Hi, I am new to enzyme kinetics having been an ordinary little botanist for years on end and now faced with some assay work. I was wondering if someone could help me by explaining the relationship between absorption, time and rate of the reaction. I have time on x axis, absorption on Y but need to figure out the velocity! Sorry if this seems like a ridiculous question and you prolly think I am stupid. I have tried textbooks but its a bit tricky when you have never done any biochem at all. Thanks to anyone who does reply:)

need more detail to help you really.

You need to get a slope of absorbence at a particular substrate conc. Then vary the conc. You can then calculate the rate, but you also need to know things like extinction coefficients for the products being formed etc.

Hey, thank you.
I have varied the concentrations and have spectra for those. How should I do an abs slope? Can I just draw a straight line through and calculate the gradient between 2 points? I dont have the extinction coefficient for the product only for the substrate at a particular wavelength unless thats what it means. I feel like a total idiot.

thank you for your help!

hi, Tam, you will never draw a line by just two data points, I would suggest at least 5. and for the time based kinetics, take the points in minutes, and the spectrophotometer you have will probable let you do the time based scan, then you just take the initial slope of the scan.
and for the extinction coefficient, what you need to know first is the product of your reaction which gives the abs, then you can find the extinction coefficient in any organic chemistry handbook.

hope that will help

Hi,

U should draw a curve between the time(on x-axis) and Product (on the y-axis) and take the slope which will be the activity of ur enzyme.