Standard enzyme assay procedure for pyruvate kinase 1. You are provided with a \

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Standard enzyme assay procedure for pyruvate kinase 1. You are provided with a "stock solution", which contains lactate dehydrogenase coupling enzyme), Mg,K, fructose-1,6-bisphosphate (an activator of pyruvate kinase), and NADH-this should be kept on ice. You are also provided with the substrates for the pyruvate kinase reaction, ADP and phosphoenol pyruvate (PEP) also kept on ice Set up the spectrophotometer assay cuvettes as follows Stock Solution ADP (150 mM) Phosphoenol pyruvate (150 mM)20 Water Total volume 720 uL 20 ul l 220 980 pal bate the assay solution at room temperature for 10 min. This allows the assay to come up to temperature. IMPORTANT: The rate of a catalysed reaction is dependent on temperature so this needs to be constant, i.e. do not start the reaction until the assay solution reaches room temperature. 2. Incu 3. Blank the spectrophotometer with distilled water Start the reaction by adding 20 HL of appropriately diluted enzyme (see note below) Mix rapidly but thoroughly by inverting the cuvette, and replacing it in the spectrophotometer as soon as possible. Record absorbance (As4o) at 15 second intervals for as long as required. Record readings for up to 3 min (or until the reaction is complete). The maximum rate should be no more than 0.05 absorbance units per 15'3econds. A quick plot of your readings will give you a better idea of the course of the reaction than a table of numbers. IMPORTANT: Get a demonstrator to check your first graph before you carry on. 4. NOTE: It will be difficult to initially gauge how to dilute your enzyme so that you get meaningful results. If you add too much enzyme, the rate of reaction will be too fast to get an initial rate. If you do not have enough enzyme, the rate will be very slow. For the crude lysate, initially try a 100x and 200x dilution. Record your data in the table on the following page, and plot your data with the absorbance measurement on the Y-axis and time (in seconds) on the X-axis. Repeat the experiment with different dilutions of enzyme until you have a useable change of absorbance. Check with your demonstrator to confirm that your data are useful. rate of When you have identified a concentration of enzyme that is appropriate, make duplicate measurements 17

Explanation / Answer

1. Yes, the concentration of the lactate dehydrogenase in this assay matter. This is so because, it is difficult to find out the loss of phosphophenol puyravate using puryate kinase . Hence, the enzyme lactate dehydrogenase is coupled with puryate kinase to measure the activity of it.  

2. Factors likely to contribute in the errors are:

a .Temperature

b. Setting of spectrometer

c. Dilution of enzyme

d . pH of enzyme

By changing the dilution of enzyme with the above variables, we can check the variables responsible for error and seeing the value in spectrophotometer.

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