thiobiotin, does not significantly change the characteristics of the molecule with respect to reverse-phasechromatography; however, the guanidino-like 2’4minobiotin is protonated (p& = 12) and this analog elutes fairly rapidly. Changesin the 2’position do affect the ionic nature of the molecule enough to provide quite different retention times on the anionexchange column.
1121 Fluorometric By
Assays for Avidin and Biotin NARGEW
and D. S. SMITH
Several methods have been describedfor the determination of biotin, a growth factor. These include microbiological, calorimetric, and enzymatic methods.14 Competitive protein binding assays which exploit the biotin-binding property of avidin and employ a labeled-biotin ligand as tracer have also been developed. Radioisotopes,2-6coenzymes,7q8 enzymes,9and fluorogenic enzyme substrateslOhave been used for labeling of biotin. A spectrophotometric method” involves biotin displacementof a dye from the avidin binding site. Biotin-induced changesin the absorption” or fluorescence’*properties of avidin form the basis of particularly simple biotin assays. Avidin, the egg-white protein, can easily be determined by a variety of binding assay techniques.1,2,7J3 There has been much interest in exploitation of the avidin-biotin interaction in immunochemical assay systems.14J5 When working with fluores’ D. B. McCormick and L. D. Wright, this series, Vol. 18, Part A, p. 379. 2 D. B. McCormick and L. D. Wright, this series, Vol. 62, Part D, p. 279. 3 K. Groningsson and L. Jansson, J. Pharm. Sci. 68, 364 (1979). 4 S. Haarasiha, Anal. Biochem. 87, 306 (1978). 5 T. Horsburgh and D. Gompertz, Clin. Chim. Acta 82, 215 (1978). 6 R. Rettenmaier, Anal. Chim. Acta ll3, 107 (1980). ’ R. J. Carrico, J. E. Christner, R. C. Boguslaski, and K. K. Yeung, Anal. Biochem. 72,271 (1976). s H. R. Schroeder, R. J. Carrico, R. C. Boguslaski, and J. E. Christner, Anal. Biochem. 72, 283 (1976). 9 C. R. Gebauer and G. A. Rechnitz, Anal. Biochem. 103, 280 (1980). I0 J. F. Burd, R. J. Carrico, M. C. Fetter, R. T. Buckler, R. D. Johnson, R. C. Boguslaski, and J. E. Christner, Anal. Biochem. 77, 56 (1977). I1 N. M. Green, this series, Vol. 18, Part A, p. 418. I2 H. J. Lin and J. F. Kirsch, Anal. Biochem. 81, 442 (1977). I3 N. M. Green, Adu. Protein Chem. 29, 85 (1975). I4 S. M. Costello, R. T. Felix, and R. W. Giese, Clin. Chem. (Winston-Salem, N.C.) 25,1572 (1979). Is J. L. Guesdon, T. Temynck, and S. Avrameas, J. Histochem. Cytochem. 27,113l (1979). METHODS
Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form resewed.
cein-labeled avidin, we noticed that the fluorescence of the labeled protein was enhanced upon binding of its specific ligand, biotin. This observation enabled the development of simple assays for avidin and biotin that are more sensitive and practical than the previously described fluorometric assays based on the intrinsic fluorescence of avidin.r2 Materials and Methods Fluorescein isothiocyanate isomer I (FITC), avidin, and d-biotin were obtained from Sigma (Poole, Dorset, United Kingdom). Triton X-100 was obtained from BDH (Poole, Dorset, United Kingdom). Preparation of Fluorescein-Labeled Avidin Avidin and FITC were reacted in sodium bicarbonate buffer (50 mM, pH 9.0) at molar ratios of 1: 1, 1: 4, 1: 6, and 1: 10 by adding 250~~1 aliquots of avidin solution (10 g/liter) to 25, 100, 150, and 250 ~1 of FITC solution (580 mg/liter), respectively. Reaction mixtures were incubated at room temperature overnight, applied to columns of Sephadex G-25 fine grade (1.2 x 20 cm), and eluted with bicarbonate buffer. The entire labeled protein peak, identified by its color, was collected from each column, and was well separated from minimal amounts of unreacted FITC . Labeled proteins were designated according to the molar ratio of FITC to protein in the reaction mixture, assuming a molecular weight of 66,000 for avidin.t3 Thus, FITCr-avidin, FIT&avidin, FIT&,-avidin, and FITCr,-,-avidin were prepared and stored at -18” until use. Concentrations of labeled proteins are given on the basis of their protein content, assuming total recovery from the original reaction mixtures. Fluorometry A Perkin-Elmer Model 1000 ratio-recording filter fluorometer was used, which was equipped with broad-band interference filters types FITC (440-490 nm bandpass) in the excitation path, and DB2 (510-600 nm bandpass) in the emission path. Both filters were from Barr and Stroud (Anniesland, Glasgow, United Kingdom). Because of the high light throughput of the FITC filter, it was necessary to limit the excitation beam to avoid overloading the reference detector of the fluorometer; this was achieved by placing a black plastic plate with a central circular aperture of IO-mm diameter in the excitation filter holder. The continuous wavelength filter of the Model 1000 was wound out of the emission beam and not used.
All fluorometric measurements in this fluorometer were made using disposable polystyrene test tubes (75 x 11 mm, No. 55.478 from Walter Sarstedt, Leicester, United Kingdom). To enable measurements on test tubes, an adapter unit with the same external dimensions as a conventional fluorometer cuvette was made. A 45mm length of half-inch (12.7 mm) square aluminum rod was drilled out to a diameter of 11 mm down most of its long axis. Slits (30 x 5 mm) were cut in each face to allow entry and exit of light beams. The adapter was anodized black, and then permanently placed in the cuvette holder of the fluorometer. To accommodate the height of the test tubes, the normal sample compartment lid of the fluorometer was replaced with a standard Perkin-Elmer accessory unit (Ittrich sample cover). Total fluorescence signals were recorded in arbitrary units and included a constant buffer background of 4.5 units. On the intensity scale chosen, a 10 nmol/liter solution of fluorescein gave a total signal of 125 units. Relationship between Fluorescence Intensity of Avidin-Labeled Products and Molar Labeling Ratio Fluorescein-labeled avidin products were diluted in phosphate-Triton buffer (100 mM sodium phosphate, pH 7.5, containing 0.1% v/v Triton X100) to a protein concentration of 4 mg/liter. To 100 ~1 of each in assay tubes was added 1.1 ml of phosphate-Triton buffer, the tube contents mixed, and fluorescence intensities were then measured. As demonstrated in Fig. 1, fluorescence of fluorescein-labeled avidin products did not show a linear relationship with the degree of labeling and was maximal at a molar labeling ratio, of FITC to avidin, of 6:l. The FITC+avidin-labeled product was chosen for further studies. Assay of Biotin To duplicate tubes containing biotin solutions (600 ~1) of various concentrations in phosphate-Triton buffer was added 600 ~1 of fluoresceinlabeled avidin solution (1 mg protein/liter) in the same buffer. After 15 min incubation at room temperature, fluorescence was measured by placing each tube into the fluorometer. Preliminary kinetic studies had demonstrated that binding reactions were complete within 5 min. The fluorescence of fluorescein-labeled avidin was enhanced by biotin to a maximum extent of about 2-fold, which enabled construction of a standard dose-response curve for the assay of biotin (Fig. 2). The mini-
90 8 i-5 g 70k! 9 2
MOLAR RATIO FITC:AVIDIN FIG.
1. Dependence of fluorescence of fluorescein-labeled avidin on molar labeling ratio.
ma1 detectable dose at the 95% confidence leveli was 0.5 ng of biotin. Similar fluorescence enhancement was noted using labeled protein preparations obtained by reacting FITC and avidin at molar ratios of 1 : 1 or 6: 1. Assay of Avidin To duplicate tubes containing avidin solutions (300 ~1) of various concentrations in phosphate-Triton buffer was added 600 ~1 of fluoresceinlabeled avidin solution (1 mg protein/liter), followed by 300 ~1 of biotin solution (16 pg/liter) in the same buffer. After 15 min incubation at room temperature, fluorescence was measured as above. The assay established for avidin was based on competition between the labeled and unlabeled protein for binding of a limited amount of biotin (Fig. 3). The minimal detectable dose was 0.04 pg of avidin. Discussion Since there is no gross change in the structure of avidin upon binding of biotin,i3 the relatively large increase in fluorescence of fluoresceinlabeled avidin was unexpected. However, biotin binding to dansyl-avidin has been foundI to lead to 40% reduction in emission intensity; this observation, together with fluorescence polarization results, was interpreted as suggesting local displacement of the dansyl groups into a more I6 D. Rodbard, Anal. Biochem. 90, 1 (1978).
8 goi5 b=i
8 3 LL 80 70 -w
. .' IA" '
FIG. 2. Typical standard curve for fluorometric assay of biotin, utilizing biotin-induced enhancement of the fluorescence of fluorescein-labeled avidin.
aqueous environment where they had greater rotational freedom and less interaction with the protein structure. This finding would be consistent with a biotin-induced enhancement in the fluorescence of fluorescein groups attached to similar sites on the avidin molecule. The quenching of the protein fluorescence of avidin upon binding of biotin has been used as the basis of existing fluorometric assays for avidin and biotin.r2 The biotin assay is comparatively insensitive, with a usefully applicable range between 20 and 120 ng of the analyte (a 6-fold span).
\ I 0 0.02
I I 0.05 0.1
FIG. 3. Typical standard curve for fluorometric assay of avidin, utilizing competition between the labeled and unlabeled avidin for binding of biotin.
BIOTIN AND DERIVATIVES
Proteins or other materials fluorescing in the ultraviolet may cause interference. The use of fluorescein-labeled avidin offers several advantages. These include increased sensitivity due to high quantum efficiency of fluorescein; absence of interference from proteins, since fluorometry is performed at visible wavelengths; and finally the fact that end point measurement can easily and directly be made in disposable plastic tubes used throughout the assay, again because of the visible-wavelength fluorometry. The labeled avidin fluorometric assays offer a sensitivity about 20-fold better than the protein fluorescence method, and comparable with that of radio-assays using tracers of low specific activity,‘J and with that of the binding assays employing other types of nonisotopic labeled reactants.‘-I0 Although microbiological assays,‘J2J3 enzymatic assays,4 and radioassays employing tracers of high specific activity2,5*6can be between 10 and 100 times more sensitive, they are generally more complex than the described fluorometric methods. Similar to many binding assays, the fluorometric assays provide a comparatively limited useful range. The biotin assay covers about a 4-fold useful range between 1.5 and 6 ng of analyte (Fig. 2) and the avidin assay about a IO-fold range between 0.06 and 0.6 pg (Fig. 3). All radioassays’J*5,6involve a separation step in which avidin-bound and free labeled biotin are separated by a variety of techniques. In the nonisotopic competitive-binding assays,‘-I0 such a step is not required since binding to avidin changes the signal from the labeled biotin. The labeled avidin fluorometric assays described above provide an unusual instance of a nonisotopic nonseparation binding assay system in which the binding protein, rather than the ligand, is labeled.
Assay for d-Biotin Cellulose Disks
By L. GOLDSTEIN,~.
and G. COHEN
Most biotin assay procedures in current use either exploit the absolute growth requirement of certain bacterial and yeast strains for d-biotin, or else depend on the extremely high affinity of avidin for this vitamin.‘-I5 Although the above two basic approaches to biotin quantitation are comparable in terms of specificity, sensitivity, and reproducibility, the binding assays, particularly those employing immobilized avidin, are faster and METHODS
Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.