Enzymes have activity and concentration. which assay is more reliable in case I want to study the role of some enzyme on some clinical case. Is it determining its activity or its concentration and why?
Both are equally important. The real question here is how can you study activity and concentration, i.e. what analytical tools do you have? So i think the question is not properly phrased yet and you need to look closer into your case. Do you have any hints that the activity of the enzyme could be different in different patients? Do you have the possibility to determine the enzyme activity? Are there interfering compounds, activity loss during sample preparation, storage, etc. There are many uncertainties here, so that i would assume that researchers in most cases go for a concentration based assay (at least initially).
Dr. Markus Sack, thank you for your answer and your interesting questions. i have another question here; does ELISA technique represent a good method to determine the concentration of CYP enzymes?
Sorry, i can only give you some generic answers for that as (1) i am not familiar with the particular assay you have in mind and (2) you have not provided enough specific details for your target. In principle ELISA has both the sensitivity and specificity to be suited as an assay for quantification of your target enzyme. However, you need the right antibody reagents and you need to qualify the assay. Or you need to search the web and find a company that sells a qualified / validated kit.
Enzyme activity should be determined by the reliable RP-HPLC-photometric method (Please see file; J Chrom B rat BIN LIP Km). Chemical synthesis of artificial substrate may be necessary by your own hands, since commercial substrate-compounds may sometimes be suddenly stopped selling (my unpublished experience).
Protein concentration should be determined by the reliable and sensitive HPLC-Surf-SEC-photometric method (please see file; HPLC-Surf-SEC protein determination method). Surfactant of PGC (Polyethylene-glycol 1000 monocetyl ether; Anapoe-58; Brij-58) available from Anatrace Products LLC, Maumee, OH, USA or FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan should be utilized un this HPLC method.
These two HPLC methods are uniquely quantitative.
I think there are many reliable ways to determine the quantity and activity of cytochrome P450s. In your case, quite a lot of background information is missing.
Do you want to measure the activity/concentration from a biological fluid, cell culture etc or the enzyme is already purified?
Do you know the substrate/products of your enzyme?
In regard of ELISA, a specific antibody should react with your enzyme and give a fluorophore which can be measured. Based on this, you can quantifiy your enzyme from various of samples. As I am aware, you cannot measure the activity with ELISA.
Activity can be measured by using spectrometers (fluorescence, UV/VIS), calorimetry etc. It depends which is the substrate/product or the interacting partners of your enzymes.
I am sure that there should be quite a lot of literature about this topic.
Hopefully, it gave you some ideas to work on your issue.
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