1. Guanidinium Denaturation of Alkaline Phosphatase
Spencer Fosnot, Erick Karlsrud, Marvin O’Ketch, Gavin Young
University of Arizona, Biochemistry 463a

2. Fluorescence Spectroscopy

3. Amino Acid Fluorophores

4. Important Reagents Chemical denaturant
Guanidine Hydrochloride
Tris(2-carboxyethyl)phosphine
(TCEP)
Chemical denaturant
Selectively reduces disulfide linkages

5. Alkaline Phosphatase Hydrolysis of phosphate esters Two active sites
Homodimer
2 intramolecular disulfides per monomer
2 active sites
Magnesium and 2 Zinc ions
Hydrophobic and Hydrogen interactions between monomers.

6. AP Active Site Detail

7. 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.

8. Materials and Methods Guanidinium hydrochloride:
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

9. Previous Studies Pace et. al. (2000)
Delta g of solvation
We also did Urea, but didn’t work
Curve structure very similar
This is equation (six parameters)
𝑦= 𝑦 𝐹 + 𝑚 𝐹 𝐺𝐷𝑁 + 𝑦 𝑈 + 𝑚 𝑈 𝐺𝐷𝑁 𝑒 − Δ𝐺 𝐻 2 𝑂 −𝑚 𝐺𝐷𝑁 𝑅𝑇 𝑒 − Δ𝐺 𝐻 2 𝑂 −𝑚 𝐺𝐷𝑁 𝑅𝑇

10. Previous Studies
Asgeirsson et. al. (2005)

11. Results Fluorescence Spectrum of Guanidine Denaturation
Dilutions prepared 0-6, protein added, fluorescence measured from
As GDN goes up, the max emission decreases
*No red shift*, lambda max all the same

12. Results Guanidine Denaturation Curve
Plot each max against its corresponding conc
Normalized
Logistic-type curve, eqn later (too many parameters to show)
Follows a typical denaturation curve

13. Results Fluorescence Spectrum in the Presence of TCEP
Repeat same experiment, but tcep added

14. Results Guanidine Denaturation Curve in the Presence of TCEP
Note slopes at ends of graph -> GDN initially stabilizes prot
Urea-H-bond backbone
GDN-planar molecules “stack”
GDN has been shown to stack on top of hydrophobic residues -> increased packing -> increase in fluorecence

15. Results TCEP’s Influence Over AP Denaturation Two main differences:
-critical point shifts left
-delta g (h2o) drops
Increase in delta g (“is an estimate of the conformational stability of a protein that assumes that the linear dependence continues to 0 M denaturant”) NOT delta g (unfolding rxn)
The higher delta g(h2o), the stable the protein and more resistant to unfolding -> TCEP decreases stability
Keep in mind this is for 25 C

16. Results Summary λmax at 319 nm
GDN
GDN + TCEP
λmax at 319 nm
Maximum fluorescence increased up until 3M
Decreased sharply until 5M
ΔG(H2O) = 10.5kcal•mol-1
λmax at 319 nm
Maximum fluorescence increased up until 1.5M
Decreased sharply until 4M
ΔG(H2O) = kcal•mol-1

17. 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

18. 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, Print.
B. Asgeirsson and K. Guojonsdottir (2005) Biochim. Biophys. Acta 1764, Reversible Inactivation of Alkaline Phosphatase from Atlantic Cod in Urea.