Biotin Binding Assay Protocol

The biotin-binding activity of streptavidin is determined using a modification of the dye-binding assay of Green (1970). One unit will bind one microgram of d-biotin at pH 7.0.

Reagents Required

Procedure

1. Adjust spectrophotometer to read at 500 nm.
2. To two tubes labeled A and B add as follows:
   
   

   Label	A (mL)	B (mL)
   Streptavidin Sample	0.05	0.05
   Phosphate Buffer	0.5	0.5
   HABA Stock	0.1	0.1
   Biotin Stock	-	0.25
   H2O	0.35	0.1
   Total Volume	1.0	1.0

3. After mixing, zero the spectrophotometer with water and read the absorbances in tubes A and B.
4. Calculations:
   
   (10⁶ µg/g)(A-B)MV 141(A-B) Units/mg = ____________ = ____________ E(Cv) C
   (10⁶ µg/g)(A-B)MV 141(A-B) Units/mg = ____________ = ____________ E(Cv) C
   (10⁶ µg/g)(A-B)MV 141(A-B) Units/mg = ____________ = ____________ E(Cv) C
   
   M = Formula weight of d-biotin (244 g/mole)
   V = Volume of assay in liters (0.001 L)
   v = Volume of streptavidin sample in milliliters (0.05 mL as written)
   C = Concentration of streptavidin in sample (mg/mL)
   E = Net molar extinction coefficient of HABA-streptavidin complex at 500 nm (34,500 M-1)

Suggestions for Use

Bayer (1989) reports that streptavidin may form aggregates under certain conditions. Streptavidin is highly soluble under alkaline conditions (pH > 8.5). Streptavidin is often supplied lyophilized. Under these conditions there is a tendency for the material to aggregate if it is redissolved in water or other low ionic strength buffers at neutral or acidic pH. As a convenience to customers, Streptavidin has been lyophilized from a dilute sodium chloride solution at mildly alkaline pH. This material is readily soluble in water. The activity of the material recovered after reconstitution under these conditions is undiminished. We recommend dissolving streptavidin in de-ionized water or, preferably, 1.0 mM sodium bicarbonate buffer (pH 9) at twice the desired final protein concentration. The protein may then be diluted with an equal volume of 2x buffer to produce a stock solution. Upon standing some turbidity may develop in certain buffers. Centrifugation will usually yield a clear solution with negligible loss of streptavidin.

References

• Agaraña, C. E., Kuntz, I. D., Birken, S., Axel, R. and C. R. Cantor. Molecular cloning and nucleotide sequence of the streptavidin gene. Nucleic Acids Res. 14: 871-882 (1986).
• Bayer, E. A., Ben-Hur, H., Hiller, Y. and M. Wilchek. Postsecretory modifications of streptavidin. Biochem. J. 259: 369-376 (1989).
• Bayer, E. A., Ben-Hur, H., Gitlin, G. and M. Wilchek. An improved method for the single-step purification of streptavidin. J.Biochem. Biophys. Meth. 13:103-112 (1986).
• Brower, M. S., Brakel, C. L. and K. Garry. Immunodetection of streptavidin-acid phosphatase complex on western blots. Analyt. Biochem. 147:382-386 (1985).
• Cartun, R. W. and C. A. Pedersen. An immunocytochemical technique offering increased sensitivity and lowered cost with a streptavidin-horseradish peroxidase conjugate. J. Histotechnology 12:273-277 (1989).
• Chaiet, L. and E. J. Wolf. The properties of streptavidin, a biotin-binding protein produced by Streptomyces. Arch. Biochem. Biophys. 108:1-5 (1964).
• Gitlin, G., Bayer, E. A. and M. Wilchek. Studies on the biotin-binding site of streptavidin. Biochem. J. 256:279-282 (1988).
• Green, N. M. Spectrophotometric determination of avidin and biotin in Methods in Enzymology, Vol. XVIII (McCormick, D. B. and L. D. Wright, eds.) Academic Press, New York, pg. 418 (1970).
• Green, N. M. Avidin. Adv. Protein Chem. 29:85-133 (1975).
• Hofmann, K., Wood, Sara, Brinton, C. C., Montbeller, J. A. and F. M. Finn. Iminobiotin affinity columns and their application to retrieval of streptavidin. Proc. Natl. Acad. Sci. USA 77:4666-4668 (1980).
• Leary, J. J., Brigati, D. J. and D. C. Ward. Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots. Proc. Natl. Acad. Sci. USA 80:4045-4049 (1983).
• Moxley, G. Heightened sensitivity of quantitative ELISA for IgM Rheumatoid Factor with the use of the biotin-streptavidin system. Am. J. Clin. Path. 92: 630-636 (1989).
• Nikolau, B. J., Wurtele, E. S. and P. K. Stumpf. Use of streptavidin to detect biotin-containing proteins in plants. Analyt. Biochem. 149:448-453 (1985).
• Sano, T. and C. R. Cantor. Cooperative biotin binding by streptavidin. J. Biol. Chem. 265:3369-3373 (1990)
• Sharma, H. and C. Tihon. Determination of avidin and streptavidin by a modified Bradford method. Analyt. Biochem. 170:135-139 (1988).
• Suter, M., Cazin, J., Jr., Butler J. E. and D. M. Mock. Isolation and characterization of highly purified streptavidin obtained in a two-step purification procedure from Streptomyces avidinii grown in a synthetic medium. J. Immunol. Methods 113:83-91 (1988).
• Weber, P. C., Ohlendorf, D. H., Wendoloski, J. J. and F. R. Salem. Structural origins of high-affinity biotin binding to streptavidin. Science 243:85-88 (1989).