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Effect of substrate concentration and enzyme inhibitor on enzyme activity

Effect of substrate concentration and enzyme inhibitor on enzyme activity. Determination of Alkaline Phosphatase activity. Introduction :. Phosphatases are enzymes which catalyze the splitting off phosphoric acid from nonphosphoric esters. Two common types are estimated in serum:

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Effect of substrate concentration and enzyme inhibitor on enzyme activity

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  1. Effect of substrate concentration and enzyme inhibitor on enzyme activity Determination of Alkaline Phosphatase activity

  2. Introduction: • Phosphatases are enzymes which catalyze the splitting off phosphoric acid from nonphosphoric esters. • Two common types are estimated in serum: • Alkaline Phosphatase (optimum pH 10) • Acid Phosphatase (optimum pH 5 – 6)

  3. Alkaline Phosphatase • Purified forms from different sources undergo 3 types of activity: • 1- Hydrolytic R-O- P + HOH ROH + H3PO4 • 2- Phosphotransferase R-O- P + R`-OH R- OH + R`- O- P • 3- Pyrophosphatase: R-O- P -O- P -O- R` + HOH R-O- P + R`-O- P

  4. Sources: • Osteoblasts in the bone • Bile canaliculi in liver • Small intestinal epithelium • Proximal tubules in the kidney. • The placenta • Lactating breasts In all these sites, it seems to be involved in the transport of phosphate across membrane • ALP of normal serum is mainly derived from liver (the bone isozyme is absent)

  5. ALP requires metal ions : • Mg2+, Zn2+ and to a lesser extent Mn2+ • Inhibitors of ALP: • Cu2+, Hg2+, EDTA (chelating Mg2+), Phosphate & some amino acids e.g. L-phenylalanine

  6. Principle of the test: • ALP from human serum will hydrolyze the artificial substrate, disodium phenyl phosphate to phenol which will react with 4-aminoantipyrine in the alkaline, oxidizing agents giving a red purple color which can be measured at 520 nm

  7. Objective of the test • In this experiment we will investigate the effect of changing substrate concentration on enzyme activity in presence and in absence of the inhibitor (inorganic phosphate) to identify the type of inhibition.

  8. PROCEDURE: Into 10 test tubes, add the following (in ml):

  9. Calculate ALP activity (v) for each substrate concentration using the following formula: • ALP activity (v) = X 10 = mg of phenol produced/100 ml serum in 15 minutes = King Armistrong (KA) units/100 ml O.D test – O.D control O.D Standard – O.D blank

  10. Calculation of substrate concentration: Substrate concentration = 0.01 M Substrate M. Wt. = 254 Substrate concentration = 0.01 X 254 = 2.54 [S] = substrate conc. X volume = 2.54 X volume

  11. 1. Calculation: • O.D. B = 0.0 O.D. C = 0.04 • O.D. S = [S] = 2.54 X volume -I +I

  12. 2. Type of inhibition:Using 1/V and 1/[S], draw the Linweaver-Burk plot to calculate the Km and Vmax of the reaction in presence and absence of the inhibitor • 1/Vmax(-I) =  Vmax (-I) = • - 1/Km (-I) =  Km (-I) = • 1/Vmax(+I) = Vmax (+I) = • - 1/Km (+I) = Km (+I) =

  13. Vmax is the same in presence of a competitive inhibitor Competitive Inhibitor 1/vi 1/vi + Inhibitor 1/Vmax’ Apparent Km is increased in presence of a competitive Inhibitor No Inhibitor No Inhibitor 1/Vmax - 1/Km - 1/Km1/[S] Non - Competitive Inhibitor Competitive Inhibitor 1/vi + Inhibitor No Inhibitor 1/Vmax - 1/Km1/[S] Uncompetitive Inhibitor

  14. 3. Calculation of the percentage inhibition: V - Vi • % inhibition = ---------------- X 100 V where V = rate without inhibitor, Vi = rate with inhibitor • 4. Does the % inhibition change as [S] increase? • Do the results of this calculation confirm your • conclusions as to the type of inhibition?

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