Dear Catia Mota

to provide more reilable answer i need to know more detail about your enzimatic assay.

In general the activity is the rate with an enzyme is able to convert a certain substrate and the colorimetric or fluorimetric assay follow this convertion by measuring or the increase in the value associated to the formation of the product or the decrease of the value associated with the diseappearing of the substrate.

Now if in your assay, you monitor the increase in the time of an absorbance at 450nm, probably it detect the formation of the product during the time and to associate the absorbance value to an amount, you need to perform a calibration line with this product.

In this way you will know how much product (in umol) correspond to an Absorbance of 1 (for example 1mmol)

therefore if in your measure you find that 1ug of your enzime, in 1 minutes is able to induce an increase of absorbance of 0,2

you can extrapolate that in 1 minute 0,2mmol (that corresponds to 200umol) were converted from 1ug (that are 0,001mg)and therefore

your activity is 200umol/0.001mg= 200.000

dont' take in consideration the numbers that use for the example, those are causal just to explain the idea, but is it possible that your real values are completely different.

good luck

Manuele

It may have to be umol/s. This means how much product has been formed, employing a standard curve and then the enzyme. Maybe try values like U or kcat. You may just leave it as product.

If you are measuring the end product of enzyme reaction.

Prepare a standard curve using different concentrations of product . Developing colour measuring absorbance at 420nm as you are doing for product absorbance measurement.

Keep the volume same as that of the final volume in enzyme assay.

You have a particular amount of mannose in volume same as enzyme assay volume which if say ML.

say 2mg per ML.

convert it to micromoles per ML by dividing it with molecular weight of Mannose and 1000

Now say x micro mole is produced per 10 minutes of enzyme assay .

Divide this by 10 to get micro moles produced per minute Say it is Y

This is say for 0.1 ML of enzyme extract

you have to find out the soluble protein in enzyme extract by Lowry method

find mg protein in volume of extract used that is say 0.1 ML in assay. Now you have activity in 0.1 ML of extract

Protein in 0.1 mg of extract divide activity by protein

unit you get for enzyme is micro mole/min/mg protein

Knowing you are wanting to measure the Enzymatic Activity of a Mannosidase, you should be using a p-Nitrophenyl Mannopyranoside as a substrate, where the product you measure in the reaction is the P-Nitrophenol, which has a specific Extinction Coefficient. You can then simply measure the Activity of your mannosidase in the appropriate buffer system and using the appropriate Stop reagent, to obtain a Delta A405 of a test sample verus a Blank Sample and with Volume of reaction known, dilution of the Mannosidase known, Time of reaction known, and the Extinction Coefficient, obtain your desired umol/min/mg. See example in Attachment.

What you are proposing is a so-called "end-point assay", where the absorbance is measured at 2 time points, 0 min (your blank) and 60 min (your end point). The calculation of activity from the slope of the line through these points has been described by others. However, you are simply claiming that the 2 points are connected by a straight line, there is no evidence for that. Deviations from the straight line can be caused for example by substrate depletion, product inhibition or inactivation of enzyme molecules. This would seriously bias your results. Therefore, you need to plot the results of several time points (5-10, depending on how much variance is in your results), plot absorbance vs time and check for linearity, then use linear regression to calculate the slope and its error (!).

Btw, the old unit of enzyme activity (U = µmol/min) is no longer valid and should be replaced by the SI-unit katal (kat = mol/s, 1 U = 16.7 nkat).

As per the answer to Engelbert Buxbaum I hope the researcher has checked the linearity by plotting activity on Y axis and different period of time of incubation of assay on x axis and checked for linearity of the enzyme reaction by keeping other parameters fixed I.e substrate concentration and enzyme concentration and then selected the time period of incubation used by them over the period showing linearity

That is done to standardise conditions for assay

It is done even at varying substrate concentration and also foe enzyme concentration and keeping other parameters fixed.

Since the researcher is interested in knowing regarding calculation of only enzyme activity it has been described in the previous answer

Please excuse, I mis-labeled it as a End Point Assay, the assay that I was describing and the attached is a colorimetric Assay using the pNP- Substrate. This is a reaction that is run for 5 minutes for both a Blank and Test sample and stopped resulting in the Free pNP that is measured at 405nM.

Vernon L Reisenbichler Thank you for your answer! My goal is to understand if my mannosidase has a1,2 or a1,3 mannosidase activity thus I'll be using Methyl-2-alpha-D-mannopyranosyl-alpha-D-mannopyranoside and Methyl-3-alpha-D-mannopyranosyl-alpha-D-mannopyranoside as substrates. As far as I've read in previous works, if processed there will be mannose release. However, as I couldn't find D-mannose's extinction coeficient I'm trying to find the best way to proceed.

Engelbert Buxbaum Thank you so much for your input! As I am following a protocol from a work published in 2009, I also think that introducing more time points would make my results more reliable. I didn't know that nanomol per min per mg was no longer being used so it's good to know! Thank you again!