ASSAY OF Km VALUE OF ALKALINE PHOSPHATASE

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ASSAY OF Km VALUE OF ALKALINE PHOSPHATASE EXPERIMENT 4 ASSAY OF Km VALUE OF ALKALINE PHOSPHATASE 2012-09

Aims 1. To understand the effect of the changes of substrate concentrations on the velocity of enzyme-catalyzed reaction. 2. To learn the principle and methods of determining Km value and its role at enzyme kinetics.

velocity of enzyme-catalyzed reaction Principle 〔 E 〕 T B C velocity of enzyme-catalyzed reaction 〔 S 〕 A pH D E F activator inhibitor

Rectangular hyperbola

Michaelis-Menten equation Km is Michaelis constant When V= Vmax/2, Km=[S] the units of Km are the same as substrate concentration’s( mol/L)

Lineweaver-Burk double-reciprocal plot 1 V = Km Vmax × [S] +

pH10

Mixing , incubate in 37℃ water bath for 5 mins. Protocol 1. Add reagents according to the following table. (ml) No. 1 2 3 4 5 6 7 Blank 0.02mol/L substrate 0.05 0.10 0.2 0.4 0.60 0.80 1.00 0.00 pH10 carbonate buffer 0.90 dH2O 0.95 0.40 0.20 Mixing , incubate in 37℃ water bath for 5 mins. Enzyme (0.5mg/ml) 0.1 Mixing immediately, incubate in 37℃ water bath for 15mins exactly.。 NaOH 1.0 4-aminoantipyrene K3Fe(CN)6 2.0 Mixing, place at room temperature for 10 mins, and record A510 A510 8

Conc. of substrate mmol/L Calculation of data Conc. of substrate mmol/L 0.50 1.0 2.0 4.0 6.0 8.0 10 1/[S] 2 1 0.5 0.25 0.167 0.125 0.10 1/ A510 Making a plot with 1/[S] as abscissa, 1/A510 as ordinate 9

According –1/Km, calculate the value of Km Calculation According –1/Km, calculate the value of Km Double reciprocal graph

Demand 1. Two persons cooperate for the experiment 2. Enzyme solution used here is toxic, so carefully to handle it 3. The amount of E and S transferred should be exact 4. The incubation time should be exact as 15 minutes

Next week work To design a project about experiment research To find the optimum pH for saliva amylase How to do? Which method shoul be used? What is the principle? Which kind of solution should be used? What is concentration of a reagent?