Transcript Denaturation of Alkaline Phosphatase by Urea

Denaturation of Alkaline Phosphatase by
urea at elevated temperatures
DuyenVo, Blake Tye, and Allison Morley
Bioc 463a Spring 2012
Objectives
•Find the minimum temperature at which the addition of
urea denatures alkaline phosphatase (AP)
•Determine the rate of denaturation at the minimum
temperature
Background Information
•Urea is a common denaturing agent
•It disrupts noncovalent bonds in proteins
•The [urea] needed to denature some proteins is dependent
upon pH, temperature, and added salts
•Some proteins, such as lysozyme, is completely resistant
to denaturation at RT
• But becomes largely unfolded if the temperature is
raised
Background Information
•AP is a stable protein with noncovalent and disulfide
bonds
•AP is highly, thermally stable
•According to past experiments (Fall 2010), all AP activity
was lost when temperatures as high as 95 oC was reached
•Chloride has been found to disrupt the thermal stability of
AP leading to assisted denaturation upon heating
•AP is essentially resistant to denaturation by urea at RT
Previous Experiments
Max Fluorescence λ=328 vs. [urea]
Fall 2011 class:
At RT, no
denaturation
was observed
even with 8M
urea
140000
Fluorescence
120000
100000
80000
60000
40000
20000
0
0
1
2
3
4
5
[urea] (M)
6
7
8
9
Previous Experiments
50
40
Ellipticity
30
0 Molar
4 Molar
230
240
8 Molar
20
10
0
-10 200
210
220
-20
-30
-40
-50
Wavelength (nm)
250
260
Lack of denaturation
was confirmed by CD
in the presence of
MgCl2 and MgSO4
Fluorescence
•Fluorescence is the emission of light by a compound which
has absorbed light
• Usually emitted light has longer wavelength (and lower
energy) than the absorbed light
•Emission occurs from relaxation of excited electrons the
ground state.
•Fluorimeter: Reads the emission of a fluorophore by
photon count at a fixed excitation wavelength
AP Fluorescence
•Fluorescence in AP is due to tryptophan and tyrosine side
chains
• Tryptophan residues have a much higher and wider
emission band than tyrosine
•Excitation is similar to absorbance
• Usually we would excite at 280 nm, but due to
interference with tyrosine, we instead performed
excitation at 295 nm
Methods
Fluorescence Instrument Configuration
•Excitation: 295nm
• Tyrosine residues are not excited at 295 nm but
tryptophan residues are, so at the chosen excitation
wavelength we would not get interference from
tyrosine
•Emission:300nm-400nm
Methods
Samples:
1. Tris buffer, pH 7.4 (control – water Raman band)
2. 96.2 uM AP in Tris buffer, pH 7.4 (control – no urea)
3. 96.2 uM AP in 8M urea with Tris buffer, pH 7.4
Methods
1. Scan an emission spectrum of sample 1 from 300-400
nm
2. Heat up Tris buffer (1 mL) to 55 °C in instrument, then
inject 40 uL of AP to give sample 2
3. Scan an emission spectrum from 300-400 nm every 10
minutes
Methods
4. Heat up 8M urea (1 mL) to 55 °C in instrument, then
inject 40 uL of AP to give sample 3
5. Take a spectrum from 300-400 nm every 10 minutes
6. Repeat steps 2-6 for 70, 80, and 90 °C
Results
• With the addition of urea:
• At temperatures 70 oC and below, no denaturation
of AP was observed
• Unexpectedly, at 80 oC, the intensity of max
emission increased relative to the control
• At 90 oC, the intensity of max emission gradually
decreased over time
Excitation scan of free Trp
Buffer “Blank”
25000
20000
# hv count
Water Raman Band
15000
10000
5000
0
290
300
310
320
330
340
350
Wavelength (nm)
360
370
380
390
No denaturation at 70 oC
Odd observations at 80°C
600000
500000
80-w/o-10
# hv count
400000
80-w/o-20
80-w/o-30
300000
80-w/urea-10
80-w/urea-20
80-w/urea-30
200000
80-w/urea-30
80-w/urea-40
100000
0
290
310
330
350
Wavelength (nm)
370
390
Denaturation at 90°C
450000
400000
350000
90-W/urea-10
# hv count
300000
90-w/urea-20
90-W/urea-30
250000
90-w/urea-40
90-w/urea-50
200000
150000
100000
50000
0
290
310
330
350
Wavelength (nm)
370
390
Rate of denaturation at 90°C
420000
370000
# hv count
y = -4497.8x + 427848
320000
270000
220000
170000
10
20
30
Time (min)
40
50
Results
• Observing the overlap of intensities at 80 oC with and
without urea, the change in intensity was most likely
due to experimental errors (lower [AP])
• Denaturation of AP over time at 90 oC
• Slight red shift
• Could not be verified that denaturation was due to
urea and not just temperature due to lack of control
• Rate: -4500 hv count/ min
Conclusions
• Although denaturation of AP with urea was observed at
90 oC, since a control was not used, conclusions could
not be verified
• Discrepancies remain that incomplete transition to the
denatured state cannot be observed for proteins that
contain disulfide bonds
• Native conformation is still stabilized by disulfide
bonds
Future directions
• Run a control of AP in just buffer at 90 oC at 10 min
intervals before observing denaturation with urea at 90
oC
• Run a similar experiment, but instead use both TCEP
and urea in order to observe complete denaturation
• Experiment with different pH and added salts other
than ones already done of course
References
 Tanford, C. (1968). Protein denaturation. Advances in Protein
Chemistry, 23, 121-282.
 Coleman, J.E. (1992). Structure and mechanism of alkaline
phosphatase. Annual Reviews, 21, 441-483.
 Garen, A., Levinthal, C. (1959). A fine-structure genetic and
chemical study of the enzyme alkaline phosphatase of E. Coli
I. Purification and characterization of alkaline phosphatase.
Biochimica et Biophysica Acta, 38, 470-483.