calculating substrate Enzyme kinetics lab . I don\'t understand how to fill out
ID: 93412 • Letter: C
Question
calculating substrate
Enzyme kinetics lab . I don't understand how to fill out the chart for [S]....at the bottom
Tube #
Reaction
Cocktail l
0.1% BSA
Enzyme (ul)
Mass enzyme (mg)
Time
A410
1
400
100l
0
0
15 min
0.00
2
400
80
20
.0046
15
0.088
3
400
60
40
.0092
15
.0091
4
400
40
60
.0138
15
.112
5
400
20
80
.0184
15
.118
6
400
0
100
.023
15
.128
Y=4.6646x+0.0359
R2=0.745
Determine Absorbance of Products
Set spectrophotometer to 410 nm.
Fill a cuvette with water, zero the spectrophotometer.
Measure the absorbance of each sample and record in Table 2.
Data Analysis
Prepare a graph of mg of acid phosphatase versus absorbance of product.
The zero absorbance, zero enzyme point is a real data point and should be included in the graph.
Determine the best-fit line, either using a computer program or by hand drawing.
Pick a point on your graph to use in calculation of specific activity. A calculated point is more accurate than any single data point. The coordinates of this point (X,Y) will be used in the following calculation.
We chose point 3, x=0.0092 y =0.091
Congratulations! You’ve determined the specific activity of the enzyme!
The Mega-equation for the above calculation is:
.0091 /1.88x10^4L/molcm x 0.005L/15min x 106mol/mole x 1 / 0.0092mg protein x 1/1cm =1.75
Tube #
-S mix
+S mix
0.1%BSA
Enzyme l
Time min
A410
1
320
80
100
0
15
0
2
388
12
0
100
15
.122
3
380
20
0
100
15
.159
4
360
40
0
100
15
.244
5
320
80
0
100
15
.332
6
200
200
0
100
15
.449
7
0
400
0
100
15
.522
Tube #
[S]
1/[S]
Specific activity= velocity
1/V
1
2
3
4
5
6
7
Tube #
Reaction
Cocktail l
0.1% BSA
Enzyme (ul)
Mass enzyme (mg)
Time
A410
1
400
100l
0
0
15 min
0.00
2
400
80
20
.0046
15
0.088
3
400
60
40
.0092
15
.0091
4
400
40
60
.0138
15
.112
5
400
20
80
.0184
15
.118
6
400
0
100
.023
15
.128
7:54 PM ooooo AT&T; LTE ggcview.usg.edu 2.30 seconds later, add 100 ulenayme to tube 2 and place in the water bath. 3 Continue for the remainder of the tubes 4 After 15 minutes, add 45mistopping reagent totube 1.30 sec later add stopping reagent totube 2 spectrophotometer Tubel has an substrate and serves as a control for spontaneous appearance of product in the absence of ename. Measure the absorbance of tubes 1-2 and necord the data in Table 2. Calculate 1/ISl for tubes 1.7 and enter into Table 3. Calculate specific activity lvelocty) forreactions Use the formula below to calculate the specifcactivity for each tube.Rememberthat multiplying the enayme concentration by the volume used gives me enryme. A wilbe unique for each tube.ifyou need, consult the step by step calculations for specific activity used in the previous lab. 15 min mg protein Specific activity initial velocity umols product formedWmin mg of protein Tube TisiExplanation / Answer
Substrate concentration [S] can be calculated from absorbance using the formula,
[S] = (A-b)/m
where, A is absorbance,
b is y intercept and
m is slope.
Given, Y=4.6646x+0.0359
Here, the value of b is 0.0359 and the value of m is 4.6646
[S] for tube 1 = (0-0.0359)/4.6646 = 0.00769 = 7.69 mM
[S] for tube 2 = (0.122 - 0.0359)/4.6646 = 0.018458 = 18.45 mM
[S] for tube 3 = (0.159 - 0.0359)/4.6646 = 0.026390 = 26.39 mM
[S] for tube 4 = (0.244 - 0.0359)/4.6646 = 0.044612 = 44.61 mM
[S] for tube 5 = (0.332 - 0.0359)/4.6646 = 0.063478 = 63.47 mM
[S] for tube 6 = (0.449 - 0.0359)/4.6646 = 0.088560 = 88.56 mM
[S] for tube 7 = (0.522 - 0.0359)/4.6646 = 0.104210 = 104.21 mM
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