The question is regarding enzyme specific activity. Is well established that the equation (Delta Abs/time(minutes))/Epsilon(M-1*cm-1)*Pathlength (cm) times the volume (here is the "problem") is used to calculate the substrate/product over time (dS/dT or dP/dT). The outcome is usually expressed in U/ml or micromol*min-1*ml.
Using the outcome in micromol*min-1*ml, dividing this value by the final concentration of the protein in mg/ml , we will have the specific activity, in U/mg or micromol*min-1*mg-1.
The volume used sometimes is just the volume of the reaction, however, some biochemists use a ratio between the reaction volume and the volume of protein inside the reaction. Which one is the most appropriate?
As Dominique Liger said, enzyme's specific activity generally is informed in International Units (U or IU before) . This units are expressed as (micromol /minute) per mg of enzyme. Then, you have take into account the amount of product (or substrate) produced (or used) in the total volume of reaction (lets say 100 microliters) by a precise amount of the studied enzyme (lets suppose here that you put 10 microliters of enzyme solution in the volume of reaction). Besides, you must know the protein concentration (mg/ml; equal to microgram/microliter) ergo you will know the mass of protein catalyzing the reaction you measured. Don`t forget the time.
Hi there,
The specific activity is expressed as the amount of product formed per minute per mg of protein. From the Beer's law you have activity expressed as concentration transformed per minute, so the volume to consider is the reaction volume to express activity as mole per minute. Then knowing the amount of enzyme used in the assay (either mL or mg of enzyme solution ) therefore you can express activity as mol/min/mL(here mL is the unit volume of enzyme solution) or as mol/min/mg just by dividing activity by either volume or mg.
Hi Leonardo. Here in the lab, we express specific activity as mol/min/mg. The volume is just considered to calculate the total activity.
As Dominique Liger said, enzyme's specific activity generally is informed in International Units (U or IU before) . This units are expressed as (micromol /minute) per mg of enzyme. Then, you have take into account the amount of product (or substrate) produced (or used) in the total volume of reaction (lets say 100 microliters) by a precise amount of the studied enzyme (lets suppose here that you put 10 microliters of enzyme solution in the volume of reaction). Besides, you must know the protein concentration (mg/ml; equal to microgram/microliter) ergo you will know the mass of protein catalyzing the reaction you measured. Don`t forget the time.
it doesn't matter as long as you use the same volume of protein concentration. You can even use the total activity and proteins, because the volume will simply cancel out.
The specific activity is simply activity per amount of protein, no matter what units you use for the activity. Let's say we have the activity in U, thus the concentration is in U/ml or U/ul or whatever best suits you and the total activity is in U. Thus for specific activity you'll have U/ml / mg/ml = U/ml or U/ul / ug/ul = U/ug or U / mg (total proteins) = U/mg. The volume I use here is of the enzyme extract, not of the reaction mixture, but technically you could use that one as well, but you'd have to calculate the amount of proteins in the reaction mixture so that seems as extra work for me.
You asked: " reaction volume and the volume of protein inside the reaction. Which one is the most appropriate?" The volume of the protein inside the reaction will eventually thin into the reaction volume. The more important parameter to think of is the total concentration of protein in the total reaction volume because of the importance of mass transport with respect to reaction volumes. Thus, I will prefer to work with the reaction volume.
Dear all, thank you for your time used answering my question. I think that would be simpler to as that to reach the unit U/ml , the volume used is the total volume of the reaction or the ratio volume of the reaction/enzyme volume add. I worked in different places and i realised that you can find this variation, leading to a different outcome. I used to use the ratio volume of the reaction/enzyme volume add. After calculate the Absorbance variation per minute/ epsilon times path length, do you multiply this outcome by the total volume or the ratio?
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