The virtually irreversible binding of the vitamin, biotin, to avidin and streptavidin is the basis for the vast array of detection schemes that have become routine in biotechnology.
The degree of biotin labeling can be critical for downstream applications. Low biotin density may result in reduced interaction with the (strept)avidin component whereas over labeling can reduce activity of the labeled molecule. Quantitation of biotin enables optimized biotinylation protocols.
For many years, the standard method for determining the extent of biotinylation has been the spectrophotometric 2-(4’ hydroxyazobenzene) benzoic acid (HABA) assay developed by Green (Meth. Enzymol. 18A:418-24). The dye HABA binds avidin with low affinity. Upon binding of biotin, HABA is displaced causing a shift of the absorbance maxima that is used to determine the number of biotins in the sample. However avidin-based assays, including the HABA assay, suffer from inaccuracies caused by steric hindrance that prevents closely spaced or “buried” biotins from accessing a binding site on avidin. To reduce this problem, samples are pre-treated with hydrolytic enzymes. Unfortunately, enzymatic degradation requires several hours with no certainty that complete digestion is attained or that steric problems are eliminated.
In recent years, mass spectrometry (MS) has been shown to be an accurate method for biotin determination on proteins by comparing the protein's mass before and after biotinylation. Unfortunately, mass determination of proteins larger than about 60 kD is problematic and assay of a large number of samples may be cost prohibitive.
An excellent alternative to avidin-based assays or MS is the Quant*Tag™ Biotin Kit from Vector Laboratories. The Quant*Tag Kit contains proprietary reagents which chemically react with biotin, yielding a visible color change quantifiable by spectrophotometry. Because steric interference is not an issue, accurate biotin quantitation is possible without digestion, saving time and reducing assay variability.
In this article, it will be shown that the Quant*Tag assay is more accurate than the HABA assay, even with sample digestion.Materials and Methods
A complete materials and methods section is available in an expanded whitepaper at www.vectorlabs.com/catalog.aspx?prodID=1641.Results and Discussion
To compare different analytical methods, biotin-labeled and unlabeled Protein A were analyzed by MS, HABA, and the Quant*Tag procedure. Figure 1a shows that unmodified Protein A is a single species with a mass of 46,761 g/mol. Figure 1b shows biotin-labeled Protein A. The expected increase in mass contributed by a single biotin label would be 338.2 g/mol. The spectrum demonstrates an increase in mass, following a normal distribution, corresponding to about 2 to 11 biotins with an average of about 6 biotins per protein A molecule.
The same biotinylated Protein A sample was tested using the Quant*Tag Biotin Kit and the HABA assay. Table 1 shows that Quant*Tag gives a result very much in agreement with MS. However, HABA, even after overnight enzymatic digestion, significantly underestimates the number of biotins.Table 1.
Because of the inaccuracy of the HABA assay compared to the Quant*Tag assay and because the results of the HABA assay were unaffected by sample degradation, it was decided to see if this situation was unique to Protein A. A variety of biotinylated proteins were analyzed by Quant*Tag and HABA. Table 2 shows that in some, but not all cases, digestion of the sample improved the accuracy of the HABA assay. However, in all measurable cases HABA underestimated the degree of biotinylation. In addition, upon addition of the HABA/avidin solution to wheat germ agglutinin and alkaline phosphatase, the proteins precipitated. Precipitation of the samples caused light scattering that interfered with spectrophotometric measurement of the sample. This precipitation is indicative of macromolecular complex formation due to crosslinking of avidin through proteins containing multiple biotins. Crosslinking by this mechanism in the digested samples shows that enzymatic digestion does not always achieve the desired goal.Table 2.
Quantification of biotins on detection reagents is critical for optimization of the biotinylation reaction. Assays based on avidin binding suffer from inaccuracy caused by steric hindrance. MS provides accuracy but protein size, heterogeneity and cost are all limiting factors. The Quant*Tag Biotin Kit offers a simple and accurate method for biotin quantitation with no sample digestion required.