More specifically, I have a simple enzymatic assay where I follow the Absorbance at 260 nm as my substrate is converted to product. Based on the literature values of extinction coefficients, there is approximately 2500 M-1cm-1 difference in extinction coefficient between the substrate and product. However, based on the total change in absorbance seen when the reaction is allowed to go to completion, I back calculate the difference in extinction coefficient to be approximately 12000 M-1cm-1. I have ruled out any contaminate by HPLC and the absorbance does not change if no enzyme is present, which rules out any adsorption issue. I have also largely ruled out any substrate adsorption to the enzyme itself.
No, I'm actually measuring the conversion of crotonoyl-CoA to Hydroxybutyryl-CoA.
You will have to excuse my ignorance, but crotonoyl-CoA and hydroxybutyryl-CoA have the same adenosine moiety that absorbs at ~260 nm.
Are you looking at changes in absorbance when they are bound by proteins?
Hi Dr. Stefansson,
I am looking at changes in absorbance of free crotonoyl-CoA as it coverts to Hydroxybutyryl-CoA. This conversion can be monitored at 260 nm, but I have found you get a much more robust signal change at 280 nm, which corresponds to the hydration of the enoyl double bond. I mistakenly stated above an extinction coefficient for this change of 2500 M-1cm-1, but it is actually 3600 M-1cm-1, however my issue as stated is the same.
Hi Brian, again, I am not familiar with this reaction, but I gather that this is an enzymatic reaction that does not occur with free reagents in solution.
As far as I know, enoyl bonds do not absorb in the UV.
Anyway, I seem to remember (from way back) that these cofactors are involved in tryptophan biosynthesis or degradation. The appearance (or disappearance) of tryptophan can really do a number on any absorbance measurements.
I will have to do more digging.
I appreciate the help and response. I attached one of the papers I am referencing, which mentions the extinction coefficient for the enoyl double bond. It is mentioned near the end of page 9510. The reaction itself is done with purified recombinant 3-hydroxyacyl-[acyl-carrier-protein] dehydratase, which I have crystalized so I am confident of its purity and proper fold. The reaction mixture contains the enzyme and crotonoyl-CoA obtained from Sigma in a TRIS buffer solution. So it is just free reagents in solution. The progress of the reaction was monitored with a spectrophotometer and also by HPLC. Thanks again.
Hi, thanks for the reference. There is this description that I find confusing
"An extinction coefficient of 3600 M-1 cm-1 at 280 nm was used for the hydration of the enoyl double bond".
Dehydrases that I know use cofactors such as NADPH, FADH to reduce double bonds, but the paper you posted didn't mention cofactors.
Am I missing something?
Enoyls are simple compounds that should not, by themselves, absorb in the ~280 nm range.
Is the absorbance change you see time-dependent after the enzyme and substrate are mixed together? is the enzyme itself a source of absorbance? Does the absorbance change when you add the enzyme but not the substrate?
Have you prepared a standard curve of substrate and product to measure the extinction coefficients yourself?
Sorry, I seem to have wasted everyone's time. I made a very stupid error and the total signal change is indeed what would be expected for my reaction. However, if either of you have any suggestions for the actual problem I am having it's that my reaction velocities and therefore specific activities and Kcat are massively higher than the reported values for a highly similar enzyme. In my haste to figure out what could be contributing to this I thought I'd discovered that my total signal was too high and perhaps there was some other erroneous reaction occurring simultaneously that was making the velocities seems higher than actual.
Hi Brian, is "reaction velocities" Vmax and is "specific activities " Km?
Hi Dr. Stefansson,
Basically, my Vmax is about 15 times higher than the expected value. What I mean by "specific activity" is the reaction velocities at specific substrate concentrations in units of nmole/min-1/mg-1. The Km value I've obtained is somewhat higher than the reported values but generally within the expected range.
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