Hi there,

Relative activity is the ratio between the activity of your sample of interest and the activity of the control sample and therefore expressed as a percentage.

There is a free guide here. It uses ATPase as an example enzyme but the basic principles of how to work out enzyme activity apply generally. Hope it helps.

https://www.innovabiosciences.com/guides/enzyme-guides/enzyme-unit-definitions-assay-design/

Hi there,

Relative activity is the ratio between the activity of your sample of interest and the activity of the control sample and therefore expressed as a percentage.

Each of the enzymes needs to be assayed by a method specific to that particular type of enzyme. I believe chromogenic substrates and methods exist for each of those enzymes. Of course, there are many sub-types within each of the three general types, so for example, there are both exo-amylases (beta-amylase, glucoamylase, etc) and endo-amylases (alpha-amylase, etc) that will act differently on each substrate.

I do not understand the question exactly. Are you trying to compare the activities of the enzymes in a cell/tissue extract?. If so you can express the activity of each enzyme in terms of protein concentration. Estimate the activity of each enzyme under saturating substrate concentrations and then measure the protein concentration of the extract. Each enzyme activity can then be expressed per mg of protein. These are the specific activities and can be useful for comparing the activity of the various enzymes in the system.

Peter

Relative activity of enzyme if you study different factors affecting enzyme for example pH or ligands to detect inhibitor or activator of that activity.

You have to follow these steps

1- Plot standard curve of substrate concentration or product concentration versus absorbance

2- Calculate the slope of that curve

3- Determine the enzyme activity now you have just absorbance and to convert into enzyme activity divide over slope then on time of incubation.

4- You will get different enzyme activities according to factor you study

5- To calculate the relative activity just divide the enzyme activity in the presence of factor over that in its absence.

Good luck

Hi

It is a little difficult to work out exactly what you need but these are my comments.

When you speak of relative activity it refers to activity relative to some standard or control. This might be the activity in the presence of series of concentrations of inhibitors compared to an uninhibited control, for example.

The enzymes you mention can be assayed by monitoring a change in absorbance using a chromogenic substrate (although other methods are possible). For a spectrophotometric assay the common output for activity measurement is in the form of Absorbance Unit change per minute (AU/min).

Dividing the "inhibited" rate by the "control" rate will give a fractional activity or relative activity.

Multiplication by 100 will convert to a percentage activity. This is typically plotted with the control indicated at 100% and the other activities as a fraction (%) of the control rate.

Thank you all for your gentle support. Actually I am working on three different enzymes purified from a plant seed. I have plotted the standard curve for each individual enzyme but I am not sure what next to do. I searched out different research papers but none of them gave the complete calculation or the way they calculated the enzyme activity. Then I started with Sir Nick Jee guide that helped me a lot. I also plotted the standard curve of all the enzyme but was in confusion what to do next.

Thank you all.

Mr. Gary and Mr. Liger, I have determined the relative activity of the effect of inhibitors on enzymes in the same way as you both have mentioned.

And last, to Thoria Donia, can you further elaborate the third point what you have mentioned.

Mr. Greg and Mr. Peter I ask you both that specific activities and enzyme activities are the same terminologies or are they different?

Thankyou

Enzyme activity is measured in Units (or sometimes katals). A Unit is typically defined as the amount of enzyme that will convert one micromole of substrate or create one micromole of product in one minute, at the optimum conditions for the enzyme. Specific activity is defined as the number of enzyme units per weight or volume of the preparation. Specific activity can be used to measure the concentration or purity of the enzyme during a purification procedure. For example, if your Units of enzyme activity per mg of protein increases, you are successfully purifying your enzyme. (For a definition of katal, see Wikipedia.)

Enzyme activity is given in units per ml (U/ml), which is the same as micromol per min per ml. i.e 1 unit = 1 umol substrate converted per min.

Specific enzyme activity is very different (usually abbreviated to 'specific activity') has it has nothing to do with volume.

Specific activity is given in U/mg. To get this value you divide the activity (U/ml) by protein concentration (mg/ml), hence U/mg.

If you dilute your enzyme 1 in 2, you have bigger volume but your specific activity has NOT changed, as both numerator and denominator in the equation above have halved. The number of units is the same too. What HAS changed though is your activity.

Thank you Greg and Gee. I am very grateful to both of you.

I would like to express my opinion. Determination of the enzyme kinetics including V or Vmax should be performed by the HPLC-photometric method (please see file; J Chrom B rat BIN LIP Km). Precise determination of V is only achievable by using sensitive and quantitative HPLC-Surfactant-SEC-photometric assay (please see file; HPLC-Surf-SEC protein determination method).

We have recently found that HPLC-photometric fucoidan determination gives c.a. 3-fold higher value than the direct-use of photometer (please see Fig. 10 of file; JCB Fucoidan transport).

I wish you good luck.

Good Afternoon all.

First, thank you for all your informations.

I work on lipolytic activity of microalgea and i search these activity in different cell fractions using a paranitrophenol substrat....i want to calculate the relative activity %. But i don't know if it is possible!!: i measured the O.D of product per min of a commerciale enzyme as "positif control" and the enzyme present in each cell fraction "my samples". And i measured the protein concentration on algea suspension not on each cell fraction.

Can i measure the relative activity % using : the paranitrophenol product concentration of the commerciale enzyme and the enzyme of my samples?

Should i measure the protein concentration of each cell fraction or it's ok if i use the protein concentration on algea suspension used for this experiment?

Thank you for your help

I think that you need the protein concentration in each cell fraction because you are determining the enzyme activity in each fraction nand I assume that you wish to compare the specific activity (units/mg protein) in each fraction.

Peter

Thank you for your answer.

I just want to compare the relative activity in each fraction....but when i saw this article i didn't know why they measure the protein concentration and how they calculate the raltive activity ?

Article Cell surface and cellular debris-associated heat-stable lipo...

I think the specific activity, we measured it just when we have a pure enzyme not the enzymes in cell fraction..is it correct?

Hello everyone,

I am looking for a lipolytic activity in different cell fractions using a chromophore substrate (50µL) on microplate reader 96 well:

Vs is the volume of my cell fraction wich contain the enzyme =50µL and Vt is the total volume of enzyme reaction = 300µL

1- I have measured the absorbance (Abs) of the product : Abs Vs time (min) " I have an absorbance for each 5 min"

2- I have prepared a standard curve : Concentration of product (µmol/L) Vs Abs

3- Then I have calculated the amount of product released (µmol/L) by the regression using equation of standard curve: Concentration of product released (µmol/L) Vs Time (min),

I found many propositions to how we calculate the Enzyme activity; my question is what should i use to calculate Enzymatic activity (U/L = µmol/L/min) ?

A - U/L= Vt/Vs * 106/ ε.l * Δ Abs/Δt : (ε is coeficient of extinction (M-1.Cm-1), (l =?? i don't know if I can use 1 Cm because I did use a microplate 96 well not a cuve), Vt=300µL, Vs=50µL

B - Or just divide each Concentration (µmol/L) by Time (min) ? Δ Concentration/Δt * Vt/Vs

I want to compare the Enzymatic activity of two Cell fractions, how should I do? Can I choose a delta of concentration/delta of time (the same points of time for two Cell fraction) ?

Thank you for your help.