The use of HPLC to assay enzymatic activities has continued to grow in popularity since publication of the first edition of this book in 1987. Papers describing methods of assay for approximately 150 different enzymes were reviewed in preparation for writing this chapter.
There are several reasons for the development of HPLC-based methods for assaying enzymatic activities. First is the desirability of accurately assessing enzymatic activity in preparations as near the biological state as possible. This often requires the use of turbid preparations, and the product to be measured must be separated from other components of the reaction mixture. However, the use of crude preparations often carries the price of needing to correct for secondary reactions. HPLC allows secondary reactions to also be measured.
Second, the resolving power of HPLC greatly exceeds that of other separation methods. An enzymatic assay that was developed to use thin-layer or paper chromatography may readily be improved by switching to HPLC. Furthermore, HPLC is automated.
Third, the hazards of handling and disposing of radioisotopes have made it desirable to find alternative methods of assay. When the assay involves fluorescent or intensely absorbing substrates or products, the sensitivity of HPLC-based assays rivals that of radiochemical assays.
Fourth, column eluates can be monitored by several means, including UV-visible absorbance, fluorescence, and electrochemical or radiochemical detection. Postcolumn mixers and reactors can readily be incorporated into the eluate stream, thus allowing modification of eluted compounds to increase sensitivity of detection. And finally, the resolving power and sensitivity of HPLC allows monitoring of subtle changes in molecules, especially macromol-ecules.
Each assay is presented according to the scheme used throughout this book. The primary reaction is introduced, followed by the methods used for separation, including stationary phase, mobile phase, and the elution protocol. The method of detection is also described.
208 survey of enzymatic activities assayed by the hplc method
The enzymatic assay is then described, including buffers and pH, the method for initiating the reaction, and the process used for termination. Next, the methods used in the preparation of the sample for HPLC analysis are described, including centrifugation, filtration, or any type of purification preceding injection into the HPLC system. For many of the assays, time span and range of protein concentration for which the reaction is linear are also indicated.
Finally, the source of the enzyme activity is mentioned, including disclosure of purification procedures that were employed.
Adoption of a published procedure for an HPLC-based enzyme assay is not always a straightforward matter. For example, most separations are obtained by reversed-phase chromatography on a C18 column. The chromatographic behavior of some compounds is sensitive to the amount of unreacted silanols present in the matrix. The extent to which silanols are "end-capped" varies from manufacturer to manufacturer, and even between lots from the same vendor. Second, the composition of the mobile phase may not be subscribed sufficiently. For example, the starting materials for preparing a phosphate buffer may not be clearly stated, and it may not be indicated whether pH adjustment was made before of after addition of organic solvent. Third, it is advisable to validate an assay whenever the enzyme source is different from the one described in the published method. It is especially important to determine the time span and protein range in which product formation is linear with time. Deviations from expected behavior can be due to competing (secondary) reactions as well as differences in properties of the enzyme assayed.
The general reference section contains additional citations that are relevant to the enzymes described, to provide readers with a more extensive survey of the HPLC assay methods that have been developed for these activities.
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