This experiment aims for a direct measure of the acetate forming activity of acetate kinase. Initiate the reaction by adding enzyme to a reaction mixture containing the acetyl phosphate and a DP substrates. Then add hydroxy lamine hydrochloride to convert the remaining acetyl phosphate to acetyl hydroxylate.
Add development solution to convert the acetyl hydroxylate to a colored ferric hydroxylate complex that can be measured. Spectro photometrically based on the amount of acetyl phosphate substrate consumed in the reaction results allow for determination of enzyme kinetic parameters. The advantage of this technique over existing methods, it's based on the direct measurement of acetyl phosphate consumption rather than de coupling of a TP production to the reduction of an A DP.Because this assay does not rely on a TP production, it can be used for a new group of acetate kinases, which produce power phosphate instead of a TP For a standard curve of six to eight points, dilute the acetyl phosphate stock solution in 100 millimolar triss to a final sample volume of 300 microliters also include a control sample without acetyl phosphate equilibrate all samples in a 37 degree Celsius heat block for one minute.
Now, add 50 microliters of two molar hydroxy lamine hydrochloride, mix by shaking three times and incubate in 60 degrees Celsius heat block for five minutes. Then add 100 microliters of development solution mix by shaking three times and allow color to develop for at least one minute at room temperature. Centrifuge the samples using plastic cuvettes for the spectrophotometer.
Measure the samples at 540 nanometers. Proceed to generate a standard curve of absorbence at 540 nanometers versus micromoles of acetyl phosphate with appropriate data analysis and graphing software. Calculate the r squared value.
Distribute 300 microliter aliquots of reaction mix to micro centrifuge tubes. Remember to include a control reaction with no enzyme equilibrate tubes for one minute at 37 degrees Celsius. If several reactions are performed, add enzyme to the tubes at specific time intervals and immediately return each tube to the 37 degree Celsius heat block.
After five minutes, add 50 microliters of two molar hydroxy lamine hydrochloride, mix and incubate five minutes at 60 degrees Celsius. Then add 100 microliters of the development solution, mix and place it room temperature for color development. Then centrifuge samples at 18, 000 times G for one minute.
Proceed to measure absorbance at 540 nanometers. Determine the amount of acetyl phosphate present by comparison to the acetyl phosphate standard curve. The data can be plotted and fit to the McKayla Cementin equation for determination of kinetic parameters.
This assay determines consumption of the acetyl phosphate substrate to measure acetate kinase activity in the direction of acetate formation. Acetyl phosphate remaining after a given time during the enzymatic reaction is converted to acetyl Oxybate and subsequently to ferric hydroxy ate complex, which has a reddish color that can be measured at 540 nanometers. This typical standard curve plotting absorbence at 540 nanometers versus micromoles acetyl phosphate has a slope of 1.2332 absorbence units per micromole acetyl phosphate.
The resulting r squared value of 0.99 indicates the data fits well with the linear equation as depicted here. This assay can be used to determine the KM for acetyl phosphate for various enzyme preparations. At five millimolar a DP with varying concentrations of acetyl phosphate purified recombinant acetate kinase from methanol Sarina thermophilic follows standard McKayla cementin like kinetics for each substrate resulting in an apparent KM of 0.27 plus or minus 0.01 millimolar acetyl phosphate.
Unlike the standard coupled assay, this direct assay can measure activity of acetate kinases that utilize substrates other than a TP.For example, in the utilization of acetyl phosphate by the pyrophosphate producing inba histolytica acetate kinase, sodium phosphate was substituted for a DP in the reaction mix. The resulting curve indicates an apparent KM value of a 0.50 plus or minus 0.006 millimolar acetyl phosphate. After watching this video, you should have a good idea about how to measure acetate kinase activity directly by determining the amount of acetyl phosphate consumed.
A key factor in obtaining reproducible results is to be consistent in each step, especially in adding enzyme and in the mixing step.
