Pre-Lab for Part I1: Calculate dilution of inhibitor Standard assay procedure fo

Question

Pre-Lab for Part I1: 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 should 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 see for 2 min. After the desired time has lapsed (usually 2 minutes), plot the absorbance (y-axis) vs time (x-axis) and determine the velogtity as the slope (AA/min) of the initial lincar 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- 1972 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 [T yrosinasetock:0.2 mg/mL; about 384 U/mL (MW- 128 kD) [Cinnamic acidak: 100 mM in 1.5 x buffer Preliminary Data Estimated Km for L-DOPA is 330 microM Esimated Km for D-DOPA is 1200 microM Estimated ICso for cinnamic acid is 1 mM

Explanation / Answer

Calculate concentration of inhibitor in the solution

Initial concentration of inhibitor = 100 mM (stock solution)

take 1 ml of inhibitor stock solution and dilute to 12 ml of final volume with buffer solution

Using,

C2 = C1 x V1/V2

with,

C1 = 100 mM

C2 = ?

V1 = 1 ml

V2 = 12 ml

So,

concentration of inhibitor in 12 ml solution = 100 mM x 1 ml/12 ml

                                                                   = 8.33 mM

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