The effect of salts and chaperone
proteins on Alkaline Phosphatase
stability
Aka: the results of 271 continuous
enzyme activity assays
Mary Klein
Dhruve Ringwala
Stephanie Korte
Spring 2011
Biochem 463A
Background
• During the purification process of AP we heated the
lysate to 80° C for 15 minutes to remove all of the
proteins not as thermally stable
• Kurokawa et al, in 2000, showed that E. coli over
expression of protein disulfide isomerase DsbC
stabilized proteins with multiple disulfide bonds in
the periplasm, including AP
• During the purification, after running a column with a
MgCl2 buffer, compared to the MgSO4 buffer used
for dialysis, enzyme activity was lost
• Poe et al, in 1993, showed that NaCl lowered the
activity of AP when compared to Na2SO4
Goals
• We want to discover if there is a protein in the
periplasm of E. coli that leads to alkaline
phosphatase’s high thermal stability and, if
that protein is lost during the purification,
which step it is lost in
• Additionally, since different buffers containing
varying salts are used through out the
purification process we want to determine if
anions can affect heat stability
Hypothesis
• Based off last semester’s data, it appears there is a
protein in the cell lysate that increases the thermal
stability of AP compared to the almost completely
pure Sigma Aldrich AP
• It is likely that the loss of disulfide bonds during
heating is what leads to the loss of activity, and
since Dsb proteins can increase the stability of these
bonds, we believe this is a possible protein that may
be present in the cell lysate but not in the purified
AP
• Additionally, we think that the presence of MgCl2 in
the buffer used in the last step of purification is what
leads to decreased activity after running a column
and therefore this anion leads to decreased thermal
2-
Materials and Methods
• Tested
– Pure Alkaline phosphatase obtained from Sigma
Aldrich diluted in MgSO4 buffer and diluted in MgCl2
buffer
– Stage 1 enzyme from E. coli cell lysate
– Stage 2 enzyme after heat denaturation
– Stage 3 enzyme after dialysis and AmSO2 precipitation
– Stage 4 enzyme after DEAE column
• Measured protein concentration using Bradford
reagent and stock BSA for a standard curve
Materials and Methods cont.
• Temperatures:
– Samples were heated for 30 min at 85, 90, and 95°
C using heat blocks
• Times:
– During the 30 minutes of heating, each sample’s
activity was measured at 5, 10, 15, 20, 25, and 30
minutes
– Activity was also measured 24 hours after heating
(samples were left at room temperature during
this time period)
Materials and Methods cont.
• Activity
– After removal from the heat block samples were
centrifuged for 10 seconds and 40 uL were removed
– This sample was added to a cuvette containing 250 uL
of 1 mM PNPP and 750 uL of Tris buffer at pH 8.0
– Activity was measured at 400 nm using the Cary50
spectrometer
• Analysis
– Activity at each time point was calculated using the
extinction coefficient 0.0175 uM-1cm-1 and divided by
activity before heating to determine percent activity
Baseline levels of Pure AP activity
In MgSO4 buffer
In MgCl2 buffer
0.1003 mg/mL
0.0878 mg/mL
Units of Activity
1,603 U/mL
835.1 U/mL
Specific Activity
15,982 U/mg
9,512 U/mg
Protein
Concentration
Purification Level of Alkaline Phosphatase
Volume
Stage
Units/mL
(mL)
1
2
3
4
60
21
3.2
4
751.4
661
3273
553.5
Total
Units
[Protein]
(mg/mL)
45085
13882
10472
2214
0.5642
0.2512
1.055
0.0954
Total
Protein
(mg)
33.85
5.275
3.033
0.3816
Specific
Activity
(U/mg)
1332
2633
3453
5802
% Yield
Purification
level
-----30.79
23.23
4.91
-----1.98
2.59
4.36
Effects of Salt on Heat Stability
120
Percent Activity
100
MgSO4-85
80
MgCl2-85
MgSO4-90
60
MgCl2-90
40
MgSO4-95
MgCl2-95
20
0
0
5
10
15
Time (min)
20
25
30
Effects of Salt on Activity Recovery
Pure AP in MgSO4 buffer
120
Percent Activity
100
80
60
40
20
0
0
5
10
15
20
25
30
Time (min)
120
Pure AP in MgCl2 buffer
100
Percent Activity
85 degrees
80
85-After 24 hours
60
90 degrees
40
90-After 24 hours
95 degrees
20
95- After 24 hours
0
0
-20
5
10
15
Time (min)
20
25
30
Heat Stability During Purification Process
85 degrees
140
120
Percent activity
100
100
80
60
40
80
60
40
20
20
0
0
0
5
10
15
20
25
30
0
5
10
Time (min)
15
20
Time (min)
95 degrees
120
100
Percent activity
Percent activity
90 degrees
120
80
60
40
20
0
0
5
10
15
Time (min)
20
25
30
Sigma
Stage 1
Stage 2
Stage 3
Stage 4
25
30
Ability to regain activity
Stage 1
120
Percent Activity
100
85 degrees
80
85-After 24 hours
60
90 degrees
40
90-After 24 hours
20
95 degrees
95- After 24 hours
0
0
5
10
15
20
Time (min)
25
30
Stage 2
120
Percent Activity
100
85 degrees
80
85-After 24 hours
60
90 degrees
40
90-After 24 hours
20
95 degrees
95- After 24 hours
0
0
5
10
15
20
Time (min)
25
30
Ability to regain activity
Stage 3
120
100
100
Percent Activity
120
80
60
40
20
80
60
40
20
0
0
0
5
10
15
Time (min)
20
25
30
0
5
10
15
Time (min)
Stage 4
120
Percent Activity
Percent Activity
Pure AP in MgSO4 buffer
100
80
60
40
20
0
0
5
10
15
Time (min)
20
25
30
85 degrees
85-After 24 hours
90 degrees
90-After 24 hours
95 degrees
95- After 24 hours
20
25
30
Further Evidence
Marker
S1 S2 S3
S4
Marker
Alkaline Phosphatase
Conclusions
• What anion is present in the buffer makes a
difference in Alkaline Phosphatase activity and
its thermal stability. Chloride anions clearly
lower the activity and ability to regain activity of
the enzyme when compared to sulfate anions.
• This may be a concern during the purification
process since different ions are used in the
buffers at different steps.
• It is possible that sulfate anions are what lead
to the thermal stability of alkaline phosphatase
at 85° C
Conclusions cont.
• We believe there is a protein present in the
cell lysate that is not present in Sigma Aldrich
purified AP that contributes to the thermal
stability of alkaline phosphatase
• It appears that this protein works optimally at
around 90 °C but is less able to function by
95° C
• It is our conclusion that we lose some of this
protein during the purification process but it is
still present in the stage 4 enzyme
• It is possible that this protein is DsbC
Remaining Questions
• How would the stage 4 enzyme’s thermal
stability and ability to regain activity be
affected if we dialyze it with a MgSO4 buffer?
• If a SDS-Page gel was run for the stage 4
enzyme, silver stained, and the bands left
excised and processed with mass
spectroscopy would they be identified as
DsbC or another protein?
• Alternatively, if a western blot was run and
probed with a DsbC antibody would a band
appear in the stage 4 lane?
References
• Kurokawa, Yoichi, Hideki Yanagi, and Takashi Yura.
"Overexpression of Protein Disulfide Isomerase
DsbC Stabilizes Multiple-Disulfide-Bonded
Recombinant Protein Produced and Transported to
the Periplasm in Escherichia Coli." Applied and
Environmental Microbiology 66.9 (2000): 3960-965.
PubMed. Web. 22 Apr. 2011.
• Poe, Richard W., Vani S. Sangadala, and John M.
Brewer. "Effects of Various Salts on the Steady-state
Enzymatic Activity of E. Coli Alkaline Phosphatase."
Journal of Inorganic Biochemistry 50.3 (1993): 17380. ScienceDirect. 12 Apr. 2001. Web. 22 Apr. 2011.