I posted the same question earlier but didn\'t post what I had already. For the
ID: 1052476 • Letter: I
Question
I posted the same question earlier but didn't post what I had already. For the dilution tab I just need help determining the vol in mL of the enzyme and buffer. The two together should equal 2mL. II. Experimental Procedure. Pre-Lab for Part I: Use preliminary data to calculate the dilutions of both L-DOPA and D-DOPA Pre-Lab for Part II: Calculate dilution of inhibitor Standard assay procedure for tyrosinase. The standard assay mixture contains the following (except when indicated otherwise): 0.1 M sodium phosphate, pH 7.0, L-DOPA, inhibitor (if required) and tryosinase enzyme, all in 3.0 mL reaction volume. The assay should be performed in the following manner Everything except the enzyme shouid be kept at room temperature. Dilute substrates in a volume of 1.0 mL in water. Inhibitors are made in assay buffer, and replace some . buffer in the assay tube The buffer is 1.5 x the concentration needed in the assay · (this makes up for substrate not being in buffer). Concentrated enzyme is diluted in 1.5 x buffer. . Combine substrate (1 ml), and buffer (2 mL- enzyme volume), vortex, check the absorbance (above zero), initiate the reaction by addition of enzyme, and invert the tube to mix (cover with parafilm). .Follow the rate every 20 sec for 2 min. . After the desired time has lapsed (usually 2 minutes), plot the absorbance (y-axis) vs time (x-axis) and determine the velocity as the slope (AA/min) of the initial linear portion of the curve. Speed and accuracy in the manipulations are of considerable importance if reproducible results are to be obtained from assays of enzymatic activity. Timing should begin immediately at the time of enzyme addition and mixing of the enzyme. The spec should be blanked with buffer and substrate alone. Stock Concentrations L-DOPA] (MM 197.2 g/mole) 8 mM in water [D-DOPA] (MM- 197.2 g/mol) 12 mM in water Assay Buffer: 1.5 x is 0.15 M sodium phosphate, pH 7.0 [Tyrosinaselock : 0.2 mg/mL; about 384 U/mL (MW = 128 kD) (Cinnamic acid] ok : 100 mM in 1.5 x buffer Preliminary Data Estimated Km for L-DOPA is 330 micro.M Esimated Km for D-DOPA is 1200 microM Estimated ICso for cinnamic acid is I mM
Explanation / Answer
As you need only 2mL total volume of buffer and enzyme so we can use following combinations
Buffer Enzyme 0.1 1.9 0.2 1.8 0.4 1.6 0.6 1.4 0.8 1.2 1 1 1.2 0.8 1.4 0.6 1.6 0.4 1.8 0.2