Hi, Ravi, your question is very interesting, indeed! It is not so easy to compare different methods, because the normalization were done per different parameter. The most relaible is to calculate activity per cell. however, in many case the calculation were done per fresh weight, per protien etc. So, you can compare only results which normlaized by same way.

Good luck!

Enzyme activity in itself is not an absolute measure (everyone defines what they want to call 'activity'). This makes comparison difficult. I would advise you stick to only one of the methods, then you can compare different samples. Different methods will always give different activities for the same sample.

We are, in most cases, interested in "specific enzyme activity", i.e. enzyme activity related to mass of protein. We found out that the values determined this way in many cases are values that do not reflect the real activity in the cell. This appears to be caused by the fact that enzyme activity measurements are performed in artificial systems. This appears to be true especially for enzymes activity that are located inside the cell. It was sugggested that these deviations are caused by the fact that the composition of the immediate surroundings of an anzyme within the cell (e.g. concentration of Ions, properties of water organization very close to the active center of the enzyme, etc) are not properly simulated by measurements in artificial systems. In conclusion, the advice of Taras Pasternak appears to be the most reasonable one.

Frank, Tara,

It seems to me that Ravi's problem is not being addressed here: he measures activity for the same sample with different methods (titrimetric, spectrophotometric, colorimetric) and gets very different activity units. Meaning if these values are normalized against the dry biomass the results will still be different for the same sample. I foresee this problem (inconsistency in using two methods to determine the same activity) in a work we are doing in two different labs, please help address it if u can.

A question for cases like these: can deviations from a mean value of enzyme activity be observed in a series of experiments with the same sample and the same method?

An answer to the question "How do you compare the results of enzyme activity estimated by different methods?" is that it is not safe from a definition and from a scientific point of view to compare activities measured by different methods.

The reason is due to the definition of one international unit of enzyme activity.

The DEFINITION is the following:

One unit is the amount of an enzyme product that will catalyze the transformation of 1 micromole of SUBSTRATE per minute under standard conditions of TEMPERATURE, OPTIMAL pH and OPTIMAL SUBSTRATE CONCENTRATION.

There are variations on all the mentioned parameters including the amount of enzyme product. If the amount of enzyme product is 100 % pure enzyme protein we call the activity specific.

For internal comparable assays so-called process substrates and process conditions are recommendable to use together with internal standard samples specific enzyme products under testing.

Enzyme activity measured by different method but using THE SAME substrate at THE SAME concentration and under THE SAME other condition (including enzyme concentration, pH, temperature, buffer) should be the same. If it's not so, you use wrong coefficient between measured parameter and product/or substrate concentration. If any of the conditions vary, you'll get different results. If you need to compare methods - you should use the same conditions. In case if enzyme concentration should be different, you can use specific acitivty, normalizing on [E]. All the other conditions should be constant, as velocity of the reaction, catalyzed by any enzyme, depends on substrate structure, substrate concentration, pH, temperature, sometimes buffer composition.

I agree with Anna Bacheva. But see for example the attached paper by Chung et al. from 1971.

Thank u all for the answer .

I always learned that "a lower Km of an enzyme for the same substrate means, a higher efficiency". Is there a flaw in that understanding?

Ravi Mohan ,

I always asked myself the same question. Different methods using different substrates lead to results of very different activities, and are often impossible to compare. I had the same problem with lipase and recently with asparaginase. Thus, we compared 4 different methods used for the quantification of asparaginase and after the reaction of each method, and the amount of product formed was quantified by HPLC. As an additional validation, I also measured the amount of substrate converted in each method. In this way I obtained a percentage value of how much each method quantified more or less when compared to a standard method (in this case was HPLC). If you have more interest, I suggest you read the article (Article A critical analysis of L-asparaginase activity quantificatio...

Hi, Agnes,

actually, you need to compare not absolutely value, but how different tretaments/mutation affect relative changes.

For example, you can compare did similar treatments changes enzyme activity in the same direction and similar magnitide.

It is quite easy to do and you can avoid problem with calculations.

My best wishes!

Yes absolute values may differ with different methods used for estimation of the same enzyme. It is always better to estimate the amount of product formed that also during initial stages of the reaction means initial velocity measurement where linear response is obtained.

.I fully agree with the answer of Taras Pasternak

Hi, Taras Pasternak

I also agree with what you said: in many cases only relative activity is enough. However, in the specific case of my publication that I commented, it was necessary to know the absolute values ​​of the enzyme since I worked with the production of the same. For this it was necessary to measure the activity of the enzyme in different fermentation media, and therefore I had to use different methods. In this case, it was extremely necessary to find a conversion factor between the different methods I used, because I needed to compare activity values, and in this case the relative activity gave me an erroneous value.

Thank you very much for the answer,

Best regards

Dear Agnes,

thanks for the answer. About absolute quantification: in this case a primary important to quabtify per cells. Some method use quantification per protein, some per FW, etc.

I think that most suitbale is to normalize per cells (or per DNA contents).

In that case comparison between different methjod will be more precise.

My best wishes!

One has to compare the method of enzyme activity by validating the method developed, sensitivity of the method and specificity of the method