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How to calculate kcat from Lineweaver-Burk plot following RFU?

Polaris
in Biochemistry and Molecular Biology

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Hi all,


I'm using SensoLyte® 520 HIV Protease Assay Kit *Fluorimetric* of Anaspec to determine the activity of my HIV-1 protease. I have some problems in calculating Kcat value. I have calculated Vmax and Km follow Lineweaver-Burk plot are 6.97 RFU/sec and 3.7 uM, respectively. These two values are roughly equivalent to the parameters that Anaspec launched. But the kcat value is many smaller than Anaspec's value. My enzyme weight is 24500. I used 200 ng enzyme/1reaction with a total volume of 30 uL. And Vmax was converted to nM using a produce standard curve in an attachment file. Please give me some suggestion to determine kcat.


Thank you


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Kcat is also called turnover number and formula goes like this: turnover number=vmax/enzyme concentration

Kcat represents maximum number of substrate molecules which enzyme can turn into product.

Edited by GnothiSeauton

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