Tryptophan-quenching experiments

Acrylamide quenching experiment:

Acrylamide quenching experiments of the protein samples in free and in membrane bound conditions were carried out using a PTI fluorimeter (Photon Technology International, USA). A cuvette with 1 cm path length was used for the fluorescence measurements. For the tryptophan fluorescence quenching experiments, an excitation wavelength of 295 nm was used to eliminate the contributions from tyrosine fluorescence. Fluorescence data were recorded using a step size of 1 nm and an integration time of 1 s. Excitation and emission slits were kept at 5 nm in each case. Emission spectra between 305 nm and 450 nm were recorded in triplicate for each experiment. Typical protein concentration was kept 10 mM for each sample. For performing the experiment, we made different sets of samples in eppendorf tubes for each protein variant and then in each set appropriate volume of protein and premade DPPC liposome solution were mixed to reach final protein: liposome, 1:200. Next, samples were incubated at room temperature for 1 hr. After that, increasing amount of acrylamide quencher (10 M stock) was added to each eppendorf tubes to reach a certain quencher concentration and volume makeup was done by the addition of reaction buffer (20   mM sodium phosphate, pH7.4) to maintain protein lipid ratio constant for each sample. All the samples were then incubated at room temperature for 10 mins and subsequently tryptophan fluorescence was measured. Each set of samples was prepared as triplicate. Necessary background corrections and inner filter effect corrections were made for each experiment. 

Acrylamide-quenching data analysis:

Assuming I and I₀ are the tryptophan fluorescence intensity of the proteins in the presence and absence of acrylamide concentration [Q], the Stern–Volmer equation (Lakowicz, 1999) can be represented as follows: 

Where, k_sv is the Stern–Volmer constant, which can be determined from the slope of the linear plot of I₀/I versus acrylamide concentrations [Q].