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Lab Assignment PDF

This document contains problems from a biochemistry lab problem set. Problem 1 involves calculating initial velocities and specific activities for two fractions of lactate dehydrogenase (LDH) precipitation based on absorbance readings over time. Problem 2 shows data from a multi-step purification of an unnamed enzyme including calculations of specific activity, fold purification, and percent yield for each fraction. Problem 3 uses a Lineweaver-Burk plot to determine the kinetic parameters Km and Vmax for an enzyme with and without an inhibitor at different concentrations, identifying the inhibition as competitive based on the results.

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46 views

Lab Assignment PDF

This document contains problems from a biochemistry lab problem set. Problem 1 involves calculating initial velocities and specific activities for two fractions of lactate dehydrogenase (LDH) precipitation based on absorbance readings over time. Problem 2 shows data from a multi-step purification of an unnamed enzyme including calculations of specific activity, fold purification, and percent yield for each fraction. Problem 3 uses a Lineweaver-Burk plot to determine the kinetic parameters Km and Vmax for an enzyme with and without an inhibitor at different concentrations, identifying the inhibition as competitive based on the results.

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michaeldalessio
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© © All Rights Reserved
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Download as PDF, TXT or read online on Scribd
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BCH 361 Lab Problem Set

Problem #1:
1.
Fraction A

Fraction B

0.3

Absorbance (340 nm)

0.225

0.15

0.075

0
0

0.75

1.5

2.25

Time (mins)
Figure 1: Absorbance Readings of Two Fractions of LDH Precipitation as Time
Progressed. Fraction A and B containing 3.0 mL of 50 mM phosphate buffer with a pH of
7.5 were eluted to collect the 70% pellet of LDH. The first thirty seconds for both fractions
were used to calculate the initial velocity.

2. Fraction A: (0.091 - 0) / (0.5 - 0) = 0.182


Fraction B: (0.066 - 0) / (0.5 - 0) = 0.132
3. Fraction A: (0.182 / 6220 M cm) x 106 uM / M x 2.9x10-3 L = 0.085 umol/min
Fraction B: (0.132 / 6220 M cm) x 106 uM / M x 2.9x10-3 L = 0.062 umol/min
4. Fraction A: (0.085 umol / min) / 0.01 L = 8.5 umol/mL/min
Fraction B: (0.062 umol / min) / 0.01 L = 6.2 umol/mL/min
5. Fraction A: (8.5 umol/mL/min) x 3 mL = 25.5 umol/mL/min
Fraction B: (6.2 umol/mL/min) x 3 mL = 18.6 umol/mL/min

Problem #3
a)
No Inhibitor

3 mM Agent A

5 mM Agent A

1 / Vo (min / mM)

0.75

0.5

0.25

-1

-0.75

-0.5

-0.25

0.25

0.5

0.75

1 / S (1 / mM)
Figure 2: A Lineweaver-Burk plot (double reciprocal plot) that shows the relationship
between an enzymes substrate and its velocity. The graph depicts an enzyme with the
presence of an inhibitor and without. The inhibitor was also studied at two concentrations.

Km = - (1 / x-int)
Max = 1 / y-int
No inhibitor: Km = - (1 / -0.37) = 2.70 mM
Vmax = 1 / 0.19 = 5.26 mM / min
3 mM Agent A: Km = - (1 / -0.28) = 3.57 mM
Vmax = 1 / 0.19 = 5.26 mM / min
5 mM Agent A: Km = - (1 / -0.21) = 4.76 mM
Vmax = 1 / 0.19 = 5.26 mM / min

b) Competitive inhibition since the Km changes while the Vmax remains the same for the three
lines.
c) = Kmapp / Km
= 4.76 mM / 2.7 mM = 1.76
= 1 +{(I) / Kl}
Kl = 5 mM / (1.76 - 1)
Kl = 6.57 mM

Problem #2
Fraction

Total
Volume
(mL)

Total
Protein
(mg)

Total
Activity (U)

Specific
Activity (U/
mg)

Fold
Purification

Percent
Yield

10.00

54.30

65697

1210

1.00

100%

12.50

23.36

42645

1826

1.51

64.9%

8.25

16.25

51281

3156

2.61

78.1%

4.30

3.35

48039

14340

11.85

73.1%

3.50

1.08

46378

42943

35.49

70.6%

Sample Calculations for Fraction 2:


Specific Activity = 42645 / 23.36 = 1826 U / mg
Fold Purification = 1826 / 1210 = 1.51
Percent Yield = (42645 / 65697) x 100% = 64.9%
The results depicted in this chart with regards to the fold purification are typical of an
enzymes purification table, but its percent yield is not. The fold purification of an enzyme
generally increases and is directly proportional to its specific activity because the enzyme
becomes more pure as the amount of protein decreases with a constant rate. Percent yield should
decline as each fraction number progresses due to the fact that more and more enzyme is lost
through each fraction, whereas this chart increases from fraction #2-3 (Berg et al, 2002)
To improve the results for this enzyme purification table, the total activity of fraction #2
would need to increase above the 51,281 U of fraction #3 and below 65,697 of fraction #1 in
order for it to gradually reduce through the five fractions. This would allow for the percent yield
to decrease accordingly.

References
Berg, Jeremy M., John Tymoczko L., and Lubert Stryer. Biochemistry. New York: W.H.
Freeman, 2002. Print.

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