It is difficult to compare the numbers from different sources. Although the biotin-binding capability of Streptavidin is defined as: 1 Unit binds 1 microgram of d-biotin at pH 7, the measured specific activity of a given lot depends on the assay method used, as well as the method used to quantitate the Streptavidin (gravimetric vs spectrophotometric).

Table 1 summarizes the published information from a number of Streptavidin suppliers and demonstrates the variance in the numbers and methods:

¹ProZyme's molecular weight is determined by SDS-PAGE gel electrophoresis (ie. the monomer, ~14 kda). It is difficult to distinguish differences between 12,000 and 15,000 daltons, which accounts for some of the variability among suppliers.

²The calculation of the theoretical maximum is:

 1000

------ x 4 x 243 = U/mg  

MW

where:

MW = molecular weight in daltons

4 = number of biotins/Streptavidin molecule (1 biotin bound/monomer)

243 = molecular weight of d-biotin

³Even HABA assay results are not necessarily comparable as HABA(1) uses a concentration of HABA dye 4 times that of HABA(2). The extinction coefficient of the Streptavidin-HABA complex in HABA(1) is 34,500 M-1, while that used in HABA(2) is 24,000. Details of ProZyme's HABA assay are given in the SA10 Streptavidin Technical Data Sheet.

⁴ProZyme uses a HABA assay as a release specification, but also reports results of the direct biotin method on our Certificates of Analysis. The table below gives some typical comparative values of biotin-binding for recent ProZyme Streptavidin lots.

We received a sample from a vendor who reports a [¹⁴C]-biotin binding. The results from the various biotin-binding assay methods were:

These assays measure the binding of biotin, a small molecule, and not the larger biotinylated proteins or peptides. Streptavidin's binding site is known to be recessed, so the use of LC-biotin and the size of the biotinylated complex raises issues of steric hindrance and accessibility of the binding site.

In conclusion, one cannot safely compare the activities of Streptavidins from different vendors on the basis of reported biotin-binding activities without taking into account whether different assay methods were used in their determinations.