QUIZ 3 MAY 12, 2017
MCB 120L S2017V2
Name:___________________________________________
Lab Section (circle): MORAND A01 LAGARIAS A02 Acid phosphatase (APase) is an enzyme that, like alkaline phosphatase studied in Exp. 3, can catalyze pnitrophenol phosphate hydrolysis. You have been asked to determine the specific activity of APase in an extract
from the newly isolated archeabacterium Thermococcus furiosus. Since you have taken MCB120L, you know
that you need to determine both protein concentration and APase enzyme activity of the T. furiosus extract.
The total volume of the extract is 2.1 ml.
T. furiosus cells are hard to obtain in quantity since it only grows near volcanic vents deep in the ocean. For this
reason, you used a Micro BCA Protein Assay in which BSA standard and Babelfish extracts were diluted into
50 µl (final volume) from which 12.5 µl was removed and diluted with 237.5 µl BCA reagent in individual
wells of a microplate. This colorimetric assay measures the change in absorbance at 562 nm. You analyzed
different amounts of your undiluted T. furiosus extract and obtained the results shown in Table 1 below. The
BCA standard curve that uses BSA as the protein standard is shown below on the right. The formula for the
best-fit line of this data is y = 0.0554x + 0.488 where y corresponds to A562 and x corresponds to µg protein.
Table 1. BCA Protein Assay
Sample Mixtures
APase
Amount of
extract
water
assayed, µl
added, µl
2
4
8
16 48
46
42
34 Assay Results
Ave A562 for 12.5 µl
µg
protein
sample mix + 237.5 µl
in assay
BCA reagent (n=3)
0.500
0.712
0.942
1.282 1. (2 pts) Complete the last column of data table above. In the box below, indicate the concentration of
APase protein in your T. furiosus extract (mg/ml) and the total protein (mg) in the T. furiosus extract. Show
your work below or on back. Conc =
Total Protein = Quiz 3 (cont) May 12, 2017V2 page 2 Name:___________________________________________
You next assayed the T. furiosus extract for APase activity at four pHs using the continuous and microplate
fixed time assays protocol identical to those you used in Experiment 3A&B, except that the fixed time was
shortened to 4 min. The assay results for 1/50 dilutions of the T. furiosus extract are shown below. Since
acidic buffers were used for these assays, the pH of the fixed time assays were measured after the 1.0 M
K2HPO4 Stop solution was added to permit correction for the ionization of p-nitrophenol product in the
microplate. This was done with a micro pH electrode and the pHs measured are shown in the Table. The
standard curve for p-nitrophenol for the microplate is shown below.
Continuous Assay (1 cm cuvette) Microplate Fixed Time Assay Assay
pH Non-enz.
control
∆A410
min–1 + Enz.
∆A410
min–1 Corr.
ΔA410
min–1 I.U. per
assay 4
5
6
7 0.0003
0.0002
0.0001
0.0002 0.0005
0.0020
0.0100
0.0753 0.0002
0.0018
0.0099
0.0751 XXX
XXX
XXX Assay
pH 4
5
6
7 Measured
pH after 1.0
M K2HPO4
Stop solution
added
7.0
7.5
8.0
8.5 Non-enz.
∆A410
(4 min)–1 + Enz.
∆A410
(4 min)–1 0.010
0.050
0.080
0.102 0.4765
0.6980
0.5728
0.4609 Corr.
ΔA410
(4 min)–1 I.U. per
assay p-Nitrophenol Standard Curve pH 9.5 A405 microplate 3.5
3.0
2.5
2.0
y = 0.0121x
R2 = 0.9979 1.5
1.0
0.5
0.0
0 50 100 150 200 250 300 p-Nitrophenol Product (uM) 2. (3 pts) Based on the continuous assay results, calculate the specific activity of the T. furiosus APase at pH 7.
Put IU per assy in Table above. Show how you calculated this value below and place your calculated specific
activity in the box below. APase sp. activity @ pH 7 = Quiz 3 (cont) May 12, 2017V2 page 3 Name:___________________________________________
3. (4 pts) Complete the Table on the previous page. In the space below (and continued on next page or back),
show how you calculated the I.U. per assay for the fixed time assays measured at pH 7. Put answers in Table on
page 1. All other calculations can be performed on back pages. 4. (1 pt) Based on your data (above), at which pH is the activity of APase the largest – pH 4, pH 5, pH 6 or pH
7 (CIRCLE ANSWER)? Justify your answer below.

 

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