Question

3. Use the below tables to complete lab calculations on the worksheet on pages 2-6 of this document. You will submit the worksheet via dropbox on Canvas by you assigned lab time the week of March 30th through April 3rd. A document with sample calculations of different concentrations is provided to you on canvas. Enzyme Kinetics Lab Buffer volume (mL) Enzyme Volume (ml) Substrate Volume (ml) TOTAL VOLUME (mL) 0.04 0.5 1.5 0.04 0.25 1.5 0.04 0.1 1.5 0.04 0.05 1.5 Time (Seconds) ABS 30 45 75 90 ABS 0.14 0.2 0.259 0.317 0.371 0.425 0.473 0.522 0.57 0.613 0.654 ABS 0.085 0.143 0.204 0.257 0.309 0.359 0.407 0.453 0.498 0.539 0.578 ABS 0.078 0.113 0.145 0.176 0.205 0.234 0.262 0.288 0.313 0.336 0.359 105 0.063 0.088 0.112 0.136 0.158 0.178 0.198 0.217 0.236 0.254 0.27) 120 135 150 165 180

Answer 1

1.

Buffer Volume (mL)	0.96	1.21	1.36	1.41
Substrate Volume (mL)	0.5	0.25	0.1	0.05
Enzyme Volume (mL)	0.04	0.04	0.04	0.04
Total Reaction Volume (mL)	1.5	1.5	1.5	1.5

2.

Time (sec)	Abs 475nm Substrate 5mM	Abs 475nm Substrate 2.5mM	Abs 475nm Substrate 1mM	Abs 475nm Substrate 0.5mM
30	0.14	0.085	0.078	0.063
60	0.259	0.204	0.145	0.112
90	0.371	0.309	0.205	0.158
120	0.473	0.407	0.262	0.198
150	0.57	0.498	0.313	0.236
180	0.654	0.578	0.359	0.27

3.

Time (sec)	Abs 475nm Substrate 5mM	Abs 475nm Substrate 2.5mM	Abs 475nm Substrate 1mM	Abs 475nm Substrate 0.5mM
90-30	0.231	0.224	0.127	0.095
120-60	0.214	0.203	0.117	0.086
150-90	0.199	0.189	0.108	0.078
180-120	0.181	0.171	0.097	0.072
Average ∆Abs(475nm)/min	0.206	0.197	0.112	0.083

4.

a. ∆Abs(475nm)/min (5 mM Substrate) = 0.206

b. ∆Abs(475nm)/min (2.5 mM Substrate) = 0.197

c. ∆Abs(475nm)/min (1 mM Substrate) = 0.112

d. ∆Abs(475nm)/min (0.5 mM Substrate) = 0.083

5. A = e C l   C = A /e = A/3600

a. [dopachrome] for 5 mM substrate reaction = 5.73 x 10-5 M/min

b. [dopachrome] for 2.5 mM substrate reaction = 5.47 x 10-5 M/min

c. [dopachrome] for 1 mM substrate reaction = 3.12 x 10-5 M/min

d. [dopachrome] for 0.5 mM substrate reaction = 2.3 x 10-5 M/min

6. mole = molar concentration x total volume of enzyme reaction mixture

a. Using 5 mM substrate:

Moles dopachrome produced per min = 3.82 x 10-8 moles/min

U moles dopachrome produced per min = 0.038 µmoles/min

b. Using 2.5 mM substrate:

Moles dopachrome produced per min = 3.65 x 10-8 moles/min

U moles dopachrome produced per min = 0.036 µmole/min

c. Using 1 mM substrate:

Moles dopachrome produced per min = 2.08 x 10-8 moles/min

U moles dopachrome produced per min =0.021 µmole/min

d. Using 0.5 mM substrate:

Moles dopachrome produced per min = 1.53 x 10-8 moles/min

U moles dopachrome produced per min = 0.015 µmole/min

7.

Vo	[S]	1/ Vo	1/[S]
µmole/min	mM
0.038	5.0	26.31579	0.2
0.036	2.5	27.77778	0.4
0.021	1.0	47.61905	1
0.015	0.5	66.66667	2

8.

y = 23.361x + 21.071 R2 = 0.9828 1N. (umol/min) ........ Linear (0) - 0.5 2 2.5 1 1.5 1/[S] MM

9. Lineweaver-Burk plot    1/V = Km/Vmax [1/S] + 1/Vmax

y-intercept = 1/Vmax = 21.07       Vmax = 0.0475 µmol/min

X-intercept = slope = 23.361

Km/Vmax    = 23.361

Km= Vmax x 23.361 =    0.0475 x 23.361 = 1.1096 mM     

Note : please give your feedback