Analysis Assignment 5

1.  Using the data of measured absorbance vs. time (min) for each substrate concentration, determine the slope of the linear portion of this curve.  The value of the slope is the initial velocity (Vo) in Abs/min.  Convert AU/min to mM/sec using the extinction coefficient you calculated for 5-TNB.  Use these values to construct a table in Excel with the following column titles.

 Trial # Sample # [E] mM [S] mM 1/[S] mM-1 Vo (mM/sec) 1/Vo (sec/mM)

Print a copy of this table

Show an example calculation for [E], [S] and Vo

2.  Plot the initial velocity as a function of substrate concentration. This is a Michaelis-Menton plot. Fit the curve the best way you can. Estimate Vmax and KM from this plot. Note: The curve may not level off to give an accurate Vmax, but please try your best.  Print a copy of this plot with proper labels.  Add additional curves for each additional substrate.

3.  Plot (1/V0) as a function of (1/[S]). This is a Lineweaver - Burk plot or a double reciprocal plot. The data should fit a straight line. Calculate Vmax and KM from this plot. Calculate kcat .   Print a copy of this plot.  Complete plots and calculations for each additional substrate.

4.  Calculate the specific activity of the enzyme.  Specific activity is often reported in units/mg, the higher the activity suggests good things about the enzyme preparation and purification.  In our case we will define a "unit" as the amount of free thiol formed (mmol) per minute at pH 9, with 0.25 mM cystine.

5.  Present you calculations in tabular format for easy comparison.  Include results from a published resource.  Include any additional appropriate observations or interpretations.  Remember our goal is not to just generate numbers, but to give these numbers meaning in terms of how the enzyme shows specificity and enhances the rate of the reaction.

6.  Turn in your analysis by Friday at 5:00.

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