Transcript Guanidinium Denaturation of Alkaline Phosphatase

Guanidinium Denaturation of Alkaline
Phosphatase
Spencer Fosnot, Erick Karlsrud, Marvin O’Ketch, Gavin Young
University of Arizona, Biochemistry 463a
Fluorescence Spectroscopy
Amino Acid Fluorophores
Important Reagents
Guanidine Hydrochloride

Chemical denaturant
Tris(2-carboxyethyl)phosphine
(TCEP)

Selectively reduces
disulfide linkages
Alkaline Phosphatase



Hydrolysis of phosphate esters
Two active sites
Homodimer


2 active sites


2 intramolecular disulfides per
monomer
Magnesium and 2 Zinc ions
Hydrophobic and Hydrogen
interactions between monomers.
AP Active Site Detail
Purpose


Utilize fluorescence spectroscopy to observe the
chemical denaturation of AP by GdnHCl
Determine effects of the reducing agent TCEP on the
denaturation of AP.
Materials and Methods
Guanidinium hydrochloride:
1.
2.
3.
4.
5.
6.
7.
Made 6M Guanidinium stock by dissolving 57g of Guanidinium in 100mL
of tris buffer
Mixed buffer and 6M Guanidinium to make 3mL samples of varying
concentrations from 0M to 6M Guanidinium going up by 0.5M each time
Put in the fridge for 24 hrs
After 24 hrs in fridge removed and added 14uL AP (50 ug/mL)
Put back in fridge for 24 hrs
after 24 more hours in the fridge we took the samples to the flourometer
and excited at (fill in)nm and recorded the flourence between (fill in)nm
Extracted data to flash drive and plotted
Guanidinium hydrochloride with (TCEP):
Same as above with tcep (5 mM) added in step 4
Previous Studies
Pace et. al. (2000)
𝑦=
𝑦𝐹 + 𝑚𝐹 𝐺𝐷𝑁
+ 𝑦𝑈 + 𝑚𝑈 𝐺𝐷𝑁
1+𝑒
−
𝑒
Δ𝐺 𝐻2 𝑂 −𝑚 𝐺𝐷𝑁
𝑅𝑇
−
Δ𝐺 𝐻2 𝑂 −𝑚 𝐺𝐷𝑁
𝑅𝑇
Previous Studies
Asgeirsson et. al. (2005)
Results
40000
60000
Increasing [GDN]
20000
Fluorescence (excited at 295 nm)
80000
Fluorescence Spectrum of Guanidine Denaturation
300
320
340
360
Wavelength (nm)
380
400
Results
0.8
0.6
0.4
0.2
0.0
Maximum Fluorescence ( = 319 nm )
1.0
Guanidine Denaturation Curve
0
1
2
3
[GDN] (M)
4
5
6
Results
80000
40000
60000
Increasing [GDN]
+ TCEP
20000
Fluorescence (excited at 295 nm)
Fluorescence Spectrum in the Presence of TCEP
300
320
340
360
Wavelength (nm)
380
400
Results
0.8
0.6
0.4
0.2
0.0
Maximum Fluorescence ( = 319 nm )
1.0
Guanidine Denaturation Curve in the Presence of TCEP
0
1
2
3
[GDN] (M) + TCEP
4
5
6
Results
1.0
TCEP’s Influence Over AP Denaturation
0.8
G(H2O) 6.5 kcal mol
1
1
0.4
0.6
GDN
0.2
GDN + TCEP
0.0
Maximum Fluorescence ( = 319 nm )
G(H2O) 10.5 kcal mol
0
1
2
3
[GDN] (M)
4
5
6
Results Summary
GDN




λmax at 319 nm
Maximum fluorescence
increased up until 3M
Decreased sharply until
5M
ΔG(H2O) =
10.5kcal•mol-1
GDN + TCEP




λmax at 319 nm
Maximum fluorescence
increased up until 1.5M
Decreased sharply until
4M
ΔG(H2O) =
6.5kcal•mol-1
Conclusions


Both methods efficiently denatured AP
TCEP promotes denaturation




Reduces disulfide linkages
Steeper slope during denaturation
Decrease of ΔG with TCEP
TCEP decreases AP structural stabilization
References and Sources

Stec, B., Holtz, K. M., and Kantrowitz, E. R. A revised mechanism for
Alkaline Phosphatase (2000) J. Mol. Biol. 299,1303–1311

Pace, N., and Shaw, K. Linear Extrapolation Method of Analyzing Solvent
Denaturation Curves (2000). Prot. Str. Function. 4:1-7

Alexander Ninfa, David Ballou, and Marilee Benore. Fundamental
Laboratory Approaches for Biochemistry and Biotechnology. 2nd ed.
Hoboken, NJ: Wiley, 2010. Print.

B. Asgeirsson and K. Guojonsdottir (2005) Biochim. Biophys. Acta 1764, 190
- 198. Reversible Inactivation of Alkaline Phosphatase from Atlantic Cod in
Urea.