Effect of substrate concentration and enzyme inhibitor on enzyme activityDetermination 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 PhosphatasePurified forms from different sources undergo 3 types of activity:1- Hydrolytic R-O- P + HOH ROH + H3PO42- Phosphotransferase R-O- P + R`-OH R- OH + R`- O- P3- Pyrophosphatase: R-O- P -O- P -O- R` + HOH R-O- P + R`-O- P

Sources:Osteoblasts in the boneBile canaliculi in liverSmall intestinal epitheliumProximal tubules in the kidney.The placenta Lactating breasts In all these sites, it seems to be involved in the transport of phosphate across membraneALP of normal serum is mainly derived from liver (the bone isozyme is absent)

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

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 testIn 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.

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

BlankStandard Test Test Without Inhibitor With Inhibitor 12345678Buffer 1.01.01.01.01.01.01.01.01.01.0Substrate--0.250.500.751.0----Substrate + Inhibitor------0.250.500.751.0Standard -1.0--------Dist. Water1.10.10.750.500.25-0.750.500.25-Serum sample--0.10.10.10.10.10.10.10.1Mix and incubate the tubes at 37C for exactly 15 min.NaOH 0.5 N0.8 0.80.80.80.80.80.80.80.80.8Na HCO3 0.5 N 1.21.21.21.21.21.21.21.21.21.24-amino-antipyrine1.01.01.01.01.01.01.01.01.01.0Potassium ferricyanide1.0 1.0 1.01.01.01.01.01.01.01.0Mix and read the tubes against blank at 520 nm

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 controlO.D Standard O.D blankCalculate ALP activity (v) for each substrate concentration using the following formula:

Calculation of substrate concentration:Substrate concentration = 0.01 MSubstrate M. Wt. = 254Substrate 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 = 0.04O.D. S = [S] = 2.54 X volume-I+I

Test tubeSub.volume(ml)[S]1/[S]O.D.T T- CV = --------- X 10 S B 1/V 10.250.6351.575 20.501.270.787 30.751.9050.525 41.002.540.394 50.250.6351.575 60.501.270.787 70.751.9050.525 81.002.540.394

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) =

3. Calculation of the percentage inhibition: V - Vi% inhibition = ---------------- X 100 V where V = rate without inhibitor, Vi = rate with inhibitor4. Does the % inhibition change as [S] increase?

Do the results of this calculation confirm your conclusions as to the type of inhibition?

[S]VVi% inhibition0.6351.271.9052.54

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