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Unformatted text preview: 45-*: 98 Experiment 18 Enzyme Kinetics of.E coliAlkalinePhosphatasePart 6Biochemistry 332L--3 26 09Last week you calculated the kinetic parameters for alkaline. phosphatase in the absence of inhibitors This week you will determine the , (inhibitory effects of one of the enzyme s products the phosphate ion usually ).referred to as inorganic phosphateMaterials. , 0 5 mM PNPP solution freshly prepared. -.0 2 M Tris HCl pH 8 01 mM sodium phosphate, Stage 4 Enzyme diluted as last weekBefore Lab1., , -Review from class the material on the Michaelis Menten equation and the.various permutations of it that correspond to different types of inhibitionRecall how to calculateKI. values2., After reading the rest of the procedure fill in the blank slots in the table. (below When filling in the column for the amount of buffer to add to bring it. ), . up to 2 7 mL don t forget that each tube will already have 0 3 mL of PNPP aswell as the volume of Pi.shown in column twoProcedure3.~/ . Dilute the Stage IV enzyme to60 mU 0 2 mL if you do not have any left.from last week4.. . . Number ten 13x100mm test tubes Place 0 3 mL of 0 5 mM PNPP in each ofthe first five tubes and add inorganic phosphate in the amounts shown below:in the second columnTubeL PinmolesPi(Buffer to add step, )5 belowTube(Buffer to add step, )11 below110.0037.2 396.1 792158.0585.2 2427.1 6423306.1133.2 0948.1 4994453.1678.1 9479.1 3475600.2222.1 810.1 25.. -. .Add enough 0 2 M Tris HCl pH 8 0 to bring the volume in each tube up to 2 7. .mL Place parafilm over the top of the tubes and mix6.[( . + . . -+ . Prepare a blank 0 3 mL of PNPP2 6 mL of 0 2 M Tris HCl0 1 mL of 10 N)]. NaOH7., , Add 200 L of enzyme to tube 1 note the time quickly cover with parafilm. , and mix Let the tube stand for exactly three minutes then proceed.immediately to step 88..Stop the reaction by adding 100 L of 10 N NaOH and mix again9..Read the absorbance at 410nm against the blank from Step 610.- . Repeat for each of tubes 2 5Be sure to zero the instrument to the.blank before adding enzyme11.-, . . Repeat the above exactly for tubes 6 10 this time using 0 9 mL of 0 5 mM. . , PNPP in each tube instead of 0 3 mL All other amounts are the same exceptyou will have to recalculate the amount of buffer required to bring the volume. ().up to 2 7 mL column 6 in the table above.12.Take a standard PNP readingCalculations13.Calculate the reaction velocity in nanomoles of product produced per minute...
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