A. Review the Process of Fluorescence
B. Effect of Solvent Polarity on ANS Fluorescence
C. Binding of ANS to BSA
D. Scatchard Plot and Calculation of Kd and n (number of ANS molecules/BSA)
Part A. Review the Process of Fluorescence
In this experiment we will probe hydrophobic binding sites on molecular surface of BSA using the fluorescent probe 1-Anilino-8-naphthalene sulfonate (ANS). ANS binds to hydrophobic pockets in enzymes. ANS is virtually nonfluorescent in aqueous solution but is highly fluorescent in nonpolar solvents. The binding of the probe to hydrophobic sites can be monitored by an increase in fluorescence intensity.
- Review reading on fluorescence spectroscopy from class.
- Research ANS and record a few common applications of this fluorescent probe.
Part B. Effect of Solvent Polarity on ANS Fluorescence
Prepare or obtain 1 mM ANS stock solution in ultrapure, filtered water. Prepare 4-6 solutions, 5 mL each, of varying concentration of ethanol in water. For example 0, 25, 50, 75, 90, 100%.
Record the emission spectrum of ANS (20 μM) in 100% ethanol using an excitation wavelength of 370nm. You will need to determine the optimum parameters for the instrument and emission range and the appropriate blank solution. Determine the wavelength of maximum emission.
Operating Instructions for BioTek Fluoresecnce plate reader
Repeat for the emission spectrum of ANS in the other solvent mixtures using the same parameters as determined in above.
Create an overlay of the various spectra to include in your notebook. Discuss the effects of solvent polarity on the emission spectrum of ANS. Does the quantum yield change? Wavelength of maximum emission?
Part C. Binding of ANS to BSA
Prepare or obtain 100 mM potassium phosphate buffer, pH 7.4.
You will measure the binding of ANS to BSA by monitoring the change fluorescence intensity as increasing amounts of ANS are added to a fixed concentration of BSA.
This can be done by one of two methods.
1) Direct titration of ANS into a solution of 10 uM BSA.
2) Prepare several tubes containing 10 uM BSA in buffer and increasing concentrations of ANS.
For procedure 2, prepare several tubes of 10 μM BSA in buffer and increasing concentrations of ANS ranging from 0 to 100 μM. It is important for the upcoming calculations that you have several points in the 0-10 μM range. Run a total of 12-15 samples in the complete range of ANS concentrations. Measure the fluorescence intensity using and excitation wavelength and emission wavelength determined previously. Plot fluorescence intensity (F) versus [ANS] in μM.
Part D. Scatchard Analysis.
We will follow the procedure shown in Möller and Denicola. Study of protein‐ligand binding by fluorescence. BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION (2002) vol. 30 (5) pp. 309-312