How do i define the exact substrate concentration for 1 U of enzyme.
Say my protein concentration after purification is 1mg/ml. If i take 500µl of it (which has 500µg) to convert the substrate...
How should I estimate how mush substrate to start with? 500µmol of substrate or 200 or 100?
Or
Should I directly have 500µl of protein in 1mg of substrate and calculate how much substrate has been converted within 1mg?
How do I know how much µmol is converted in 1 mg? explain
What you usually do is to define how much enzyme is required to convert X amount of substrate to product over something like a minute, under specific condition say 1 mg/ml substrate in your buffer at a given assay temperature.
I strongly suggest high substrate concentration as this parameter is most useful when the enzymes are saturated.
If you are using absorbance as a measure of product formation we can do the following
Say we define 1 unit as the amount of enzyme required to generate an absorbance of 1 per minute in the initial linear phase of catalysis with 2 mg/ml substrate in your buffer.
Then you should measure a few points with different enzyme concentration on the x axis and the speed of conversion on the y-axis. plot this and do a linear regression and calculate x when y = 1/minute. this is the molar concentration required for 1 unit recalculate this concentration to mg of protein and you can now tell how many units/mg of protein you have. Which is an estimate specific activity on a given substrate, though it should be stressed that proper kinetics are preferable if you can do them instead.
Hope this was helpful.
One unit of a given enzyme is the amount of enzyme that converts one micromole of a substrate to it's product per minute under standard conditions. The conditions may be temperature, pH and molarity of the assay buffer and shaking, if done.
Another way of determining the enzyme e unit is to perform reactions using known about i.e. units of a commercial enzyme, and make it to react with a chromogenic substrate to generate a coloured end product whose absorbance is measured spectrophotometrically. A reference plot is calibrated between known amount of enzyme and the absorbance. use this profile to find out unknown amount of an enzyme in a given sample or culture broth.
Xin Pin Hi
1 U (μmol/min) is defined as the amount of the enzyme that catalyzes the conversion of one micromole of substrate per minute under the specified conditions of the assay method.
The specified conditions will usually be the optimum conditions, which including but not limited to temperature, pH and substrate concentration, that yield the maximal substrate conversion rate for that particular enzyme. In some assay method, one usually takes a temperature of 25°C.
regard
Emil G P Stender "recalculate this concentration to mg of protein and you can now tell how many units/mg of protein you have."
For Eg. If substrate concentration is 3mg/ml in PBS buffer with Enzyme conc. as follows: 500µg/100µl, 750µg/100µl, 1000µg/100µl, 1250µg/100µl, 1500µg/100µl, 2000µg/100µl.
If 750µg (x axis) gave 1abs (y axis) in 1 min i.e 0.75mg therefore 1U = .75mg. If my total protein conc. is 5mg/ml I have 6.6 units/ml of enzyme?
Why is it expressed as units/mg?
Specific activity: U(µmol/min)/mg
Hi Pin,
You can define the unit by treating minimum concentration of enzyme (through maximum dilution in variable manner) which liberate maximum level of product from substrate with constant concentration and optimum level factors in enzymatic reaction. While increasing the maximum dilution of enzymes there by can make the substrate which fully saturated in the active site of the enzyme with maintaining essential factors at optimum level (pH, temperature, incubation time, etc.,). Calculating Km, Vmax & turn over value, specific activity will give potential of enzyme in catalysis
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