Low-tech or low-cost technique for quantitative estimation in enzymology

Question

If an accurate measurement of enzymologic quantities is needed, then following established methods in the field is necessary.

However, it is sometime of great usefulness to ballpark a value before committing to taking the necessary time for protein purification, or buying the necessary equipment/reagents. For such purpose a little "hack" can go a long way. This often involve using an available equipment to substitute for another (e.g. maybe I have a FACS readily availably, however no HPLC).

I am seeking examples of "unconventional" use of lab technique that can be used to determine enzymologic quantities ($K_d$, $V_{max}$, $K_i$ ...) as a first estimate -- whether you have partaked in such heresies, or have merely seen them used, possibly even in a non-formal/DIY environment.

Answer 1

If there is a way to:

• Make the replication of a common culture organism (e.g. E. coli) dependent upon the inhibitor or substrate of interest, possibly with a restrictive media.
• Express the enzyme of interest in a relatively controlled manner in such organism (e.g. well caracterized promoter in plasmid with well characterized copy number),

Then you can get good mileage out of some serial dilutions and a OD600 meter. Even better with a plate reader.

Exact protocol will depend on the quantity under consideration, but mostly will amount to substituting around in the enzyme kinetics equations until the quantity of interest is a function of the concentration, and devise a scheme to cancel out the other unknown quantities (often substrate concentration ramp + starting OD ramp + curve fitting)