Eddie Khav, Jessica Li, Jonathan Wan & Victor Arias Bioc 463a Evaluation of denaturation of alkaline phosphatase by means of fluorescence spectroscopy Eddie Khav, Jessica Li, Jonathan Wan & Victor Arias Bioc 463a

Objectives of Experiment Denature alkaline phosphatase (AP) using urea and determine the effects of urea denaturation on AP fluorescence. Urea: powerful denaturant that disrupts noncovalent bonds Denature AP using urea and tris(2-carboxyethyl)phosphine (TCEP) and determine the effects of urea denaturation on AP fluorescence. TCEP: reducing agent that breaks disulfide bonds in proteins

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 after the excited electrons return to their ground state.

Fluoremeter Reads the fluorescence of the compound Set parameters that adjust fluorescence intensity: slit width, Read spectrum from 303 to 400nm

Fluorescence of AP Fluorescence occurs in AP due to Tryptophan and Tyrosine. The fluorescence of Tryptophan is much larger and broader than the fluorescence of tryosine Fluorescence at 295nm was observed because tyrosine doesn’t fluorescence at the wavelength

Methods Control measurements: Free Trp, 8M urea, Free Trp in 8M urea Different concentrations of urea were made with a maximum concentration of 8M and a minimum concentration of 0M Stock: 8M urea AP (14mM) was denatured with the different concentrations of urea at room temperature (~25ᵒC) for ~48 hrs.

Methods Cont. Fluorescence was determined for the denatured AP at each concentration of urea using the fluorimeter

Expectations According to literature, the denaturing of AP would cause a decrease in the magnitude of fluorescence of tryptophan residues consistent with the concentration of urea. Additionally, cause a red shift on max fluorescence.

Expectations Cont. Pace, C.N. (1986)

Results

RESULTS Cont.

Methods Cont. The same amount of TCEP (70mM) was added to each concentration of urea AP (10mM this time) was reduced by TCEP and denatured by urea for ~24 hours. The fluorescence of these samples were also determined in the same manner as before.

Expectations The reduction and subsequent denaturing by TCEP was applied in order to determine the effects of TCEP in the fluorescence of AP. Due to the high denaturing environment, it was expected that the measured fluorescence will decrease.

Results Λ=317 nm Higher level of [Urea] increases k of Typ and Tyr, binding constant for binding of Urea. [Urea] =2M, ktrp=0.14, and ktyr=0.25; [Urea] =8M, ktrp=0.16 and ktyr=0.33. In protein, both the wavelength and the extent of absorption depends on the Amino Acid present and on their physical environment. **Less Tyr binded to protein means fluorescence intensity is greatest at lower wavelength **More Tyr binded to protein means fluorescence intensity is greatest at higher wavelength **(I actually don’t know if this is true) The aqueous solubility of protein increases with increasing Urea concentration. This explains the ability of theses compound to unfold protein, even though it is unclear how Urea exert their solubilizing effect

Results Cont. Decreasing emission at 317 nm, with increasing [urea]

Results Cont. Increasing emission at 328 nm, with increasing [urea]

Discussion of Results Comparison between oxidized and reduced AP displayed a decreased max fluorescence from 1.2x105 to 8.0x104. The results of the denaturement of reduced AP displayed a switch in the wavelength of max emission. Red shift was evident.

Discussion Cont. However, decrease in fluorescence was not consistent with increased concentration of urea for both oxidized and reduced samples of AP. Therefore, inconsistency caused the inability to proceed to calculate the structural stability of AP.

Suggestions for Future Experiments Limit the time before measurement to only 24 hours. Allow samples to sit at a low temperature ~4oC Modify pH of solution buffer to a pH closer to the denaturation pH.(pH:~8.0) Suggest utilization of more concentrated urea at 10 M to account for self decomposition of urea.

References Pace, C. N. "Determination and Analysis of Urea and Guanidine Hydrochloride Denaturation Curves." Methods in Enzymology 131 (1986). Print