PROTEIN BINDING1 Introduction & Background A fundamental chemical process underlying many biochemical systems is the specific binding of a ligand (L) with a receptor molecule (R). For example, insulin binds to the insulin receptor or oxygen binds to hemoglobin. In the simplest and very common case, this is a bimolecular reaction between ligand and receptor. Since most natural ligands and many drugs bind reversibly, there is a unimolecular reaction for dissociation of the ligandreceptor complex (LR). The two reactions can be written: k1 L+R LR k -1 where k1 is the second order rate constant for association and k -1 is the first order rate constant for dissociation. The condition at equilibrium is expressed by the equilibrium association constant Ka or its reciprocal, the equilibrium dissociation constant, Kd. Ka = k1 k -1 Kd = [LR] [L][R] = 1 Ka This describes a saturable system since [LR] can never attain a concentration higher than the initial concentration of receptor. Thus as the concentration of ligand is increased, the concentration of ligand-receptor complex also increases but asymptotically approaches a maximum concentration. For further analysis, it is useful to define a variable B which represents the concentration of ligand bound to the receptor, F which represents the concentration of unbound ligand or "free" ligand, and Bmax which represents the greatest attainable concentration of bound ligand: B = [LR] F = [L] Bmax = [R] + [LR] Combining equations, you can obtain the following: B K aF = = Bmax 1 + K aF 1 1 + 1 K aF = 1 1 + Kd F The equation after the first equal sign is a commonly-used form; the equations after the second and third equal signs introduce each variable only once. This may help clarify mathematical properties of these equations. Plotting B/Bmax vs. concentration of free ligand gives a hyperbolic curve. In many experiments (including the one you will do today), the free ligand is assumed to be the concentration of ligand added to the test tube. This is only true if the fraction bound to the receptor is relatively small. 1 Modified from: Anderson, A. J. A simple competitive protein-binding experiment. J. Chem. Ed. (1994) 71:994995. Algebraic transformations can yield variables that graph a straight line. The most common linear transformation for binding data is the Scatchard Equation: B = K aBmax - K aB F Bmax/Kd 1 Slope = -1/Kd B/F Bmax 2 F Scatchard Plot Although these plots are convenient for quickly evaluating, comparing, and illustrating binding data, they can trap the unwary. A few points on a Scatchard plot may suggest a nice straight line--not completely straight due to “experimental error”--when in fact the data is not good at all as seen from the hyperbolic curve. For this reason, you should ALWAYS plot both graphs. During today's experiment, you will determine the KD and Ka for both HABA prontosil (the molecules made last week) binding to bovine serum albumin (BSA). You will then investigate qualitatively aspirin as a possible inhibitor which may compete for the HABA binding site on serum albumin. Procedure I. Determine the KD and KA for HABA binding to serum albumin using your synthetic HABA. A. Make a HABA solution from your synthetic material: Dissolve 0.036 g of synthetic HABA in 50 mL of deionized water containing 0.5 mL of 3.0 M NaOH. Swirl for a few minutes to get all of the solids dissolved. Then measure out 10.0 mL of this HABA solution and dilute with 115 mL of diluent. Use this solution for your binding analysis described below. In order to determine the KD and Ka, you will measure the binding of HABA to serum albumin using several concentrations of HABA. Complete the following table (total volume = 5.2 mL). Note: HABA solution: 0.45 mM HABA and 0.3% Brij 35 detergent. Diluent: 5.6 mM phosphate buffer, pH 6.3 and 0.3% Brij 35 detergent. BSA = 60 mg/mL. Reagent 1 2 3 4 5 Working HABA soln 0.5 mL 1.0 mL 2.0 mL 4.0 mL 5.0 mL BSA solution 0.2 mL 0.2 mL 0.2 mL 0.2 mL 0.2 mL Diluent B. Prepare a reagent blank which would include HABA but no BSA for each experiment. C. Measure the A485 (absorbance at 485 nm) for each concentration of HABA. II. Repeat the above assay with the standard solutions of HABA and Prontosil. You will test a possible inhibitor: Acetylsalicylic acid (aspirin). The test concentration will be 3.2 mM. Use 4.0 mL of standard HABA, 0.2 mL of BSA and 1.0 mL of acetylsalicylic acid solution. Measure the A485 for the solution with the inhibitor present. Data Analysis Below are the data manipulations that should be done in your Results Section and/or topics that should be discussed in your Discussion Section. Be sure to read and understand the Beer’s law overview provided in the laboratory manual. 1. Determine the Bmax, KD and Ka of HABA binding to serum albumin, both for your synthetic material and the HABA standard. Assume a 1:1 binding of HABA to BSA. Does your synthetic HABA show the same characteristics as the standard HABA? a. Plot Absorbance vs [HABA] calculated molarity. Determine Bmax (with units of Absorbance), KD and Ka. b. Calculate an extinction coefficient ( in Beer's Law) of HABA-BSA complex using your Bmax data. You know the absorbance at Bmax; you know the concentration of BSA, all of which is bound at B max; you know the length of your cuvette, and thus you should be able to determine the extinction coefficient. The units of will reflect the fact that you have to give your HABA-BSA concentration in mg/mL. c. Calculate the concentration of HABA-BSA (bound complex) from your Absorbance data, using the value that you determined in part 1b. Repeat the calculation for each of the different HABA concentrations d. Plot the Scatchard Plot using the concentration of bound HABA (determined in part c). Remember to use the HABA concentration as the concentration of free ligand. Determine B max, KD and Ka using the Scatchard analysis. 2. Compare the constants that you determined in part 1a vs part 1d. Are the units the same? Which set of constants are more reliable? WHY? 3. Repeat the above analysis for Bmax and KD for the standard prontosil solution. 4. Is aspirin an inhibitor of HABA-BSA binding? Compare the shapes of the two molecules. Does your data make sense?