Basic Assay Method Switch on the spectrophotometer, set the wavelength to 340nm. Place a blank, Tris-filled cuvette in the spectrophotometer and zero using the CAL button. You just need to do this once. a) For the assay, pipette the following into a cuvette: Tris buffer, 100mM pH 7.4 2.7 mL (may differ if adding inhibitors) NADH, 6mM 0.1 mL LDH purified from heart or skeletal muscle 0.1 mL b) Mix well with a pastette and place in the spectrophotometer. Select start on the laptop. c) Add to the cuvette 0.1ml, 30mM sodium pyruvate, pH 7.4; mix well immediately with a pastette and record for 3 min. d) The easiest way to measure a rate from a recording is to note the absorbances at two points separated by one minute (make sure the recording is linear over that section) then take one absorbance from the other to give Ab change/min. Investigations We have available to us… Two LDH isoenzymes (bovine muscle and bovine heart) Oxalic and oxamic acids (potential inhibitors of the enzyme) Buffers of varying pH Each group of 3 students will be assigned one of the following questions … How do the Km values for muscle and heart compare? How do the pH optima for muscle and heart compare? Is oxalic acid an inhibitor of muscle LDH and, if so, what is its Ki? Is oxalic acid an inhibitor of heart LDH and, if so, what is its Ki? Is oxamic acid an inhibitor of muscle LDH and, if so, what is its Ki? Is oxamic acid an inhibitor of heart LDH and, if so, what is its Ki? Analysis Rate calculations (all investigations) In the reaction system used, a fixed amount of enzyme (typically 0.1 ml of stock) was followed in a fixed final volume (3 ml) and other factors affecting the rate of the reaction were varied (eg pH, substrate concentration, inhibitor
Basic Assay Method Switch on the spectrophotometer, set the wavelength to 340nm. Place a blank, Tris-filled cuvette in the spectrophotometer and zero using the CAL button. You just need to do this once. a) For the assay, pipette the following into a cuvette: Tris buffer, 100mM pH 7.4 2.7 mL (may differ if adding inhibitors) NADH, 6mM 0.1 mL LDH purified from heart or skeletal muscle 0.1 mL b) Mix well with a pastette and place in the spectrophotometer. Select start on the laptop. c) Add to the cuvette 0.1ml, 30mM sodium pyruvate, pH 7.4; mix well immediately with a pastette and record for 3 min. d) The easiest way to measure a rate from a recording is to note the absorbances at two points separated by one minute (make sure the recording is linear over that section) then take one absorbance from the other to give Ab change/min. Investigations We have available to us… Two LDH isoenzymes (bovine muscle and bovine heart) Oxalic and oxamic acids (potential inhibitors of the enzyme) Buffers of varying pH Each group of 3 students will be assigned one of the following questions … How do the Km values for muscle and heart compare? How do the pH optima for muscle and heart compare? Is oxalic acid an inhibitor of muscle LDH and, if so, what is its Ki? Is oxalic acid an inhibitor of heart LDH and, if so, what is its Ki? Is oxamic acid an inhibitor of muscle LDH and, if so, what is its Ki? Is oxamic acid an inhibitor of heart LDH and, if so, what is its Ki? Analysis Rate calculations (all investigations) In the reaction system used, a fixed amount of enzyme (typically 0.1 ml of stock) was followed in a fixed final volume (3 ml) and other factors affecting the rate of the reaction were varied (eg pH, substrate concentration, inhibitor
EnzymeDataAnalysis.docx