Principle of Kinetic technique for enzyme testing
Basics of kinetic technique:-
The main goal of the enzymatic assay is to measure the activity of the enzyme not the concentration because the enzymatic reaction is affected by many factors. In this test, the substrates are applied for the enzyme under testing and measure the amount of substrate disappeared or decreased per time intervals or measure the amount of products appeared or increased per time intervals.
Enzymatic reaction depends on some factors; such as:-
- Temperature:- The higher temperature increases the kinetic energy of the substrates and enzyme molecules, leading to increasing the chance for colliding between them and thus increase the rate of the reaction. And the low temperature does opposite action.
- pH:- Hydrogen ions (H+) and hydroxide ions (OH–) change the allosteric site of the enzyme (i.e. the site used to catalyze the reaction) leading to inhibition of the enzyme activity. Each enzyme has a specific Optimum PH at which its activity is optimum.
- Concentration of the enzyme or substrate concentration:- increasing the enzyme concentration (while the substrate concentration is stable) increases the rate of the reaction. Also, increasing the concentration of the substrate (while the enzyme conc. is stable) will increase the rate of the reaction.
From these factors, you can conclude that if the enzyme concentration is high and the substrate concentration is constant, the reaction will be finished quickly and if it was low, the reaction finished slowly. And if the enzyme concentration is high and the temperature and/or pH is not suitable for enzyme activity, the reaction will be slow or will not occur. So, this is what meant by the activity of an enzyme. Therefore, we measure the activity of the enzyme, not its concentration because the enzyme may be there but inactive.
So, to detect the real increment of the enzyme concentration or activity, we should make other factors constant because if they are not constants, the activity is changed and you will get the wrong results of your tests. This means that:-
- The test should be performed at a constant temperature at the optimal temperature of the enzyme.
- The test should be performed at a constant pH at the optimal pH value of the enzyme.
- The concentration of the substrate provided by the reagent should be constant because if the enzyme concentration is high, its activity will be high and hence the reaction will be ended quickly and vice versa.
But how we measure the activity of an enzyme?? This can be done by 2 ways:-
- Measuring the rate of the substrate disappeared or decreased from the reaction per time intervals. Or
- Measuring the rate of products appeared or increased in the reaction per time intervals.
Way 2 is the more accurate. WHY? Because measuring or detecting small changes in products, when its concentration is zero, is easier to measure than detecting small changes when the substrate concentration is decreased.
Note that the kinetic method depends on the absorption at a wavelength in the UV region.
How to measure or calculate it practically?
We know that we measure the activity of an enzyme, not a concentration. Also, we know that we measure the activity of an enzyme by measuring the rate of the product appeared or increased from the reaction per time intervals. This means that there is not any standard solution for the activity of an enzyme. So, we can’t calculate the enzyme activity by the same equation used to calculate the analyte concentration in the colorimetric technique click here.
We will measure the absorbance of products per time intervals. To measure the absorbance per time intervals, you should measure the absorbance more than once per time intervals. Usually, 3 readings of absorbance are measured through 3 mins; one reading every one minute. In each reading, we take ΔA (absorbance difference). Let’s take Alanine Aminotransferase test (ALT test) as an example.
In each reading, the ΔA/min = (Asample/min) – (Ablank/min) but the absorbance value we get from the instrument is for the sample because we make the instrument to read the absorbance of the blank zero. So, ΔA/min = Asample – 0 = Asample
After you get the 3 absorabnce values, you will take the average of the ΔA in the 3 readings:-
The ALT activity is calculated in the sample from the next equation:-
(ε) is the molar absorptivity of NADH at 340nm; equals 6300 (i.e. measuring the amount of light absorbed per mole when the light pass through a path length of 1 cm)
(l) is the cuvette path length; 1 cm
(Vt) the total volume of the reaction; 1.1ml (1.0 reagent +0.10 sample) at 30oC or 1.05ml (1.0 reagent + 0.05 sample) at 37oC
(Vs) the sample volume; 0.1 at 30oC or 0.05ml at 37oC
(106) 1000 to convert from mol to μl.
Could you prove this equation?
The proof is very simple. Do you remember Beer-lamert law? Which is
Absorbance = ε b C
Where (ε) is the molar absorptivity, (b) is the cuvette path length; 1 cm and (C) is the concentration the analyte of activity of the enzyme
Let’s replace (C) with ALT activity and (b) with(l). So,
Now, we should take into account the dilution factor. Do we dilute the sample? The answer is yes we do. Explain how? When you add the reagent to the sample you actually dilute it and you should consider this in your equation. Think what is the difference between dilution with distilled water and your reagent???!!! Of course nothing. BUT HOW CAN WE CALCULATE IT? Click here to see how to make dilutions and calculate the dilution factor.
The dilution factor will be the total volume (Vt) divided by the volume of the sample (Vs). Then the equation will be:-
In the equation above, we see that there is 106 in the numenator. WHY? To know why, you should conclude the unit of measuring the ALT activity. The unit is one international unit per liter U/L.
The units of measuring ALT activity is one international unit (U) per liter. U is the amount of enzyme that transforms 1 µmol of substrate per minute, in standard conditions. Let’s see the unit conversions in the following equation to understand this.
Let’s see the units of each item in the equation:-
- (No units) for absorbance.
- (ml) fro Volume of the sample or the total volume is measured by ml.
- (cm) for (l)
- (liter mol-1 cm-1) for (ε)
So, we should multiply the final equation by 106
Where μmol/min equals one international unit (U), then the measuring unit will be U/L.
AST test (Aspartate Aminotransferase)