How to plan a classical enzyme-substrate reaction ???

16 October 2019 1 7K Report

Hello everyone!

I am mostly a structural biologist who did not get a chance yet to delve into a classical biochemical enzyme-substrate reaction.

So as things would have it, I will have to perform one now.

I have an NAD dehydrogenase enzyme and I have a substrate for it. As I understand, I have to monitor the decrease in the absorbance at 340 nm as NADH will be converted to NAD+

I plan to use the 96 microwell plate reader for this purpose.

How will I go about measuring the Vmax, Km, Kcat and the rate of reaction?

Also, how will I utilize the absorbance to obtain the rate of reaction? How about the enzyme and the substrate concentration?

I will be more than happy to get some help on this!

Thanks,

Nishit

Danial Yousefi

I suggest you to consult the literature regarding how to assay your particular enzyme, and details such as how to start and stop the reaction it catalyzes, as well as what to use as your negative control.

But in general, you need to measure the absorbance for samples of the enzymatic reaction that started with several different concentrations of the substrate and stopped at several different time intervals. The relationship between absorbance and concentration can be obtained by measuring the absorbance for a series of standard solutions of the substance you are going to measure; in your case, NADH. By the way, you may measure the peak of absorbance for your NADH sample experimentally using the particular spectrometer you are going to use. You need to know [E]0 as well, the concentration of the active enzyme at the beginning of the reaction.

Assuming that your reaction follows the Michaelis-Menten kinetics, the kinetic parameters can be easily calculated by data analysis software such as GraphPad Prism and SigmaPlot, which provide user-friendly wizards to obtain these parameters.

Badges
Science topic

More Nishit Goradia's questions See All

How to use C18 column to detect the presence of NADH/NAD+ in my protein and its mutants?

Dear Scientific community, I have a protein (tetramer) which has a NADH bound to it (confirmed from the crystal structure) when it is purified from Bacteria. Additionally, I have two mutants of...

13 January 2019 9,330 3 View

What techniques can I use to determine the miscibility of polymer blends?

The term miscibility refers to the single-phase state in thermodynamics. I do not mean the compatibility of different components. To determine the miscibility I know several techniques such as...

03 March 2021 4,107 4 View

Your thoughts on the spherical densiometer to estimate relative species canopy cover?

Hi biologists and biogeomorphologists, I want to characterize vegetation plots on a transect that follows a successionnal gradient from a river bed to a mature forest. Ecologically, I am...

26 February 2021 5,720 2 View

What kind of institutional environment do entrepreneurs need to achieve happiness?

Entrepreneurs are stressful, and happiness is a scarce resource. Happiness is crucial to business development.In addition to the work characteristics, individual characteristics have an effect on...

25 February 2021 876 2 View

Any interests in participating to an experiment in space?

Hello, The UN Office of Outer Space Affairs has a program called access to space for all that enables institutions to have hands on expertise related to access to space. There are three elements...

22 February 2021 7,980 3 View

How much costs direct reduction of nitrogen on farmland in terms of lower crop yield?

Dear research fellows, I wonder if someone knows how much direct reduction of nitrogen on farmland costs in terms of lower crop yield expressed as EUR/kg nitrogen (or similar)? I understand that...

15 February 2021 1,323 3 View

It is easier to knockdown a gene than overexpress a gene when generate a stable cell line?

I am just so confused that some people tell me knockdown is easier than overexpression when generating a stable cell line. I think it depends on the gene. Cells won't be happy whether we try to...

15 February 2021 1,370 2 View

For acid treated montmorillonite clays what will be the result of surface area?

The organo montmorillonite nanoclays modified with octadecylamine were used for the experiment. After acid treatment, the surface area will be increase. However, in my experiment, the surface area...

10 February 2021 1,581 3 View

How can I quantify the color of a solution?

I know colorimetry has been the standard way by way of measuring the concentration of a particular compound (solute) in a colored solution (solvent or measure quantities of a particular compound...

08 February 2021 2,673 1 View

Which is the appropriate statistic test to examine the relation between students' affective state and academic performance?

Which test should I use to compare students' mood effects on academic performance? I want to compare mood measured on a likert scale (from point 1= very sad to point= 5 very happy) to three...

04 February 2021 1,907 5 View

What are advantages of NELF model?

I am trying to simulate permeation of gases in polymers. For this reson normally i use the clasic models like dual sorption model but newly i foun dthe non equilibrium lattice fluid model. It...

03 February 2021 1,203 3 View