A Simple Competitive Protein-Binding Experiment Alexander J. Anderson School of Life Science, Queensiand University of Technology, GPO Box 2434,Brisbane 4001 Australia The binding of ligands to proteins is exploited widely in modern biochemistry, both i n research and i n applied areas such a s chemical pathology One form in which proteinllgand binding is used commonly is in comprtitiv(~prut(:inbinding assays, such a s radioimmunoassay and other types of immunoassays. Competitive protein-binding assays are used for measurement of a wide range of hormones, drugs, and other antigens ( I ) and for study of the binding of ligands to cell surface receptors (2). However, practical assays of this type are usually too expensive, technically demanding and potentially hazardous for routine use by inexperienced students i n undergraduate teaching laboratories, due to the expense of the antibodies and the labelled ligands, the requirement for specialized equipment, the need to separate the hound and free forms of the labelled ligand prior to measurement, and the handling and disposal of radioactive isotopes. This experiment was developed for teaching the concept of competitive protein-binding assays, but with the use of inexpensive reagents and commonly available laboratory equipment in place of specialized immunoassay reagents and radiation counting equipment. The experiment is based on the known interference by salicylate in the binding of 2-(4'-bydroxyazobenzene)benzoic acid (HABA) to serum albumin (3). In a competitive protein-binding assay the concentration of a binding protein and a labelled ligand is kept constant, and the amount of labelled ligand bound to the protein decreases with the increasing concentration of a n unlabelled ligand. In the uresent svstem. constant concentrations of HABA and serum alhumln are m a ~ n l a ~ n e and d , the concentrat~onof .;;ll~c\.l;lte1s vaned over a set ranee. The HA13Acan thus hc rega;ded a s the labelled ligand,and the salicylate a s the unlabelled ligand, and the experiment is actually a n assay of salicylate concentrations. The bound HABAis measured conveniently by the absorbance of the albumin-HABAcomplex a t 485 nm, where the difference i n absorbance between the free and bound forms of HABA is maximal. This avoids the necessity of separating the bound HABA from the free HABAprior to measuring the concentration of the bound HABA.
lutions (0.33.0mmol/L) can be prepared by suitable dilution of the standard. /+xedure By 1:2 serial dilution of the 6.4mmol/L salicylate standard with the diluent, prepare additional standards of 3.2, 1.6,0.8,0.4, 0.2,and 0.1 mmol/L. Prepare a reagent blank from 4.0 mL of working HABA reagent and 1.2mL of the diluent. The zero reaction (Bo, the amount of HABA bound i n t h e absence of salicylate) contains 4.0 mL working HABA reagent, 0.2mL of the BSA solution and 1.0mL of the diluent. Standard and unknown reactions contain 4.0 mL working HABA reagent, 0.2 mL of the BSA solution and 1.0mL of the appropriate standard (0.1to 6.4mmol/L) or the unknown salicylate solution. Use the reagent blank to set zero absorbance on the spectrophotometer a t 485 nm. Read the absorbances of all other tubes and calculate %B& for each standard and unknown tube, by expressing the absorbance of each standard and unknown reaction a s a percentage of the absorbance of the zero reaction (Bo).From the standard reaction values, prepare a plot of %B& versus the logarithm of the salicylate concentration. This calibration plot (see figure) is then used to interpolate the concentration of the unknown salicylate solutions. Results and Discussion This experiment is used in teaching second-year undergraduate students i n this university and performs very reliably. I t can be completed easily within one hour, and the only technical competence required is the use of pipets. The figure shows an actual calibration curve obtained by a student during a normal laboratory class, and this is typical of the results obtained by all students. Deviations from
Experimental Details Reagents The stock buffer (75mmoVL phosphate pH 6.3)i s prepared by dissolving 2.35g of KzHPO*and 8.37gof KHzP04 i n 1 L of HzO. A stock solution of HABA (nominally 6 mmol/L) is prepared by stirring 0.145 g of HABAand 1 mL of 1 molL NaOH i n 25 mL of water for 60 min, filtering if necessary and diluting the filtrate to 100 mL. Care should be taken i n handling the 1 mol/L NaOHsolution. The working HABAreagent consists of 75 mL stock HABA, 925 mL of stock buffer and 3 mL Brij 35 detergent, while the diluent is prepared from 75 mL H20, 925 mL of stock buffe r and 3 mL Brij 35 detergent. The actual concentration of HABA i s not critical and need not be exactlv known. The I~inrlingprotcw is a solut~onof bovine serum albumin IUSA ill wdtcr at 60 m-r ml... and the sodium s;llicyl;itt~ standard (6.4-o~/L) consists of 0.512 sodium salic$late dissolved in 500 mL of the diluent. Salicylate unknown so-
994
Journal of Chemical Education
log [salicylate] (mol/L) Cal oraton plo~ofooBB, versustne logarltnm of me concentraton of salcy ate, for lhe assay of salfcylaleconcenlrallons oy compettve protein binding to bovine serum albumin with P(C-hydroxyazobenZene)benzOicacid as the ligand.
the expected results, for both the calibration curve and the values for the concentration of the unknown salicylate solution, can almost always be traced to technical errors on the part of the students. Though the concentration of HABAin the reagent is not critical (the absorbance of the zero reaction (Bo) is typically 0.5 to 0.7 with different batches of the HABAreageut), the concentrations of the salicylate standards and unknowns give best results if they are maintained within the range quoted above. I t is also unnecessary to use purified BSA. The experiment works well with bovine serum i n place of the BSA solution, but the absorbance values are somewhat lower. This can be corrected by increasing the volume of protein solution to 0.3 mL. One advantage of this experiment is that the students need no advanced techniques or equipment, nor special laboratory facilities such a s are necessary for radioactive isotope handling and disposal. Another advantage i s that there is no need to separate the bound and free forms of the labelled ligand (here represented by HABA), because only
the bound form is measured by reading the absorbance a t 485 nm. Separation of the bound and free forms of the labelled ligand is always necessary in radioimmunoassays and related methods ( I )and can contribute technical difflculties for inexperienced students. This experiment can be extended if desired to measure the binding constant of salicylate to BSA. If the binding constant of HABA to BSA is known (41, then the binding constant for saliwlate can be evaluated from [Ilso,the conthat causes a 50'; wduction in the cmtration of x;~licyl;~te binding of 11.-A to the protein 15 Literature Cited 1. Markowit%,H.; Jiang, N. S. InFundamnlolsofClrnicol Chemistry:Tern,N.W, Ed.: Saunders: F'hiladelphia. 1976: pp 286290. 2. Lewt8ki.A. Science 1988.241.800-808. 3. C.; Olitzky I.;inkp&, J.A. In Clinical Chmisfry P~lnclpl~sondTPehnicr: Henly, RJ.;Cannon,D. C.; Winkelman. J. A., Eds; Hatper and Row: Hagerstown. M" , 4 7 6.,"din ...-, 4. Practical methods for d a m i n a t i o n of protein-ligand binding eonstan* are discussed by Metnler D. E. Biochzmiatry;Academic Press: New York, 1977; pp 187-
cannon:^. 3 QO
5. Voet .D.; Voet, J. G.Bzahambiv; Wiley: New York. 1990;p 1153. A"".
Volume 71 Number 11 November 1994
995
