how to convert RFU/sec of my Vmax in mol/min/mg of protein?? - (Feb/15/2013 )

Hi everybody!!
I'm doing some enzymatic kinetic studies and I need your help!!I'm using a FRET based assay to evaluate the strenght of some potential inhibitors against HIV-1 protease.
I wanted to measure the Km and the Vmax of HIV-1 protease for my peptidic substrate http://www.sigmaaldr...atalog/product/sigma/h0790?lang=it&region=IT and I performed different enzymatic reaction at 8 substrate concentrations. I used graphpad to obtain Km and Vmax, but the Vmax is expressed as RFU/sec and I know I should convert it as mol/min/mg of protein. How can I do this?
I think I should have a calibration curve of my product but I'm not sure...

I tried to perform a calibration curve using the anthranilic acid, but at the end the concentration I obtained was negative....Where is the mistake????

thanks a lot

Claire

Yes you need to have a calibration curve that would tell you 1 RFU means how many moles? Let say you got X rfu for Y moles then these y moles will be divided by the time to get you the vmax at 2Km concentration. And calibration curve can be generated with any compound that gives RFU. If you are geting negative readings for some values, may be you are doing something wrong. Or you can omit those values next time when you do it again to generate the calibration curve.

But I should use the peptidic fragment the enzyme generate to make the calibration curve, or can I use only the fluorophore (in this case the anthranilic acid)?

In my view you can use any fluorophore because its relative fluorescence unit. Like for radioactivity assays (for instance c14) we make a calibration curve with the counts generated by any radioactive compound having c14. So basically you need to know How much moles are there in particular RFU to calculate vmax and km.

claire84 on Fri Feb 15 13:50:38 2013 said:

Don't too concerned with the absolute values of the standard curve, rather just focus on the rate of the change. An increase in x uM product causes an increase in fluorescent signal of y; therefore when the enzyme causes a change in fluorescence of y per min the rate will be x uM/min.

claire84 on Tue Feb 19 09:02:18 2013 said:

Fluorescent readings can be susceptible to their microenvironment, which is the basis of FRET assays, so ideally you would use the peptide fragment to generate the standard curve but I suspect that it may not be readily available so your approach will be acceptable. It might be possible to check by letting the reaction proceed to completion and hence checking if the change in fluorescence is about the same as you would calculate from the standard curve for a given amount of substrate.