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.

Thank you everyone!

I must repeatedly say that HPLC-enzyme assay should be utilized in enzyme kinetic analysis (please see file; J Chrom B rat BIN LIP Km). Direct-use of photometer gives 1/3-fold smaller values as compared to HPLC-photometric method (please see Fig. 10 of the file; JCB Fucoidan Transport). This is only due to direct-use of photometer without purification, which violences against the Lambert-Beer's law.

Direct use of biological specimens can be achieved to get enzyme kinetic parameters, when final enzyme- or homogenate-concentration is less than 0.1 mg protein/mL (please see file again; J Chrom B rat BIN LIP Km). This precise dilution of proteins can uniquely be achievable by using sensitive nd reliable HPLC-photometric-protein determination method (please see file; HPLC-Surf-SEC protein determination method).

By the way, you should simultaneously measure the decrease of substrate and increase of product. This task can uniquely be achievable by HPLC-enzyme assay method (please see file; Release of anchored). Therefore, I have safely published lipase/lipoamidase papers using rat brain homogenate and purified enzymes (please see files; Purify Brain LIP and Multiple hydrolase LIP).

If you have no HPLC system, then you can use HPLC as follows; i.e., Lease company is present, and please visit this Web site. ORIX Rentec Corporation http://www.orixrentec.jp/en/index.html I have recently borrowed an HPLC system from this company at 120,000 Yen (1,091 USD) per month (please see file again; JCB Fucoidan transport, though I have not mentioned the method to obtain the HPLC system in this paper).

Amended on 7 October 2018

Further, phospholipase seems to be important to maintain the membrane structure adequately. Thus, I have searched gene expression of it in my human protein database (please see file; HepG2 Fucoidan).

Fetal hepatocyte Hc has Membrane-associated phosphatidic acid-selective phospholipase A1-beta/Lipase member I at 3.9 μg/mg of cell protein, and has Genome polyprotein (Dengue virus/DENV) at 6.9 μg/mg of cell protein. Lipoamidase is not present in this cell, and lipoamidase seems to be not under the control of virus.

Hepatoma HepG2 (cultured without fucoidan) has Lipoamidase/human milk lipoamidase/CEL/BSSL at 15.0 μg/mg of cell protein. This hepatoma cell has no phospholipase.

Healed hepatocyte HepG2 (cultured with fucoidan) has Lipoamidase/human milk lipoamidase/CEL/Carboxyl ester lipase/BSSL/Bile salt-activated lipase at 7.4 μg/mg of cell protein. This healed cell has Platelet-activating factor acetylhydrolase IB subunit alpha/Platelet-activating factor acetylhydrolase/PAF-AH at 0.9 μg/mg of cell protein. This healed cell has activating virus of Influenza A virus/FluA and FluC at 3.62, Dengue virus/DENV at 5.2, and Human herpesvirus/HHV (except BoHV) at 13.9 μg/mg of cell protein, respectively. Total up-regulating virus is present at 22.72 μg/mg of cell protein. This cell has repressing virus of Mumps virus/MuV at 2.2, and Variola virus/VARV at 0.65 μg/mg of cell protein, respectively. Total repressing virus is present at 2.85 μg/mg of cell protein. The activating virus is higher than repressing virus at 19.87 μg/mg of cell protein. This case is not clear for hegemony of virus.

Liver tissue and serum has no serine-type lipoamidase. Therefore, serum lipoamidase activity is due to thiol-type human serum biotinidase/lipoamidase.

Liver tissue (with pseudo-liver cancer) has Platelet-activating factor (PAF) acetylhydrolase 2, cytoplasmic/Serine dependent phospholipase A2 at 1.9 μg/mg of tissue protein. This tissue has activating virus of Herpes simplex virus 1/HHV-1 at 40.4, Human cytomegalovirus/HHV-5 at 2.1, Dengue virus/DENV at 5.2, and FluA plus FluC at 3.62 μg/mg of tissue protein, respectively. Total activating virus becomes to be 51.32 μg/mg of tissue protein. Repressing virus is present ; i.e., Mumps virus/MuV at 1.8, VACV at 14.2, and Measles virus/MeV at 52.7 μg/mg of tissue protein, respectively. Total repressing virus becomes to be 68.7 μg/mg of tissue protein. Repressing power of MeV seems to be week.

LC tissue (with leprosy) has no phospholipase.

This tissue has activation virus of Flus at 8.3, and HHVs at 19.6 μg/mg of tissue protein, respectively. Total activating virus becomes to be 27.9 μg/mg of tissue protein.

This tissue has repressing virus of VACV at 20.0, Sendai virus/SeV/HVJ at 13.3 μg/mg of tissue protein, respectively. Total repressing virus becomes to be 33.3 μg/mg of tissue protein. Repressing power of VACV and SeV seems to have a hegemony, and repressing power becomes to be 5.4 μg/mg of tissue protein higher that activatining virus.

HCC tissue (with PBC) has Phospholipase C-beta-2/PLC-beta-2 at 18.2 μg/mg of tissue protein. This tissue has activating virus of Newcastle disease virus/NDV at 7.5, FluA plus FluB at 6.8, and HHVs at 10.2 μg/mg of tissue protein, respectively. Total activating virus becomes to be 24.5 μg/mg of tissue protein. Repressing virus of Vaccinia virus/VACV is present at 8.6 μg/mg of tissue protein. The inducting power is larger at 15.9 μg/mg of tissue protein.

LC tissue (named as No.6) has Phospholipase C-beta-1/PLC-I at 2.1, Phospholipase C-beta-2/PLC-beta-2 at 0.55, and Phospholipase C-gamma-2/PLC-IV at 12.2 μg/mg of tissue protein, respectively. Total phospholipase becomes to be 14.85 μg/mg of tissue protein. This tissue has activating virus of HHVs at 36.3, FluA at 1.7, and DENV at 0.31 μg/mg of tissue protein, respectively. Total activating virus becomes to be 38.31 μg/mg of tissue protein. Repressing virus is present as follows; i.e., Simian virus 41/SV41at 20.1, and Vaccinia virus/VACV at 7.7 μg/mg of tissue protein, respectively. Total repressing virus is present at 27.8 μg/mg of tissue protein. Avtivaion power is present at 10.51 μg/mg of tissue protein.

HCC tissue (No.6) has Esterase D/S-formylglutathione hydrolase at 0.11, Pancreatic triacylglycerol lipase/Pancreatic lipase at 0.58, and Proactivator polypeptide/Prosaposin at 38.5 μg/mg of tissue protein, respectively. Total phospholipase becomes to be 39.91 μg/mg of tissue protein.

This tissue has activating virus of HHVs at 34.0, Flus at 6.97, and DENV at 19.8 μg/mg of tissue protein, respectively. Total activating virus becomes to be 60.77 μg/mg of tissue protein. This tissue has repressing virus of Murine respirovirus/Sendai virus/SeV at 4.9, and Vaccinia virus/VACV at 24.5 μg/mg of tissue protein, respectively. Total repressing virus becomes to be 29.4 μg/mg of tissue protein. Activaing power is present at 31.37 μg/mg of tissue protein.

Serum of Gait disorder patient has Membrane-associated phosphatidic acid-selective phospholipase A1-alpha/Lipase member H at 5.8 μg/mg of serum protein. This serum has activating virus of Human herpesvirus 6/HHV-6 at 4.4 μg/mg of serum protein.

Serum of light Gait disorder patient has PAF acetylhydrolase/Platelet-activating factor acetylhydrolase IB subunit gamma at 2.6 μg/mg of serum protein. This serum has activating virus of Human herpesvirus 6/HHV-6 at 2.1 μg/mg of serum protein.

It is interesting that Gait disorder may be linked to the appearence of phospholipase in serum, and the presence of HHV-6. Therefore, membranes of cerebellum of the gait disorder patients seem to be injured.

Further, it seems that cancer tissues have higher phospholipases than non-cancer tissue. Thus, the reduction of membrane structures in cancer cells is the true marker of cancer, and up-regulations of cancer marker protein are not present. Up-regulation of Fucosylation to glycoproteins is another important cancer marker, though fucosylation decreases membrane structures is not proven yet. This finding in phospholipases is consistent to the paper "HepG2 Fucoidan".

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

Remark: You will surely find useful information in the paper mentioned by Patrick. However, using a kinetic method as you are doing makes more sure you are measuring in the linear range. Though I often use end-point methods also, one just has to be careful.