Determination of Alkaline Phosphatase 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: Alkaline Phosphatase (optimum pH 10) Acid Phosphatase (optimum pH 5 – 6)
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
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)
ALP requires metal ions : Inhibitors of ALP: Mg2+, Zn2+ and to a lesser extent Mn2+ Inhibitors of ALP: Cu2+, Hg2+, EDTA (chelating Mg2+), Phosphate & some amino acids e.g. L-phenylalanine
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
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.
Into 10 test tubes, add the following (in ml): PROCEDURE: Into 10 test tubes, add the following (in ml): Blank Standard Test Test Without Inhibitor With Inhibitor 1 2 3 4 5 6 7 8 Buffer 1.0 Substrate - 0.25 0.50 0.75 Substrate + Inhibitor Dist. Water 1.1 0.1 Serum sample Mix and incubate the tubes at 37C for exactly 15 min. NaOH 0.5 N 0.8 Na HCO3 0.5 N 1.2 4-amino-antipyrine Potassium ferricyanide Mix and read the tubes against blank at 520 nm
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
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
1. Calculation: O.D. B = 0.0 O.D. C = 0.04 O.D. S = [S] = 2.54 X volume Test tube Sub.volume (ml) [S] 1/[S] O.D.T T- C V = --------- X 10 S – B 1/V 1 0.25 0.635 1.575 2 0.50 1.27 0.787 3 0.75 1.905 0.525 4 1.00 2.54 0.394 5 6 7 8 -I +I
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) =
Non - Competitive Inhibitor 1/vi Competitive Inhibitor No - 1/Km Vmax is the same in presence of a competitive inhibitor Competitive Inhibitor Apparent Km is increased in presence of a competitive Inhibitor Non - Competitive Inhibitor 1/vi 1/Vmax - 1/Km 1/[S] No Inhibitor + Inhibitor 1/Vmax’ 1/vi 1/Vmax - 1/Km 1/[S] No Inhibitor + Inhibitor Uncompetitive Inhibitor
3. Calculation of the percentage inhibition: V - Vi % inhibition = ---------------- X 100 V where V = rate without inhibitor, Vi = rate with inhibitor [S] V Vi % inhibition 0.635 1.27 1.905 2.54 4. Does the % inhibition change as [S] increase? Do the results of this calculation confirm your conclusions as to the type of inhibition?