I used NOBA, a chromogenic substrate for phospholipases, to measure (for 120 min) the absorbance (at 405nm) of PLA2 in crude venoms from different snake species. Later, I plot the absorbance versus time curve. As far as I know, I would need the exact concentration of the PLA2 present in each venom, but I don't have that information. Is there another way to calculate each PLA2 activity?
Technically, the absorbance versus time gives me already a clue, but I tested several venoms and the more I add the less "beautiful" my graph becomes so that's why I was hoping to calculate the PLA2 activity and plot it as a histogram to make it more visible.
Hi Victor,
I would consider repeating the same experiment, but with a dilution range of pure PLA2 concentrations. You can then use that data to establish a standard curve of PLA2 activity at different concentrations and then compare your sample data to those.
Hi Stefan Kühn,
Thank you for your answer. The issue is, that I don't have any purified PLA2 from the crude venoms. I just want to have an idea regarding the activity of PLA2 based on this simple assay.
Hi Victor, the activities of purified enzymes are expressed as Km and Vmax.
For crude mixtures the best you can probably do is to calculate enzyme activity as units per mg of crude preparation.
1 unit = 1umol of substrate cleaved per minute.
Hi there,
Without any further data you can't calculate the PLA2 content of each venom. First make sure the absorbance does vary linearly during the 120' (if so final absorbance/120' is the initial velocity of the reaction). If not estimate the velocity on the time lap during which absorbance increases linearly (you absolutely need initial velocities!). All you can do after is to plot velocity of the reaction versus the volume of venom introduced in the assay (if everything is right you should get a linear relationship) and then calculate the velocity per volume unit of venom (it is the slope of the plot). These latter activities may be compared provided you are sure they are strictly PLA2 dependent and provided there is no inhibition/activation process in any of your crude venoms. An other possibility is to determine the protein content of the crude venoms and to calculate specific activities (velocity/mg of protein in the assay). They may be compared as well.
Hi Victor,
I'm not entirely familiar with your particular assay however, after some thinking, I would agree with Steingrimur. If you can determine how many umol of your substrate are consumed per minute you can determine the activity for each sample. Further, you could even normalise it to mg protein for each sample to obtain a U/mg(protein) value. To determine your umol substrate being cleaved, I would suggest establishing a dilution range of your substrate and measuring each absorbance value thereof. You can then use the curve of those values (absorbance vs. umol) to determine the umol of your samples.
I must solemnly say that nobody cares. Spectrophotometers have been used to study enzyme kinetics for 50+ years.
Further, phospholipases (plural) do not maintain membrane structures adequately because they cleave the phosphate bonds of phosphatidyl serine (PS), -choline (PC), -ethanolamine (PE) leaving behind triglycerides and fatty acids.
Hi, first you have to calculate the slope (velocity :∆abs/min) at the linear region. then if you know the extinction coefficient value (epsilon: e) of your product you can apply the Beer Lambert law(Abs= e c l),to convert it to μmol / min.Likewise ,you can also compare activities by unit (U) defined as the amount of enzyme that catalyzes the conversion of 1 micro-mole of substrate per minute,or even the specific activities by determining the proteins content using for example bradford method
Although,we can compare between the activities of your crude extracts ,the extraction method as well as the presence ofendogenous inhibitors can negatively affect the apparent enzyme activities .A purification is highly recommended to avoid this problem.
I agree with Farouk that you can calculate enzyme activities in the way he describes, using Lambert Beer's law (you can look op the extinction coefficient of the end product of your substrate, and you have to calculate the optical path length in case you use a microtiterplate reader. There are some aspects that you have to take care about:
1° Check linearity of each sample visually and select the proper linear range to calculate enzyme activity. This linear range can be influenced by enzyme lability, substrate consumption, exceeding the spectrophotometer's linear range etc.
2° Check different dilutions of your sample. Endogeous inhibitors or adsorption of enzyme on recipient walls can be detected.
3° Express as U/L of enzyme activity.
4° Make sure that you use substrate concentrations at least 5X above Km. It is to prefer that you first validate your assay! At least check activity using different concentrations of substrate and make a Michaelis-Menten plot.
5° Is your preparation stable for such a long time? Check by pre-incubating your sample and see what happens with the activity.
Hi,
I agree with Steingrimur , Dominique, Farouk and Robert. For further details you may want to read the original paper about the substrate:
Matthew Holzer, Stephen P. Mackessy,
An aqueous endpoint assay of snake venom phospholipase A2,
Toxicon,
Volume 34, Issue 10,
1996,
Pages 1149-1155,
ISSN 0041-0101,
https://doi.org/10.1016/0041-0101(96)00057-8.
cheers,
Patrick
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