Dear Luna,

Both measurements are indirect, as with many enzymatic activities with unknown real substrates/products. Of course, one can always calculate "real" concentrations using appropriate extinction coefficients from recorded kinetics of absorbance. In the case of both POX and PPO we need to add the amount of enzymatic extract representing either known amout of extracted protein or fresh (sometimes dry) mass of tissues to the respective reaction mixture. Then we can follow increase of absorbance at the respective wavelength due to formation of coloured product. After that, we can calculate the amount of product formed per unit of time per unit of mass (or protein) using known extinction coefficients. This is in general, usually each researcher forms his/her own protocol in detail how to achieve this.

Dear Gederts Ievinsh

Thank you very much for your reply.

To consider the substrate concentration, different researchers has used different concentration (For example: 10mM, 20mM, 40mM) even for the same commodity (Mushroom or apple). Is it okay to take any one of this concentration ?

If I need to check for the suitable catechol substrate concentration (in the case of PPO ), standard reference need to be taken as far as I know. Which shall be appropriate reference standard enzyme ?

I hope, my question is clear to you.

Many thanks

Best Regards

Luna

A way to make the standard curve in terms of product concentration is as follows. Prepare solutions with a range of substrate concentrations. Add a sufficient quantity of the purified enzyme, obtained commercially if possible, so that 100% of the substrate is converted to product in a reasonable amount of time. This will be seen as the progress curve leveling off. The change in absorbance representing 100% conversion for each substrate concentration is plotted against the substrate concentration. This is the standard curve.

Polyphenol oxidase

http://www.worthington-biochem.com/ty/cat.html

Peroxidase

http://www.worthington-biochem.com/hpo/cat.html

http://www.worthington-biochem.com/ty/cat.html

Dear Luna,

As for other enzyme activity measurements, we need assay conditions yielding maximum activity. Therefore, we need to use saturable concentration of all substrates. In practice, it can be measured by means of preliminary experiments with equal amounts of enzyme extract, using increasing concentrations of the substrate. Further on, the first concentration yielding maximum enzyme velocity needs to be used. In the case of POX where we have two substrates, this needs to be done in two steps. First we can detrermine appropriate concentration for H2O2 at constant concentration of phenolic substrate (guaicaol?) and then repeat the same for guaicaol with constant concentration of H2O2. One more preliminary experiment is necessary for each new plant object, involving different amounts of enzyme extract at constant substrate concentrations. When ploting these activities expressed on a fresh mass or a protein basis, one needs to get linear response which is only logical. In the case if this relationship is not linear (uusally due to inhibitors in the extract), it is necessary to change extraction buffer or other conditions to eliminate possible inhibitors or even use some Sephadex purification to eliminate low molecular mass compounds. Usually many researchers do not bother themselves with these preliminary experiments but just use some average levels from the literature.

Hi Gederts Ievinsh, Adam B Shapiro, Ashok Kumar Nadda

I appreciate all of your effort to make me clear.

Thank you so much

Best Regards

Luna

Hello !!

I found this formula to calculate enzyme activity 

Activity (U.ml–1) = [(AFsample – AIsample) – (AFblank – AIblank)]/(0.001 × t)

where, AFsample is the final absorption of the sample, AIsample is the

initial absorption of the sample, AFblank is the final absorption of

the control, AIblank is the initial absorption of the control, and t

is the reaction time in minutes.

Is it necessary to make a standard curve or not ? How reliable it would be to follow this formula to calculate the activity without taking into account other factors ?

Reference article attached!!

Looking forward to your suggestions

Best Regards

Luna

In many cases, the "unit" of enzyme activity is based upon an arbitrary definition. In the case of the formula, such an arbitrary unit definition was given in the paper as follows: "One unit of polyphenol-oxidase (U) was defined as the amount of enzyme necessary to cause an increase of 0.001 in the absorption for each minute of reaction time."

There is another meaning of "unit" in enzymology: one micromole of product formed per minute. If you want to convert the absorbance change unit to the micromoles of product unit, you need to find out how absorbance is related to product concentration. This can be done either by making a standard curve or by using the extinction coefficient, if it is already known, for the product.

Dear Adam,

Thank you for your explanation. The problem for me is I am using multi-plate reader instead of a spectrophotometer.  The length of well is unknown as such for spectrophotometer, path length of cuvette is 1 cm. Can I use extinction coefficient available in the literature to calculate the product concentration? 

Many Thanks

You could use the extinction coefficient from the literature if you knew the path length, which in the case of a plate reader is the height of the liquid column at the center of the well. In some models of clear plate, you can measure this in wells at the edge of the plate. Remember from Beer's Law that the absorbance is equal to the concentration x the extinction coefficient x the path length. Extinction coefficients have distance units (usually cm) in the denominator because of this.

Dear Adam 

Thanks a lot!!

Best Regards

Luna

hello i want to ask

how to make standard solution curve for quinone ? what is concentration range to made ? iam so confused

thanks

Choose the desired wavelength. Typically, this is the peak wavelength of the absorbance spectrum. Prepare a range of concentrations in the desired solvent that give absorbances that are within the dynamic range of the spectrophotometer. This may be between 0.1 and 2, for example. To find the concentration range for a new compound, I suggest making a set of 2-fold serial dilutions, allowing you to cover a wide range of concentrations with a small number of samples. This will help you establish the right order of magnitude for the concentrations that can be measured with the instrument. Do not exceed an absorbance of about 2, because that is near the limit of the dynamic range for most spectrophotometers, although some can go higher. The range of concentrations used for the standard curve should be those that are relevant to your experiment, and should be within the dynamic range established previously. The concentrations used for the standards should be evenly spaced, like 0, 10, 20, 30 etc. If everything is working properly, the result should be well fit with a straight line. The slope of the line is the extinction coefficient in Beer's Law.