Fluorescence Quenching of Human Serum Albumin by Caffeine
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1 CHEM 411L Instrumental Analysis Laboratory Revision 2.1 Fluorescence Quenching of Human Serum Albumin by Caffeine In this laboratory exercise we will examine the fluorescence of Human Serum Albumin (HSA) and its quenching by Caffeine. In molecular fluorescence spectroscopy an analyte is stimulated by excitation photons and then responds by fluorescing; emitting longer wavelength photons. The emitted photons are detected by a spectrometer, generating a signal that can be analyzed. It would be expected that the response signal should be linearly proportional to the analyte concentration. And this is generally true over a wide range of analyte concentrations. However, due to limitations in the technique, non-linearities do set in at higher analyte concentrations. Additionally, other species present in the analyte's solution matrix can quench the analyte's fluorescence signal. In the present case, HSA's fluorescence signal is quenched by the presence of Caffeine. Both static and dynamic quenching processes are observed for this system. Although quenching diminishes an analyte's fluorescence signal, for the present case, we can use quenching data to determine binding parameters for the interaction of Caffeine with HSA. Luminescence involves emission of photons from excited atoms or molecules. Fluorescence and Phosphorescence, both luminescent processes, involve emission of photons from systems that have been excited by absorption of photons. In molecular Fluorescence Spectroscopy, an analyte molecule first absorbs a photon (excitation, E excite ) that leaves the analyte in an electronically and vibrationally excited state. At this point, the molecule rapidly looses excess vibrational energy by non-radiatively relaxing to the ground vibrational level of the excited electronic state. This occurs because energy is transfered to solvent molecules as the analyte molecule jostles against them. This relaxation process is very efficient and very rapid. Now, the molecule can fluoresce (E relax ). Or, the molecule can undergo a non-radiative transition (Internal Conversion) to the ground state. Molecules undergoing Internal Conversion transit to the Ground State without emitting radiation.
2 P a g e 2 This is an efficient relaxation process when higher vibrational states of the Ground Electronic State overlap with lower vibrational states of the Excited Electronic State. Because of non-radiative relaxation in the electronically excited state, excitation energy is always greater than relaxation energy. E excite > E relax (Eq. 1) Since the energy of photons involved in these transitions is inversely related to the photons' wavelength: E photon = h c / (Eq. 2) (c is the speed of light and h is Planck s constant), the wavelength of an exciting photon is always shorter than that of a photon emitted during relaxation: excite < relax (Eq. 3) Fluorescence spectra can be measured using a Spectrofluorometer. Light from a source is dispersed and an excitation wavelength is selected using a monochrometer. The excitation radiation impinges upon the sample, which then begins to fluoresce. Fluorescent radiation is itself dispersed by a monochrometer and the spectrum is measured using an appropriate detector. In an actual spectrofluorometer, the dispersing element is usually a diffraction grating. In simpler fluorometers, wavelength selection is accomplished using filters.
3 P a g e 3 The Flourescent Intensity (F) of an analyte solution will be proportional to the radiant Power absorbed by the sample (P o -P): Inserting Beer s Law: F = K (P o P) (Eq. 4) P/P o = 10 -bc (Eq. 5) and expanding the exponential term, gives us: F = K P o {2.3bc (2.3bc) 2 /2 -...} (Eq. 6) Provided the sample Absorbance is relatively low, we can truncate the expansion after the first order terms: F = 2.3 K P o bc (Eq. 7) When P o is constant, we see the Fluorescent Intensity is proportional to the Concentration of the analyte: F = K c (Eq. 8) This, then, provides a method for quantifying the amount of analyte in a system based on fluorescence measurements. A few words of caution. If the concentration of the analyte is high enough, higher order expansion terms become important and the relationship between F and c is no longer linear. And, if the concentration becomes very high, the system begins to absorb its own emitted radiation, causing a decrease in fluorescence intensity and as a result severe non-linearities set in. Fluorescence spectroscopy is much more sensitive than corresponding Absorbance spectroscopic techniques. This is because light emitted against a dark background (fluorescence) is much easier to detect than a slight dimming of intensity against a light background (absorbance). However, fluorescence techniques are severely limited by the number of analytes that actually fluoresce. Most systems shed their excitation energy via radiationless pathways. Structurally, molecules that possess unsubstituted aromatic rings or other structurally rigid elements have a propensity for fluorescing. Fused-ring heterocycles also fluoresce nicely. In our case, we will be examining the fluorescence of Human Serum Albumin (HSA). Albumins constitute a family of globular proteins commonly found in blood serum. They are involved in the transport of fatty acids, bind cations and buffer the ph of their solution. HSA is a monomeric 67 kdalton protein consisting of 585 amino acid residues and comprises ~50% of all plasma protein.
4 P a g e 4 Crystal Structure of Human Serum Albumin Protein fluorescence is typically due to the presence of three amino acid residues: Tryptophan, Tyrosine and Phenylalanine. (Note the planarity of these amino acids' side chain rings.) Tryptophan Tyrosine Phenylalanine Typically, fluorescence due to the presence of Tryptophan will dominate any protein's spectrum. Fluorescence Spectra of Tryptophan (Dashed) and 18:1 Tyrosine-Tryptophan Mix (Solid Color) Biophysical Chemistry Cantor and Schimmelt The fluorescence parameters for typical biomolecule constituents are given by Cantor and Schimmel:
5 P a g e 5 As mentioned above, other species in solution can act as quenching agents (Q) and diminish the fluorescence of the fluorescing analyte. In Dynamic Quenching, the Quencher absorbs radiation non-radiatively from the excited analyte (S*) causing a decrease in the fluorescence intensity detected by the instrument's detector. S + h excite S* (excitation) S* + Q S + Q (quenching) It can be shown the ratio of unquneched-to-quenched fluorescent intensities (F o /F) is related to the quenching agent total concentration [Q] Tot via the Stern-Volmer Relation: F o /F = 1 + K [Q] Tot (Eq. 9) where K is the Stern-Volmer constant. K is related to both the fluorescence lifetime of the fluorescer and the rate constant for the quenching process. Note, F o /F is linear in the concentration of the quencher. If a Stern-Volmer plot, a plot of F o /F vs. [Q], is non-linear, a second quenching process is occurring. An upward curvature to the plot is indicative of static quenching. Static quenching occurs when the quencher complexes with the fluorescer before excitation can occur. The complex has unique properties which may include being non-fluorescent. When static quenching occurs, the Stern- Volmer relation must be modified to
6 P a g e 6 include both quenching processes: F o /F = (1 + K D [Q] Tot ) (1 + K S [Q] Tot ) (Eq. 10) K D and K S are the Dynamic and Static Stern-Volmer Constants, respectively. Data concerning static fluorescence quenching can be used to determine the "binding parameters" of the Quencher-Fluorescer complexation. We begin by defining the fraction of the fluorescence that is quenched in terms of measurable quantities: q = (F o - F) / F o (Eq. 11) This is related to the fraction of the fluorescers that are quenched: q = (Eq. 12) where QS represents the quencher-fluorescer complex and [QS] is its concentration. [S] Tot is the concentration of fluorescer before complexation occurs. The dissociation equilibrium for the QS complex is written as: QS Q + S If [S] is the concentration of unbound fluorescer, then it is true that: So, we can write: [S] Tot = [QS] + [S] (Eq. 13) q = (Eq. 14) In terms of the QS Dissociation Constant K d : K d = (Eq. 15) we have: [QS] = (Eq. 16) and thus can write: q = = (Eq. 17)
7 P a g e 7 Rearranging this equation gives us a relationship between q/[q] and q that is known as the Scatchard Equation: (Eq. 18) If the fluorescer can independently bind n quenchers, then the Scatchard Equation is modified to give: (Eq. 19) Thus, a plot of q/[q] vs. q, a Scatchard Plot, should yield a straight line with a slope of 1/K d and an intercept of n/k d. If the fluorescer has multiple and distinct types of binding sites for Q, then the Scatchard analysis becomes significantly more complex. Two types of binding sites will yield a Biphasic Scatchard Plot. A discussion of this case is beyond the scope of the current project. Thus, an analysis of quenching data can yield information about binding of the quencher to a fluorescer. In this lab we will quantify the quenching of the fluorescence of HSA by Caffeine.
8 P a g e 8 We will then show both dynamic and static quenching processes are occurring. Finally we will perform a Scatchard analysis of the quenching data. This analysis will yield the number of binding sites on the HSA macromolecule for the Caffeine ligand as well as the Caffeine-HSA dissociation constant. All fluorescence measurements will be made using the Photon Technology International system pictured below. You should examine this Spectrofluorometer and identify all the major components that comprise the system.
9 P a g e 9 Procedure Data for HSA Molecular Weight = g/mol Extinction Coefficient = M -1 cm -1 at 278 nm Week 1 1. Your laboratory instructor will provide you with a Phosphate buffer (ph = 7.20) with which to solubilize the powdered HSA. Prepare ~10 ml of HSA solution at about a concentration of 1mg/mL using this buffer. (This solution may need to be diluted 1:2 in order to obtain a measurable absorbance and emission signal. You will need to make a few measurements before deciding whether or not this dilution needs to be performed.) 2. Now obtain an absorbance spectrum for your HSA solution using the Department's UV- VIS spectrometer Your spectrum should cover the range of nm. Be sure to use matched Quartz cuvettes as Glass cuvettes absorb in the UV. (Be very careful with these cuvettes as Quartz shatters easily when the cuvette is jarred against something.) Determine the excitation max for HSA from the absorbance spectrum. Use the measured asorbance at 278 nm to determine the concentration of the HSA solution using Beer's Law; A 278 = 278 bc. 3. Obtain a fluorescence spectrum for your HAS solution using the Department's Photon Technology International spectrometer. You must use a Quartz fluorescence cuvette for this spectrum. (Same caution as above.) Use your excitation max as a guide in determining the wavelength range for the fluorescence spectrum. Determine the emission max for HSA from the emission spectrum. 4. Store your remaining HSA solution in a refrigerator for use during week 2 of the lab exercise. 4. Develop a plan for completing the experiment's of week 2 exercises. Week 2 Week 2 of this experiment will be run as an "Open" lab. The experiment will take about 1 hour to complete, if you are prepared when coming to the laboratory. Your laboratory instructor will have a sign-up sheet for available time slots. 1. Determine the range over which the fluorescence of the HSA solution is linear. To do this, perform serial dilutions of the stock HSA solution and measure the fluorescence F at each
10 P a g e 10 dilution. Dilutions should be performed using the Phosphate buffer. Keep in mind you only need 3mL of solution to fill the spectrometer cell. 2. Measure the quenching of the HSA fluorescence by Caffeine. Do this by first preparing stock solutions of Caffeine (~5.8 x 10-3 M) and HSA (~1.5 x 10-5 M). The HSA stock solution should be prepared using the remaining HSA solution from week 1. Then use variable volumes of the Caffeine solution with a constant volume of the HSA solution to prepare the series of Caffeine-HAS solutions needed for the Scatchard analysis. Bring all the solutions to the same volume by adding additional phosphate buffer. Keep in mind you only need 3mL of solution to fill a spectrometer cell. Also, you need to make a measurement of the HSA absent any Caffeine to determine F o.
11 P a g e 11 Data Analysis You will prepare a full laboratory report for this exercise. Your report should include the following: Both absorbance and fluorescence spectra for HSA. A plot of F vs. [HAS] and a discussion of the range of the linearity and causes for observed non-linearities. A Stern-Volmer plot: F o /F vs. [Caff] Tot. It should also include a discussion of the quenching processes which occur when Caffeine quenches HSA fluorescence. A plot of K app vs. [Caff] Tot, where K app is the Apparent Stern-Volmer Constant. This is given by K app = (K D + K S ) + K D K S [Caff] Tot = (F o /F - 1)/[Caff] Tot. Comment on your results and why K app is defined in this manner. A plot of q vs. [Caff] Tot / [HSA] Tot. Comment on your results. A Scatchard Plot; q/[caff] vs. q. Comment on your results. Note: [Caff] represents the free concentration of the quencher [Q]. This can be determined from measureable quantities in general via, [Q] Tot = [Q] + [QS] or [Q] = [Q] Tot - [QS] which by (eq. 12) yields, So, [Q] = [Q] Tot - q [S] Tot [Caff] = [Caff] Tot - q [HAS] Tot Your determination of K d and n for HAS-Caffeine complexation. Error estimates should be included. Comment on your results. Further, comment on the legitimacy of using a Linear Least Squares Analysis to determine these parameters.
12 P a g e 12 References Cantor, Charles R. and Schimmel, Paul R. "Biophysical Chemistry, Part II: Techniques for the Study of Biological Structure and Function" W.H. Freeman and Company, San Francisco. Montero, Maria Teresa; Hernandez, Jordi and Estelrich, Joan "Fluorescence Quenching of Albumin. A spectrofluorimetric experiment" Biochemical Education 18 (1990) 99. Skoog, Douglas A., Holler, F. James and Crouch, Stanley R. (2007) " Principles of Instrumental Analysis, 6 th Ed." Thomson, Belmont, California.
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