Nareg Boyadjian, Kenny Childers, Rebecca
Luiten and Lauren O’Neill



The goal of this experiment was to explore
the denaturation of Alkaline Phosphatase
using Urea and Guanidinium HCl
To understand the effect of MgSO4 and MgCl2
in buffers on the denaturation
To collect data on the unfolding of the
protein using Circular Dichroism.

Responsible for removing phosphates from
many different molecules
 R-O-PO3H- + H2O


ROH + H2PO4-
In E. coli, it is located in the periplasmic
space
It is very stable and resistant to inactivation,
denaturation and degradation




Three metal ions (two Zn2+ and
one Mg2+)
Two disulfide
bonds
Homodimer
Uses Serine 102
for a nucleophilic
attack on the phosphate

Urea
 Direct - blocks intramolecular hydrogen bonds by
binding to the polar(peptide groups) regions of the
protein
 Indirect – introduces non-polar residues to the solvent
which diminishes the hydrophobic effect of the protein

Guanidinium Hydrochloride
 It interacts with the planar residues by stacking them
and disrupting the secondary structure




Used to investigate the secondary structure of
proteins
Helps determine the fraction of secondary
structures for varying denaturant
concentrations
Measures the extent of the absorption
between the left and right polarized light
Molar Ellipticity = circular dichroism
corrected for concentration
 [θ] = θ x 100 / ( l x N x c)
http://upload.wikimedia.org/wikipedia/en/4/4b/Cir
http://www.cryst.bbk.ac.uk/PPS2/course/section8/ss-960531_21.html

General description of each material used:
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Urea Powder
Guanidine Hydrochloride Powder
Alkaline Phosphatase From E. Coli
Beakers
Magnetic Stir Bar
Pipettes
MgSO4 Buffer
MgCl2 Buffer
Epindorff Tubes
Circular Dichroism Equipment
CD Cells

The Procedure That We Performed:
◦ Make up a 8M stock solution of Urea in each buffer. Molecular weight of Urea is ~ 60,
multiplying this by 8 gives the amount of grams (480) needed in 1 Liter solution. Using
only 5 mL solution volume, weigh out 2.4 g of Urea powder, and put into beaker with 5 mL
of MgSO4. Do this in another beaker with another 2.4 g of powder and this time 5 mL of
MgCl.
◦ Next, make up a 6M stock solution of Guanidine Hydrochloride in each buffer. Molecular
weight is ~ 95.5, multiplying this by 6 gives the amount of grams (573) needed in 1 Liter
solution. Using only 2.5 mL solution volume, weigh out 1.433 g of Guanidine
Hydrochloride powder, and put into beaker with 2.5 mL of MgSO4. Do this in another
beaker with another 1.433 g of powder and this time 2.5 mL of MgCl.
◦ Make up samples of varying denaturant concentration:
0,0.5,1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5 and 8M for Urea samples. Use 0,1,2,4 and
6M for Guanidine Hydrochloride samples. Store each sample in labeled epi tubes. In order
to make these concentrations at a total volume of 350 uL (enough for cuvette), simply
follow these calculations:
Volume of Stock Denaturant = (Desired Concentration)
* 350 uL
(Stock Concentration)
Volume of Protein = (Desired Concentration)
* 350 uL
(Stock Concentration)
Volume of Buffer = 350 uL – (Volume of Stock Denaturant + Volume of Protein)
Note: Volume of Protein will remain constant because the desired concentration is constant.
In our case, our desired constant concentration was 5 uM, and the stock protein
concentration was 108 uM. This gave a constant protein volume of 16.2 uL.

CD Equipment Usage:
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Purge the system with Nitrogen gas for 15 minutes and ALWAYS maintain a minimum flow of 810 Liters/minute and a pressure minimum of 50 psi. Record yourself into the log book (black
folder).
After the 15 minutes, turn on the gray lamp cooling box behind the monitor.
Press the orange power button for the lamp power supply.
While making sure that NO OTHER COMPONENTS ARE ON, including the computer and main
power switch under the table, ignite the lamp by pressing and holding the ignite button until the
green/blue light on top of the chamber turns on.
When the wattage meter reads ~ 150 watts on the lamp box, turn on the main power switch and
computer and go to the Global Works program. Follow the instructions of the instructor and the
orange folder.
Record a full spectrum of the buffer as a baseline every time you are working with a new buffer,
including the first buffer. Use an integration time of 10 seconds for the wavelength range 200260 nm. Take 3 scans and manually average the observed values.
Record full spectra for 0,4 and 8M Urea samples as well as 0 and 6M Guanidine Hydrochloride
samples while following the buffer baseline rule stated above.
Record spectra from 220-224 nm with an integration time of 5 seconds for all other
concentration samples (ie: 1,2..etc.).
Make sure to save all spectra that you record and transfer to your own computer for analysis.
To turn off the equipment: turn off the lamp power supply, but leave on the lamp cooling box
and Nitrogen gas flow for 15 minutes. Turn off the computer and main power switch. Store all
cuvettes in 25% Ethanol in 1M HCl. After the 15 minutes is up, turn off Nitrogen gas flow and the
lamp cooling box. Log out of the log book.
50
40
0 Molar
4 Molar
8 Molar
30
20
Ellipticity
10
0
200
210
220
230
-10
-20
-30
-40
-50
Wavelength (nm)
240
250
260
0
2
4
6
0
8
0
-5
-2000
-10
-4000
-20
-25
-30
-35
-40
2
4
6
-6000
-15
Molar Ellipticity (deg cm^2/dmol)
Ellipticity (millidegrees)
0
[Urea] M
Ellipticity vs. Urea Concentration
at 222 nm
-8000
-10000
-12000
-14000
-16000
-18000
[Urea] M
Molar Ellipticity vs. Urea
Concentration at 222nm
8
50
40
0 Molar
30
4 Molar
8 Molar
20
Ellipticity
10
0
200
210
220
230
-10
-20
-30
-40
-50
Wavelength (nm)
240
250
260
0
0
2
4
6
8
0
-5
-2000
-10
-4000
Molar Ellipticity (deg cm^2/dmol)
Ellipticity (millidegrees)
0
-15
-20
-25
-30
-35
-40
1
2
3
4
5
6
7
-6000
-8000
-10000
-12000
-14000
[Urea] M
Ellipticity vs. Urea Concentration
at 222 nm
-16000
[Urea] M
Molar Ellipticity vs. Urea
Concentration at 222nm
8
50
0 Molar
40
6 Molar
30
20
Ellipticity
10
0
200
210
220
230
-10
-20
-30
-40
-50
Wavelength (nm)
240
250
260
0
0
0
2
4
6
Molar Ellipticity (deg cm^2/dmol)
Ellipticity (Millidegrees)
2
4
-2000
-5
-10
-15
-20
-25
0
-4000
-6000
-8000
-10000
-12000
[GmdHCl] M
Ellipticity vs. GmdHCl
Concentration at 222 nm
[GmdHCl] M
Molar Ellipticity vs. GmdHCl
Concentration at 222nm
6
50
0 Molar
40
6 Molar
30
20
Ellipticity
10
0
200
210
220
230
-10
-20
-30
-40
-50
Wavelength (nm)
240
250
260
0
0
2
4
Ellipticity (Millidegrees)
-5
-10
-15
-20
-25
6
0
Molar Ellipticity (deg cm^2/dmol)
0
[GmdHCl] M
Ellipticity vs. GmdHCl
Concentration at 222 nm
2
4
-2000
-4000
-6000
-8000
-10000
-12000
[GmdHCl] M
Molar Ellipticity vs. GmdHCl
Concentration at 222nm
6



Neither Urea or Guanidinium HCl were strong
enough to denature the α-helix fully.
The usage MgCl2 or MgSO4 had inconclusive
effects on the overall denaturation of the
protein.
Further exploration would be needed to
determine the concentration of GmdHCl or
Urea needed to fully denature the protein.

Combine heat denaturation with chemical
denaturation

Try higher concentrations of denaturant

Try different buffers

Try using DTT and/or other chemicals (to
break disulfide bonds)




Garen, A., and C. Levinthal. "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 (1960):
470-83. Print.
Coleman, J. E. "Structure and Mechanism of Alkaline
Phosphatase." Annual Review of Biophysics and
Biomolecular Structure 21.1 (1992): 441-83. Print.
Stec, Buguslaw, Kathleen M. Holtz, and Evan R. Kantrowitz.
"A Revised Mechanism for He Alkaline Phosphatase
Reaction Involving Three Metal Ions." Web. 01 Nov. 2011.
<http://www.ncbi.nlm.nih.gov/pubmed/2010919>.
Pace, C.N. "Determination and Analysis or Urea and
Guanidine Hydrochloride Danaturation Curves." Methods in
Enzymology 131 (1986): 266-80. Print.