Calculating specific enzyme activity - (Jan/19/2007 )
I've been using a 4-MU assay to measure enzyme activity. I have converted RFU units to 4-MU per uM/ug produced and would like to know how to calculate specific (?) activity. I know it has something to do with the thickness of the cuvette and some arbituary number but am not sure of the formula.
specific activity is units of activity/amount of enzyme (usually expressed in mg), ie. U/mg. that is what you present in your question.
The general formula for enzyme activity from rate of change of absorbance is:
rate of change of abs per minute X (total reaction volume/(MA/1000) X 1000/(volume sample used)
The MA term is the molar absorptivity of the molecule being monitored (NADPH?) I think this is the arbitrary number you mention in your question. It is in fact specific for each molecule and wavelength. You'll remember in Beer's Law that absorbance = eCL (e is the absorption co-efficient, C is concentration and L is length of the cuvette.
By including the MOLAR absorption and 1000/sample volume you end up with international units per LITRE which are common units. Enzymology is riddled with different units due to poor standardisation earlier on. Check with your boss or the journal you want to publish in what units they require.
Hope that helps.
Hi- to calculate the MA i need a standard curve of concentration of my product (4-MU salt) v RFU at the correct absorbance. The slope of this (y0 + a) will equal the MA. What units do I need to use or do units not matter? I come up with a figure of 33 which converts RFU into salt concentration in uM. Also why does this formula not include the thickness of the cuvette like Beer's law?
as i said in my previous post, specific activity is defined as units of activity per unit of enzyme. in your first post you say that you already have this value. therefore you already have your specific activity, even if you don't recognize it.
as for your question about the path length in the calculation of MA, that is factored out by your use of a standard curve using the same or matching cuvette as for the sample.
It does mate. The equation uses absorbance per unit time Absorbance is eCL. e is the co-efficient for that molecule at that wavelength. C is the concentration. And L is the length, or thickness of the cuvette. Like MDFenko says, you have it perhaps without realising it. Units always matter. You just need to check which system your boss wants.
ofcourse u made a 4MU standard curve. can u please teach me ur substrate dilution series to make this curve. i used a dilution series of 50. 25, 12.5, 6.25, 3.12, 1.56 umol/ml. i measured sensitivity at 25
what about u???