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

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.

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: 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) 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 1+ 𝑒 − Δ𝐺 𝐻 2 𝑂 −𝑚 𝐺𝐷𝑁 𝑅𝑇

Previous Studies Asgeirsson et. al. (2005)

Results Fluorescence Spectrum of Guanidine Denaturation Dilutions prepared 0-6, protein added, fluorescence measured from 300-400 As GDN goes up, the max emission decreases *No red shift*, lambda max all the same

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

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

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

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

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) = 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.