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BMI_Lab5_EnzymeKinetics - Biomed Lab#4 Enzyme Kinetics...

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Unformatted text preview: Biomed Lab #4 Enzyme Kinetics Objectives To learn proper use of a micropipettor To quantify the pnitrophenol (pNP) formed by the enzymatic reaction of acid phosphatase using the substrate p nitrophenol phosphate (pNPP). To understand enzyme kinetics and determine the Km and Vmax of acid phosphatase enzyme. Introduction Enzymes are proteins used in biological reactions Substrate (S) + Enzyme (E) ES complex E + Product (P) http://en.wikipedia.org/wiki/Enzyme Introduction Catalyst accelerate chemical reactions by a million times Renewable are not permanently changed or used up Optimal conditions require certain pH, Temp substrate concentration Specificity for substrate Acid phosphatase Works in acidic conditions to hydrolyse phosphate linked molecules (lysosomes) Amylase Glucose (duodenum) Introduction Approximately 3000 enzymes classified Grouped into six main classes according to the type of reaction catalysed Hydrolases Cleavage reaction in presence of water Phosphatases Hydrolyse ATP (Acid Phosphatase) Glycosidases Hydrolyse sugars ( alpha Amylase) Effects of Substrate Concentration With a fixed amount of enzyme, an increase in the quantity of substrate [S] will initially increase the rate of reaction [V0] since more substrate will bind to more enzyme. This is known as Initial Velocity increase in substrate shows a proportional increase in reaction velocity (rate of formation of product) Effects of Substrate Concentration If you continue to add substrate without changing the enzyme concentration, all the enzyme sites will get saturated with substrate, till there is no free enzyme for the substrate to bind. The reaction velocity has reached a maximum velocity [Vmax] increase in substrate NO proportional increase in velocity (rate of formation of product) The Michealis Menten Equation When the velocity (Vo) of an enzyme catalyzed reaction is plotted against the concentration of the substrate [S], a rectangular hyperbola is obtained. The Vmax, Km and [S] values can be substituted in the equation for a hyperbola to derive the Michealis Menten Equation Vo = V max [S] Km + [S] (no need to memorize the formula) Initial velocity Steady state Note: The reaction velocity will continue at a steady state until it starts to decline as the substrate is used up (enzyme sites become available but substrate has decreased) Part A: Proper use of micropipettor Today's Experiment : Pipetting stop and ejection stop Setting the volumes correctly 1ml = 1000 ml Tip attachment and Tip release button Pipetting and releasing solutions Sources of error Tip disposal Part B: Hydrolysis of pNpPO4 with Acid Phosphatase enzyme Today's Experiment : OH Acid Phosphatase enzyme NO2 NaOH O- NO2 Part B: Enzyme Assay Today's Experiment Label 6 test tubes 1 through 6 Follow instructions & Table 1 listed in lab manual. First incubation 15 mins with pNPP and citrate buffer to bring mixture to optimal temperature Add enzyme and incubate for reaction to occur for 30 mins in a 370C in incubator Read absorbance at 400nm in the spectrophotometer. Part C pNitrophenol Standard Curve Today's Experiment: OH NaOH O- NO2 NO2 Note: Last week you determined max ionized p nitrophenol at basic pH (400nm?) Part C pNitrophenol Standard Curve Today's Experiment: Label 7 test tubes 1 through 7 Follow instructions & Table 2 listed in lab manual. Read absorbance at 400nm in the spectrophotometer. Plot standard curve. Absorbance vs concentraion of pNitro phenol Find the pnitrophenol formed in Part B from this standard curve by extrapolating for the absorbances Standard curve for phenol Conc for known abs P-Nitrophenol concentration (mM) Today's Experiment Part B: Determination of Km Vmax PNitrophenol ( M) / 30mins Vmax Km PNitroPhenyl Phosphate concentration (mM) ...
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