effect of substrate concentration and enzyme inhibitor on enzyme activity determination of alkaline...
TRANSCRIPT
Effect of substrate concentration and
enzyme inhibitor on enzyme activityDetermination of
Alkaline Phosphatase activity
IntroductionIntroduction::
Phosphatases are enzymes which Phosphatases are enzymes which catalyze the splitting off phosphoric catalyze the splitting off phosphoric acid from nonphosphoric esters.acid from nonphosphoric esters.
Two common types are estimated in Two common types are estimated in serum:serum:
Alkaline PhosphataseAlkaline Phosphatase (optimum pH 10) (optimum pH 10) Acid PhosphataseAcid Phosphatase (optimum pH 5 – 6) (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 :ALP requires metal ions : MgMg2+2+, Zn, Zn2+ 2+ and to a lesser extent Mnand to a lesser extent Mn2+2+
Inhibitors of ALP:Inhibitors of ALP: CuCu2+2+, Hg, Hg2+2+, EDTA (chelating Mg, EDTA (chelating Mg2+2+), ),
Phosphate & some amino acids e.g. L-Phosphate & some amino acids e.g. L-phenylalanine phenylalanine
Principle of the test:Principle of the test: ALP from human serum will hydrolyze ALP from human serum will hydrolyze
the artificial substrate, the artificial substrate, disodium phenyl disodium phenyl phosphatephosphate to to phenol phenol which will react which will react with with 4-aminoantipyrine4-aminoantipyrine in the alkaline, in the alkaline, oxidizing agents giving a oxidizing agents giving a red purple red purple color color which can be measured at which can be measured at 520 nm520 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.
Blank Standard Test Test Without Inhibitor With Inhibitor
1 2 3 4 5 6 7 8
Buffer 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Substrate - - 0.25 0.50 0.75 1.0 - - - -
Substrate + Inhibitor - - - - - - 0.25 0.50 0.75 1.0
Standard - 1.0 - - - - - - - -
Dist. Water 1.1 0.1 0.75 0.50 0.25 - 0.75 0.50 0.25 -
Serum sample - - 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Mix and incubate the tubes at 37C for exactly 15 min.
NaOH 0.5 N 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8
Na HCO3 0.5 N 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2
4-amino-antipyrine 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Potassium ferricyanide 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Mix and read the tubes against blank at 520 nm
PROCEDURE: Into 10 test tubes, add the following (in ml):
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 test – O.D control
O.D Standard – O.D blankO.D Standard – O.D blank
Calculate ALP activity (v) for each substrate concentration using the following formula:
Calculation of substrate concentration:
Substrate concentration = 0.01 MSubstrate concentration = 0.01 M
Substrate M. Wt. = 254Substrate M. Wt. = 254
Substrate concentration = 0.01 X 254 = Substrate concentration = 0.01 X 254 = 2.542.54
[S] = substrate conc. X volume[S] = substrate conc. X volume
= 2.54 X volume= 2.54 X volume
1. Calculation:1. Calculation: O.D. BO.D. B = 0.0 = 0.0 O.D. CO.D. C = 0.04 = 0.04 O.D. SO.D. S = = [S] = 2.54 X volume[S] = 2.54 X volume
Test tube
Sub.volume(ml)
[S] 1/[S] O.D.T T- CV = --------- 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 0.25 0.635 1.575
6 0.50 1.27 0.787
7 0.75 1.905 0.525
8 1.00 2.54 0.394
-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
1/Vmax
- 1/Km 1/[S]
No Inhibitor
+ Inhibitor
1/Vmax’
1/vi
CompetitiveInhibitor
NoInhibitor
- 1/Km
Vmax is the same in
presence of a
competitive inhibitor
Competitive Inhibitor
Apparent Km is increased in presence
of a competitive Inhibitor
1/vi
1/Vmax
- 1/Km 1/[S]
No Inhibitor
+ Inhibitor
Uncompetitive Inhibitor
3. Calculation of the percentage inhibition:3. Calculation of the percentage inhibition: V - ViV - Vi % inhibition = ---------------- X 100 % inhibition = ---------------- X 100
V V
where V = rate without inhibitor, Vi = rate with inhibitor 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?
5. Do the results of this calculation confirm your conclusions as to the type of inhibition?