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Effect of substrate

concentration and
enzyme inhibitor on
enzyme activity
Determination of
Alkaline Phosphatase
activity

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

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

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) . 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.

g. ALP requires metal ions :  Mg2+. Hg2+. EDTA (chelating Mg2+). Zn2+ and to a lesser extent Mn2+  Inhibitors of ALP:  Cu2+. Phosphate & some amino acids e. Lphenylalanine .

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

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. .

0 1.25 - 0.1 0.8 Na HCO3 0.0 1.0 1.2 1.0 Standard - 1.0 1.0 Substrate - - 0.0 1.5 N 0.50 0.0 1.PROCEDURE: Into 10 test tubes.75 0.25 - - - 0.0 1.2 1.0 1.1 0.8 0.1 0.0 1.0 1.0 1.8 0.5 N 1.0 1.0 1.8 0.0 1.75 1.1 0.2 4-amino-antipyrine 1.8 0.0 1.0 Mix and read the tubes against blank at 520 nm .0 1.0 1.0 1.2 1.0 1.0 1.75 0.0 1.8 0.50 0.0 1.0 1.1 0.8 0.50 0.0 1. NaOH 0. Water Serum sample Mix and incubate the tubes at 37C for exactly 15 min.2 1.2 1.8 0.0 - - - - Substrate + Inhibitor - - - - - - 0.1 0.0 1.25 0.0 - - - - - - - - 1.8 0.2 1.1 0.25 0.8 0.50 0.0 1.2 1.75 1.0 1.0 Potassium ferricyanide 1.2 1. add the following (in ml): Blank Buffer Standard Test Without Inhibitor Inhibitor Test With 1 2 3 4 5 6 7 8 1.1 0.1 0.2 1.1 Dist.

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

54 [S] = substrate conc. Wt. = 254 Substrate concentration = 0.Calculation of substrate concentration: Substrate concentration = 0.01 X 254 = 2. X volume = 2.54 X volume  .01 M Substrate M.

50 1.D.787 7 0.00 2.525 8 1.787 3 0.75 1.27 0.394 5 0.00 2.1.D.635 1.54 0. S = O.D.75 1.D.394 O.04 [S] = 2.525 4 1.27 0.C V = --------.54 X volume Sub.50 1.575 6 0.905 0.905 0.25 0.54 0.0 O. B = 0.25 0. C = 0.635 1.volume (ml) [S] 1/[S] 1 0.575 2 0.X 10 S–B 1/V .T T. Calculation:   +I -I Test tube O.

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

1/Km 1/[S] Non .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 Uncompetitive Inhibitor 1/[S] .Competitive Inhibitor Competitive Inhibitor 1/vi + Inhibitor No Inhibitor 1/Vmax .1/Km .

Calculation of the percentage inhibition:  V .Vi % inhibition = ---------------.905 2. Vi = rate with inhibitor [S] V Vi % inhibition 0.3.X 100 V where V = rate without inhibitor.635 1.27 1. Do the results of this calculation confirm your conclusions as to the type of inhibition? . Does the % inhibition change as [S] increase? 5.54 4.