i have performed the enzyme assay by taking different concentrations (0.5-5%) of 0.5 ml substrate along with 0.5ml enzyme and have the spectrophotometric data for it. kindly suggest what should i do next
You should derive Vo from the linear part of the different curves and then use nonlinear regression to fit the data to a Michaelis-Menten curve.
That said, there are lots of pitfalls and assumptions to be made along the way that can schew your results one way or another. It would be great if you could get ahold of an enzyme kinetics textbook and/or interact with an experienced labmate or mentor to make sure you understand properly the procedure. Enzyme kinetics is one of those things where a step-by-step recipe seldom applies...
Hi Vaishali,
The first step in doing any enzyme kinetics measurement to estimate the affinity of an enzyme for its substrate is to make sure that the enzyme is in catalytic amount, i.e., 0.5 ml of enzyme doesn't indicate how much enzyme was actually present in terms of its concentration ( nM or uM). Further, the substrate should also be specified in terms of concentration( nM, uM or mM). Now, having converted your volume and % notations into concentration terms across both substrate and enzyme, make sure that the enzyme is atleast 100 times less than the lowest substrate concentration to achieve the Michaelis-Menten conditions. If everything is alright till here, use the extinction coefficient of your product and employ Beer Lambert's law to estimate the concentration of the amount of product formed from the absorbance reading. The law states that
A= (epsilon) X C X l
where A is absorbance, epsilon is the Molar extinction coefficient( units M-1 cm-1), C is concentration ( units Molar) and l is the length ( units cm).
Plot the concentration in terms of either amount of product formed per mole of enzyme (kcat) or per mg ( specific activity) as a function of substrate concentration. Use any of the non-linear fitting algorithms to fit the resultant data points to Michaelis-Menten equation.
v= (Vmax X [S])/(Km+[S])
Hi Vaishali, I commend you if you are trying to figure this out by yourself, but if you have a biochemistry teacher that asks you to do M&M graphs without giving the proper background in molarity calculations and spectroscopy, he should be fired on the spot!
Bluntly, if this what your university has to offer to their students in terms of biochemistry------it is horrible and you should go elsewhere.
I tend towards Stefansson opinion...
On the other hand I suggest you have a look at:
Biochemistry. 2011 Oct 4; 50(39): 8264–8269.
Published online 2011 Sep 9. doi: 10.1021/bi201284u
PMCID: PMC3381512
NIHMSID: NIHMS324930
The Original Michaelis Constant: Translation of the 1913 Michaelis-Menten Paper
thank u sir @ Babak Andi. it seems doable.....i m grateful that u took the tym to elaborately answer my query
Point 5 in Andi`s answer is not correct. At constant substrate concentration the rate of an enzyme catalyzed reaction increases linearly with the enzyme content. Point 2 is correct buts the enzyme content can only be given in M or µM per L, when the active site content can be determined or when it is very pure, and the molecular weight and the water content of the “dry" enzyme are known.
I strongly agree with the answers of Stefansson and Ghisla.
To solve your problem, in case your teachers or the used textbooks cannot help you directly, I suggest the following. Such a procedure should be teached and trained in all university science departments. The students must learn to solve problems alone using generally available literature and digital resources.
Has your library a textbook on enzyme kinetics, consult it. When not:
Search in Google for enzyme kinetics, there you will find information that will help you. But use only the information based on original scientific literature, given by references to papers in scientific journals (some of these may be open access). There you will find different graphical procedures to determine Vmax and K m (Lineweaver-Burk, Eadie-Hofstee etc) and their pros and cons.
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