Download Optical Spectroscopy Lab Report: Fluorescence Quenching in Physics 598OS (Fall 06) and more Lab Reports Physics in PDF only on Docsity! Physics 598OS Optical Spectroscopy (Fall 06) Clegg/Chao/Liu - 1 - Lab 7: Fluorescence Quenching - Questions Experiment I - Ionic Quenching of Fluorescein by Iodide The data consists of the 30 intensity values that you recorded for the 5 sets of 6 samples. You will prepare a Stern-Volmer plot for each of the 5 sets of data. That is, a plot I0/I vs. [Q]. (Q1) The first three data sets (ICA, ICB, ICC) correspond to low concentrations of KI. For each Stern-Volmer plot, perform a linear fit with the intercept set to 1.0. Turn in a graph of these three data sets and include the values of Ksv in the plot. Why does Ksv increase with increasing ionic strength? Because of the small steps and overall low iodide concentration for these set of samples, we wind up expecting less than 3% changes in intensity per step by samples 4 and 5. As such, we measured intensities with some rather large error bars. Samples ICB2 and ICC2 were consistent outliers, so you can exclude them from your fit. Try to get the best fits you can for your Stern- Volmer constants. Note there is a physical reason that the ICB and ICC data are close to overlapping – this is related to the dependence of Ksv on ionic strength. You should understand the expected dependence – we do see this with our data, albeit with large error bars. If you don’t know what to expect, talk to me! (Q2) The KI data set should not be linear. Why does the data show an upward curvature? The ICH samples (this was on p5 of the experiment instruction handout and misprinted as ICC in the table) should be similar to the KI data set, but be linear. Why should this be true? Turn in a graph showing these two data sets. Note that your data sets should be quite good for this part (the concentrations and steps are much larger). Physics 598OS Optical Spectroscopy (Fall 06) Clegg/Chao/Liu - 2 - Experiment II - Quenching of Tryptophan Fluorescence in Lysozyme by Acrylamide – Fractional Accessibility to Quenchers Recall that for the second part of the lab, we wish to explore one way to use fluorescence quenching to study protein structure. Lysozyme is a protein found e.g., in secretions such as tears, as well as in egg white. It contains 6 residues of the fluorescent amino acid tryptophan 4 are exposed and 2 are buried inside the protein. • For a good reference, see Eftink and Ghiron, “Exposure of Tryptophanyl Residues in Proteins. Quantitative Determination by Fluorescence Quenching Studies”, Biochemistry 15 (1976) 672. • You can check out the lysozyme structure at http://www.rcsb.org/pdb/explore.do?structureId=2HS9 Here you can use the various display options such as WebMol or JMol to highlight the tryptophan residues and see which are buried and which are exposed. Here is a three dimension plot of the protein structure of chicken egg white lysozyme. The whole protein has been drawn using a van der Waals surface. Each tryptophan residue has then been drawn with the bonds drawing method using an exaggerated radius. The yellow and blue tryptophan residues are buried while the other 4 are exposed. 3d structure of chicken egg-white lysozyme showing the tryptophan residues. This structure has been rendered using VMD [Humphrey, W., Dalke, A. and Schulten, K., "VMD - Visual Molecular Dynamics", J. Molec. Graphics, 1996, vol. 14, pp. 33-38. http://www.ks.uiuc.edu/Research/vmd/] The PDB file used is 2HS9 [Von Dreele, R.B. Multipattern Rietveld refinement with protein powder data: an approach to higher resolution To be Published, http://www.rcsb.org/pdb/explore.do?structureId=2HS9]
