Question

Sample #	2.5 mM NPP (mL)	NaCitrate buffer (mL)	water (mL)
1	0.10	2	2.4
2	0.20	2	2.3
3	0.30	2	2.2

A) make a plot of Absorbance 405 nm vs time for each substrate concentration, displaying all 3 samples on the same plot.

B) fit each data set to a trendline

C) using the slopes from the trendlines in part B and the slope from your standard curve, convert your slopes into enzyme velocity values with units of μM/min.

D) Assuming that you added 1 mL of enzyme to the samples, calculate the value of [S] for each sample. Show your V, [S], 1/V and 1/[S] values.

time in min and absorbance values at 405 nm

time	1	2	3
0	0.02410886	0.06550155	0.06248211
10	0.0701	0.11111	0.21395879
20	0.1214782	0.16430943	0.2915791
30	0.16052195	0.2222222	0.30016227

Answer 1

A) Plot of absorbance vs time for the three samples is as shown below:

B) Fitting the data set to a linear regression gives the following equations:

Sample 1: y = 0.0046 x + 0.025

Sample 2: y = 0.0052 x + 0.0623

Sample 3: y = 0.0079 x + 0.0984

C) Slopes for the three samples are:

Sample 1: 0.0046

Sample 2: 0.0052

sample 3 : 0.0079

D) Enzyme velocity = slope from each sample/molar extinction coefficient

Information regarding the molar extinction coeeficient which can be obtained from the standard curve is required for further calculations.