[stevet]

I'm trying to run some MM assays on an enzyme with a novel substrate - the nature of the substrates/products means that the assays have to be monitored by HPLC, after enzyme denaturing by heat.

The product of the reaction is, however inherently unstable to heating. A known, structurally similar compound (S-Adenosylmethionine) is known to decompose on heating into a mixture of adenine and methylthiodeoxyadensine at the pH of the assay and this decomposition goes to completion after heating for 30 minutes.

Can I determine the Km of for the enzyme by monitoring the formation of the breakdown products of the novel product even though I have no data to back up its decomposition pathways, but rather assume it behaves similarly to SAM? If not (which I think is the case) would you have any suggestions as to a way around this problem?

[Yggdrasil]

Is there an alternative way to inactivate the enzyme?  For example, you could add in some organic solvent, SDS or urea to denature the enzyme.  Alternatively, if the enzyme requires a divalent cation cofactor, you could add EDTA to chelate the metal ions.

[Babcock_Hall]

Is your product stable to acid?  I used to precipitate protein with perchloric acid, then neutralize and precipitate out potassium perchlorate on ice.  This allowed me to analyze GDP bound to a monomeric G-protein.

[stevet]

There is an alternate - previous studies have used ethanol to precipitate the enzyme. This will stop the reaction, but dilute my sample, which I am trying to avoid (The sample could be concentrated again using a vacuum concentrator, but I don't have access to one and doing them individually would be extremely time consuming, but is potential solution none the less..). The Km for is low, round 40μM and to accurately measure data to get this figure means I start to approach the detection limit of the HPLC system - so I am trying to avoid dilution at all costs - but thanks for the suggestion

On a slightly different, but related note - how would you describe a rate if there is no obvious way to quantify your product. The product is a novel compound, almost impossible to prepare synthetically so I cant construct a standard curve. I have tried to measure the decrease in substrate concentration (and assume Δ [ S] = -Δ[P]) but the measuring tiny changes in large product concentrations leaves me with large errors.

Any more suggestions?

Thanks again!

[Babcock_Hall]

How much dilution can you tolerate?  What functional groups are present in your product?  Is there any hope of trapping your product, meaning derivatizing it to something less labile?

[stevet]

I could deal with some dilution. I have just learned that the other HPLC system that we have has a loop that can take 5-1500μL of sample - so dilution is less of an issue now. Trapping the product is tough - Its an S-adenosylmethionine analogue (and has all the same functional groups), and trying to selectively derivatise the product without also derivitising the starting materials, is almost impossible.

I have looked into doing the measurements by ITC - and this looks promising. But I will keep looking into it.

[Babcock_Hall]

The perchloric acid precipitation I used came from this reference:  Seckler, R., Wu, G.-M., & Timasheff, S. N. (1990) J. Biol. Chem. 265, 7655-7661.  I seem to recall that I used an isocratic HPLC system, but there probably is some latitude about the mode of chromatography one uses.  I am not sure whether or not this would work for SAM.