Enzymes as analytical reagent

Enzyme analysis
Enzymology
Enzyme Technology

1. Enzymes as an A nalytical Reagent RAJKUMAR AGHERA IMSC BIOTECHNOLOGY 161127302001 Parul Institute Of Applied Sciences Parul University, P.O. Limbda , Ta: Waghodia , Vadodara 391760

2. What is Analytical Reagent? Analytical : it means to detect something with the help of the substance. Reagent : it is a compound or mixture used to detect the presence or absence of another substance By detecting change in color of compound. By detecting change in concentration of compound.

3. Why Enzymes As Analytical Reagent? Enzymes catalyses reaction Substrate specific Product specific Enzymes are extremely valuable as analytical reagent due to its specificity and selectivity to catalyses reaction.

4. Principles Of Enzyme Analysis There are three methods for enzyme analysis End-point method Kinetics method Immunoassays method

5. End-point Method This method is also called as total change and equilibrium methods. This method is only used for analysis of substrates. Analysis should be performed under conditions Where the concentration of added enzyme is high. While the concentration of substrate should be low. It is enough to be first order reaction with respect to the substrate.

6. End-point Method Initially no products are formed, so the concentration of all products at equilibrium will be dependent only on the concentration of the substrate being analyzed. Therefore, if one of these product concentration can be measured and suitable calibration experiments performed, then initial concentration of substrate may be calculated.

7. End-point Method For any reaction S-P, the rate of reaction at time T, - d[S]/ dt is given by –d[S]/ dt =k[S] , where [S] is the concentration of S at time T, and k is constant. By integration. Where [S ] is the concentration of S at zero time.

8. End-point Method For an enzyme catalyzed reaction S-P which goes to completion in the absence of any back reaction, a steady state may assumed to exist through out. Therefore rate of reaction at time T, d[S]/ dt is given by, At very low substrate concentration [S] is less than K m and so ([S]+K m )=K m , therefore,

9. End-point Method By comparison to the general rate equation for a first order reaction it is seen that K= V max / K m so that, This calculation applies only to those enzyme-catalyzed reaction which are irreversible

10. End-point Method To reach to a position of equilibrium, different methods are applied for completion. Trapping the product : It will continually disturbing the equilibrium. E.g. aldehydes and ketones are trapped by use of hydrazine. Use of analogues of second substrate : It displace the equilibrium in the direction of completion e.g. acetyl pyridine adenine dinucleotide (APAD) has this effect if used in place of NAD to oxidize substrates. Use of regenerating system : It converts a co-product to co-substrates.

11. End-point Method Pyruvate and LDH might be added to reform NAD from NADH, as in the Glutamate Dehydrogenase (GDH) system for the estimation of glutamate.

12. End-point Method An enzyme catalyzed reaction being used for substrate analysis by an end point method should have a product whose concentration can easily be determined. If this will not occur then the reaction may coupled to another to enable the analysis to be performed. A product of the primary reaction acting as substrate for the indicator reaction. E.g.

13. End-point Method Under these condition the rate of primary reaction is……

14. End-point Method This ensures that the B being formed from A is quickly converted to C. If the coupled procedure is satisfactory, the final concentration of C (or of a co-product of the indicator reaction) is a measure of the initial concentration of A.

15. Kinetic Method By using steady state kinetic methods, the initial velocity of the reaction is determined at fix temperature and pH; and it is used to calculate the concentration of substrate being investigated. At low substrate concentration, there is a linear relationship.

16. Kinetic Method Enzyme inhibition can be determined by this method.

17. Kinetic Method Irreversible inhibitors gives 100% inhibition if they are presented in excess. But reversible inhibitors do not give complete inhibition. The inhibitor concentration giving 50% inhibition, under specified conditions, is termed as I 50 value.

18. Immunoassay Method Enzymes are very useful in the detection of Ag or Ab present in the sample. For these, RIA, ELISA, EMIT techniques are used. RIA (Radio Immunoassay) The samples to be analyzed is mixed with Ab and with small amount to be labelled antigen. This allowed to come to equilibrium.

19. Immunoassay Method ELISA ( Enzyme Linked Immunosorbent Assay) The Ab is immobilized on microtiter well. A sample containing Ab is added and allowed to react with immobilized Ag.

20. Examples

21. REFRENCE ENZYMES: Biochemistry, Biotechnology, Clinical Chemistry Second Edition By: Trevor Palmer & Philip Bonner

22. Thank You