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Observation of “Hook Effects” in the Inhibition and Dose-Response Curves of Biotin Assays Based on the Interaction of Biotinylated Glucose Oxidase with (Strept)avidin Minas S. Barbarakis; Waleed G. Qaisi, Sylvia Daunert, and Leonidas G. Bachas’ Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055
The lnhibltlon of hlghly blotinylated enzymes by avidin and streptavldhhasbeonusedInthe devebpmentd homgmeow assays for blotln and other analytes. Usually, thk Inhibition occurs in a similar fashlon for both avidin and streptavidin. Specl?lcaily, the curves that relate the inhlbitlon of the enzymatic actMty with the concentratbn of avldin or struptavUn have a signoldelshape; Le., the InhiMlond the enzymebiotin conjugates Increasesgradually with increadngamounts of avldin of streptavldlhand arrlves at a plateauat hlgh Mndlng protein concentrations. However, when these two blotlnap8clflc binding proteins Interact with biotinyiated glucose oxidase a rlgnlficant dMerence In their inhlbltory action k observed. I n particular, the lnMbitkncurves have a sigmoidal shapefwstreptavldln,whMe~foravldkrexMbnamlulmm (“hook”)at low avidln concentratlons. Thk dMerenco In the reactivky d the two protdns with blotinyiated enzyme8 influences both the shape d the do$e-rosponse curve and thedetectlonlbntbd homogeneowenrymallnkedcompeWUve Mnding assays for biotin.
INTRODUCTION Binding w a y s experience wide applicability in biochemical analysis because of their ability to quantitatively determine trace levels of important physiological substances (e.g., drugs, hormones, proteins, etc.) in biological fluids.’ In general,these assays are based on the specific interaction between a biological binder and ita respective ligand (e.g., interactions between antibody and antigen, binding protein and ligand, receptor and hormone, etcJ.1 In the majority of assays, a doseresponse curve is obtained where the measured signal (fromeither alabeled binder or a labeled ligand) is amonotonic function of the concentration of the ligand. However, in several instances,a maximum in the dose-response curve has been reported, which is known with the desckiptiveterm ‘hook effect”.2 This characteristic shape of the dose-response curve has been observed at low ligand concentration and under certain conditions in a number of competitive binding assays,2+ and it has been attributed either to positive cooperativity between ligand binding sites on the same bindert.6 or to the formation of stable binder-ligand circular + Present address: Ciba Corning Diagnostics Corp., 333 Coney St., East Walpole, MA 02032. (1) Goaling, J. P. Clin. Chem. 1990,36, 1408-1427. (2) Feldkamp, C. S.;Smith,S. W. Inlmmunoassay; APractical Guide; Chan, D. W., Ed.;Academic Press: New York, 1987; pp 49-95. (3) Mataukura, S.; West, C. D.; Ichikawa, Y.; Jubiz, W.; Harada, G.; Tyler, F. H. J. Lab. Clin. Med. 1971, 77, 490-500. (4) Matauyama, H.; Ruhmann-Wennhold, A.; Nelson, D. H. Endocrinology 1971,88, 692-695. (5) Weintraub, B. D:; Rosen, J. A,; McCammon, R. L.; Perlman, R. L. Endocrinology 1973, 92, 1250-1255. (6) Bachas, L. G.; Lewis, P. F.; Meyerhoff, M. E. Anal. Chem. 1984, 56,1723-1726.
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complexes.7.8 In addition, hook effects have been observed at high ligand concentrations in immunometric and nephelometric assays.279JO In homogeneousenzyme-linked competitivebinding assays of the EMIT type, the activity of an enzyme-labeled ligand (conjugate) is inhibited upon binding to a ligand-specific binder (e.g., antibody, binding protein, lectin, etc.).” The inhibition of the enzymaticactivity (whichis a result of either steric exclusion of the substrate from the active site of the enzyme or a conformational change of the enzyme molecule12J3) can be modulated by a competition between the unlabeled ligand and the enzyme conjugate for the binding sites of the binder.14915 Conventional inhibition curves generated by plotting the percent inhibition of the enzymatic activity vs the concentration of binder have a sigmoidal shape; i.e., the inhibition of the enzyme-ligand conjugate increases gradually with increasing amounts of binder, arriving at a plateau at high binder concentrations.” In our laboratory, the biotin-specificbinding proteins avidin and streptavidin have been employed as inhibitors of biotinylated enzymes,in an effort to develop homogeneous enzymelinked competitive binding assays for biotin. In all cases, the inhibition of the enzymaticactivity by avidin and streptavidin led to the generation of inhibition curves of sigmoidal shape.”+lg However, it was found that the inhibitory action of avidin on biotinylated glucose oxidase (GOx-biotin) occurs in a biphasicmanner. In particular, upon interaction of avidin with biotinylated GOx, the curves that relate the inhibition of the enzymatic activity with the concentration of avidin exhibit a maximum at low avidin concentrations. This fact led to the construction of dose-response curves that under certain conditions also exhibited a maximum (Le., a “hook”) at low biotin concentrations. Although biphasic curves have been previously observed in homogeneoue assays,8to the best of our knowledge this is the fiist time that a hook effect is observed in an EMIT-type enzyme-linkedcompetitive binding (7) Ehrlich, P. H.; Moyle, W. R. Clin. Chem. 1984, 30, 1523-1532. (8) Skold, C.; Gibbons, I.; Could, D.; Ullman, E. J. Immunol. 1987, 138, 3408-3414. (9) Ryall, R. G.; Story, C. J.; Turner, D. R. Anal. Biochem. 1982.127, 308-315 (10) Haller, B. L.; Fuller, K. A.; Brown, W. S.;Koenig, J. W.; Eveland, B. J.; Scott, M. G. Clin. Chem. 1992,38,437-438. (11) Kabakoff, D. S.;Greenwood,H. M. InRecent Advances in Clinical Biochemistry; Alberti, K. G. M.; Price, C. P., Eds.; Livingston: London, 1981; pp 1-30. (12)Rubenstein, K. E.; Schneider, R. S.; Ullman, E. F. Biochem. Biophys. Res. Commun. 1972,47, 846-851. (13) Rowley, G. L.; Rubenstein, K. E.; Huisjen, J.; Ullman, E. F. J. B i d . Chem. 1975,250,3759-3766. (14) Ngo, T. T.; Lenhoff, H. M. Appl. Biochem. Biotechnol. 1981,6, 53-64. (15) Monroe, D. Anal. Chem. 1984,56,920A-931A. (16) Daunert, S.; Bachas, L. G.; Meyerhoff, M. E. Anal. Chim. Acta 1988,208, 43-52. (17) Kjellstr6m,TT. L., Bachas, L. G. Anal. Chem. 1989,61,1728-1732. (18) Daunert, S.; Payne, B. R.; Bachas, L. G. Anal. Chem. 1989,61, 2160-2164. (19) Barbarakis,M. S.;Daunert, S.; Bachas, L. G. Bioconjugate Chem. 1992,2, 225-229.
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assay. In addition, this article describes ways by which these yunconventional"inhibition curves can lead tosigmoidal doseresponse curves for the determination of low levels of biotin.
EXPERIMENTAL SECTION Reagents. Avidin (eggwhite, lyophilized)was obtained from Calbiochem (La Jolla, CA), and streptavidin, from InFerGene (Benicia, CAI. Glucose oxidase (GOx) (Aspergillus niger), peroxidase (horseradish),N-hydroxysuccinimidobiotin(biotinNHS), &D(+)-glUCOSe, 2,2'-azinobis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS), and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO). Anhydrous methyl sulfoxidewas a Sure/Sealproduct from Aldrich (Milwaukee,WI). The assay buffer was 0.100 M KHpPO4, pH 6.0 (the pH was adjusted with 1.0 M KOH). The dilutions of the enzyme conjugate, avidin,streptavidin,and biotin were prepared in assay buffer containing 0.10% (w/v)BSA. All solutions were prepared using deionized (Milli-QWater Purification System; Millipore, Bedford, MA) distilled water. Preparation of the GOx-Biotin Conjugates. Glucose oxidase was dialyzed in 0.100 M sodium bicarbonate buffer, pH 8.1. Glucose was added to the enzyme solution in a 100-fold excess to protect the active site during conjugation. For each conjugation,500units of enzyme were used and differentaliquots of a biotin-NHS solution (6 mg of biotin-NHS in 0.50 mL of methyl sulfoxide) were added to the reaction mixture. The conjugation reaction was run under magnetic stirring in an ice bath for 4 h. Following exhaustive dialysis against the assay buffer, the conjugates were diluted to 2.00 mL with the assay buffer. The concentration of the modified GOx was estimated from the concentration of flavin adenine dinucleotide (FAD) attached to the enzyme,assuming 2 molof FAD/mol of enzyme.20 The concentration of FAD was calculated by measuring the absorbanceat 452 nm and by using a molar absorptivity for bound FAD of 1.08 X 104 M-' cm-1.21The GOx-biotin conjugates were evaluated with respect to enzymatic activity and percent inhibition obtained. The GOx-biotin conjugate employed in this study was prepared by using an initial enzyme/biotin-NHS mole ratio of 1/200. Enzyme Activity. The activity of biotinylated GOx was determined with a Perkin-Elmer (Lambda 6) UV/vis spectrophotometer (Norwalk, CT) interfaced with an Epson (Equity I+) personal computer (EpsonAmerica,Torrance,CA). Premium grade polystyrene disposable cuvettes (Evergreen, Los Angeles, CA) were used. The substrate solution was prepared by mixing M), 250 pL of 0.10% (w/v) BSA, 1.20 mL of ABTS (1.5 X 500 pL of glucose (0.40 M), and 50 pL of horseradish peroxidase (60units/mL). All the above solutions were prepared in 0.100 M KH2P04, pH 6.0 buffer. A 50-pL aliquot of biotinylated GOx (3.6 X M) was mixed with 250 pL of the 0.10% (w/v) BSA buffer solution. From this mixture, 275 pL was added to the substrate solution before the absorbance was measured at 414 nm every 15 a. The change of the absorbance at 414 nm was a result of the oxidation of the dye ABTS by the generated H202 through the peroxidase-coupled reaction. Inhibition Studies for the GOx-Biotin System. A 250-pL aliquot of variable concentrationsof a biotin-specific binder was M) for 30 min. mixed with 50 pL of biotinylated GOx (3.6X From this mixture 275 pL was added to the substrate solution. Then, the enzymatic activity was determined as described previously. An inhibitioncurve was constructed by plotting the percent inhibition observed vs the moles of binding sites in the assay mixture. The moles of binding sites were determined by titrating each binder with biotin and using 4'-hydroxyazobenzene2-carboxylate (HABA) as the indicator.22 Dose-Response Curves. Dose-response curves were generated by incubating a constant concentration of the biotin-specific binder with varying amounts of biotin in the presence of the GOx-biotin solution. The percent inhibition obtained was (20) Tsuge, H.; Natsuaki, 0.; Ohashi, K. J. Biochem. 1975, 78, 835843. (21) Wysor, M. S.; Zollinhofer, R. E. Enzyme 1973, 14, 185-192. (22) Green, N. M. Biochem. J. 1965,94, 23-24c.
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Flgure 1. Inhlbltlon curves of the GOx-biotln conjugate (3.6 X M) obtained by ushg avldln(.)and streptavldin (0).The concentretkns of GOx-blotIn and of the blnding proteins refer to the incubation step. Error bars lndlcate *1 standard devlatbn (n = 3).
plotted vs the logarithm of the concentration of biotin in the standards.
RESULTS AND DISCUSSION The native biotin-binding proteins avidin and streptavidin have been extensively used in bioanalytical applications such as immunoassays, receptor studies, immunostaining methods, nucleic acid hybridization, affinity-based separations, etc.23-27 All these applications are based on the rapid and strong binding of these binding proteins with biotin or biotinylated compounds. The avidin-biotin system has been effectively employed in heterogeneous and homogeneous enzyme-linked binding assays. In particular, the inhibition of biotinylated enzymes by avidin was usedto develop sensitive homogeneous assays for bi0tin16-~9and other analytes.28 In previous work undertaken in our laboratory, the inhibition of biotinylated adenosine deaminase (ADA119 and glucose-6-phosphate dehydrogenase (G6PDH)Zgwith avidin and streptavidin was investigated. The inhibition curves obtained for these biotinylated enzymes with both avidin and streptavidin had a similar shape.lg~2~Specifically, the percent inhibition of the biotinylated enzymesincreased when the amount of the binding protein was increased and reached a plateau a t high concentrations of binding protein. In addition, by comparing the inhibition curves obtained for ADA-biotin, it was shown that a lower amount of streptavidin compared to avidin was necessary to induce the same degree of inhibition of the biotinylated enzyme. This superiority of streptavidin over avidin was attributed to a higher binding constant of biotin with streptavidin compared to avidin and/ or to the deeper binding pocket of streptavidin.19 In the case of GGPDH-biotin, the inhibition curves for avidin and streptavidin were identical, which was explained by the fact that it is easier to inhibit conjugates of G6PDH than of ADA." In the present study, the inhibition of GOx-biotin by avidin and streptavidin was measured at different binding protein concentrations, while the incubation time and the concentration of the enzyme conjugate were kept constant. Surprisingly,a significant difference in the shape of the inhibition curves for the two binding proteins was observed. Specifically, as it is shown in Figure 1, while the inhibition curve for ~~
~
(23) Wilchek, M.; Bayer, E. A. Anal. Biochem. 1988, 171, 1-32. (24) Bayer, E. A.; Wilchek, M. J. Chromatogr. 1990,510, 3-11. (25) Diamandis, E. P.; Christopoulos, T. K. Clin. Chem. 1991,37,62& 636. (26) Wilchek, M.; Bayer, E. A. Trends Biochem. Sci. 1989, 14, 408412. (27) Wilchek, M.; Bayer, E. A., Eds. Methods in Enzymology;Academic Press: San Diego, CA, 1990; Vol. 184. (28) Bacquet, C.; Twumasi, D. Y. Anal. Biochem. 1984,136,487-490. (29) Daunert, S.; Bachas, L. G. Unpublished work, University of Kentucky, 1992.
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Figure 2. Model of the interaction between GOx-blotln and biotlnspeciflc blndlng proteins.
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streptavidin was similar in shape to the curves obtained for the other two biotinylated enzymea (ADA-biotin and GGPDHbiotin), the curve for avidin exhibited a maximum at low avidin concentrations. Moreover, compared to streptavidin, a lower amount of avidin is necessary to inhibit the enzyme conjugate. In addition, the maximum inhibition caused by avidin is higher than the inhibitionproduced even by an excess of streptavidin. A proposed model for the interaction of GOx-biotin with avidin and streptavidin that may provide an explanation for the observed difference in the shape of the inhibition curves is shown in Figure 2. This model assumes the existence of two types of binding between the binding proteins and the enzyme conjugate. In the first type (I),only one of the binding sites is bound to the enzyme conjugate and linear complexes are formed. In the second type (111, multisite binding results in the formation of a cyclic complexthat hastwo of the binding sites bound simultaneously to two different biotin molecules on the surface of the same enzyme conjugate. Binding types I and I1may also result in the formation of complexesinvolving two binding protein molecules with one enzyme conjugate, three binding protein molecules with two enzyme conjugates, and so forth. It may be postulated, that the two types of binding have a different effect on the inhibition of the enzyme conjugate, considering that the binding protein may inhibit the conjugateto a higher extent in a multisite type of binding (type 11)than in a single-site one (type I). Consequently, it is the relative proportion of the amount of each complex formed that determines the shape of the inhibition curve. It should be noted that formation of type I1 binding has been suggested previously in studies involving the interaction of surface-bound biotin residues with avidin.30 Further, it has been demonstrated that cyclic complexes between bivalent binders, such as intact antibodies, and G6PDH (a dimeric enzyme) do form in which the two antibody binding sites bind to two identical epitopes on the two enzyme subunits.8 To further substantiate the proposed model, the dependence of the reactivation of the inhibited enzyme on the concentration of added biotin was considered. It was found that dose-response curves for biotin generated by using concentrations of avidin binding sites in the range 3 X 10-8 to 3 X M had asigmoidal shape (Figure 3). The detection limit for this “conventional”dose-response curve was found to be 6 X M biotin. When a concentration of avidin binding sites higher than 3 X 10-7M was employed, doseresponse curves of biphasic shape (Le., with a hook) were obtained. In particular, a maximum was observed at low biotin concentration (Figure 3). In contrast, the shape of the dose-response curves for biotin obtained using streptavidin as the binder were sigmoidal (Figure 3), and their shape was independent of the streptavidin/GOx-biotin molar ratio in the assay. (30) Goldstein, L.;Niv, A.; Yankofsky, S. J. Chromatogr. 1990,510, 23-39.
Figure 3. Dose-response cwves for blotlnobtalned by using the aOxM) and 3.1 X 10“ M avidln binding sites blotln conjugate (3.6 X (O),7.4 X lo-’ M avidin binding sltes (e), 1.1 X lo-’ M avidln blndlng sltes (01, and 1.9 X M streptavidin blndlng sites (0). All concentrations refer to the Incubation step. Error bars Indicate fl standard deviation (n = 3).
It should be noted that this difference in reactivity of avidin and streptavidin with biotinylated GOx was observed with several GOx-biotin conjugates prepared by using various enzyme/biotin-NHS molar ratios. The proposed model of this interaction may be discussed further by considering structural differences between the two proteins. While the individual subunits and binding sites of avidin and streptavidin have similar structures, the angular relations between subunits in the tetramer are such as to bring the openings into the pair of binding clefts located on the same side of streptavidin closer together than avidin by ca. 10 A.31 This difference in the spatial arrangement of the binding sites may favor the simultaneous binding of the same GOx-biotin moleculeto neighboringsites of avidin, but not of streptavidin. Knowledgeof the three-dimensionalarrangementof the e-NH2 groups of lysine residues on glucose oxidase (where biotin can be attached) and of the structure of the binding proteins could provide information about the probability of formation of type I1 complexes between avidin (or streptavidin) and GOx-biotin. However, although the three-dimensional structure of streptavidin is already known,32 complete crystallographic data for avidin are not available and the threedimensional structure of glucose oxidase from A. niger is unknown.33 Finally, it should be mentioned that type I1 attachment of streptavidin to the biotinylated enzyme may occur as well, but in all cases streptavidin may be able to inhibit the enzyme conjugate to the same extent (i.e., once formed, binding types I and I1 are as effective in inhibiting the modified enzyme). As shown in Figure 1, at high binding protein concentrations, streptavidin inhibits the enzyme-biotin conjugate to a higher extent than avidin. This observation is consistent with the results of previous work performed in our laboratory, where streptavidin was found to inhibit ADA-LC-biotin, a conjugate that has a seven-atom spacer arm between ADA and biotin, to a higher degree than avidin. These resulta were attributed to the deeper binding pocket of streptavidin.19 Therefore, it may be concluded that at high binding protein concentrations additional factors (i.e., depth of the binding pocket) may play a role in the inhibitory ability of the binding protein. In conclusion,the inhibition of biotinylated GOx by avidin occurs in a biphasic manner and plays an important role in (31) Green, M.N. Methods Enzymol. 1990,184, 5147. (32) Weber, P.C.;Ohlendorf, D.H.; Wendoloski, J. J.; Sdemme, F. R. Science 1989,243,8588. (33) Kalisz, H.M.;Hecht, H.; Schomburg, D.;Schmid, R. D.J.Mol. B i d . 1990,213, 207-209.
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determining the response characteristics of the developed assay for biotin. In addition, the inhibition of biotinylated GOx by avidin and streptavidin reveals differences in the reactivity of the two proteins with biotinylated enzymes. Moreover, it appears that the choice between avidin and streptavidin in homogeneous enzyme-linked competitive binding assays that are based on the inhibition of an enzymeligand conjugate has an impact on the attainable detection limits. Given the different behavior of the two binding proteins with conjugates of GOx, ADA, and GGPDH, it may
be concluded that the decision to use one binding protein instead of the other should be made on a case-to-case basis.
ACKNOWLEDGMENT This research was supported by a grant from the National Institutes of Health (Grant GM 40510). RECEIVEDfor review April 6, 1992. Revised manuscript received August 24, 1992. Accepted November 13,1992.
