of new enzymatic activities. Some generalizations about the use of HPLC methods for the analysis of enzymatic activities will be presented.
Rossomando
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Department of BioStructure and Function, University of Connecticut Health Center, Farmington 06030.
D002851:Chromatography, High Pressure Liquid; D004798:Enzymes / Q000737:chemistry*; D009097:Multienzyme Complexes / Q000737:chemistry; D013379:Substrate Specificity
D004798:Enzymes; D009097:Multienzyme Complexes