Crosslinking of Streptavidin–Biotinylated Bovine Serum Albumin Studied with Fluorescence Correlation Spectroscopy

Abstract

The strong binding between streptavidin (SA) and biotin has been used as a useful tool in the study of biological system. When SA is mixed with biotinylated bovine serum albumin (bBSA), aggregates are formed by crosslinking. We employed fluorescence correlation spectroscopy to determine the hydrodynamic radius of SA-bBSA aggregates. We found that their hydrodynamic radius depends on the molar ratio of SA to bBSA, with the maximum radius being observed for 4:1 SA to bBSA. When the molar ratio of SA to bBSA was 6:1, the size of the aggregates decreased sharply because the SA molecules surround the bBSA and as a result, crosslinking is greatly reduced. Based on the hydration radius of the aggregates, we estimate that 7 to 8 SA molecules were bound to one bBSA.

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“…Figure 4 A shows a 150s-portion of the photon count trace for EITC–strep (orange trace) at 60 nM (tetramer concentration), where it is possible to distinguish the low-intensity (due to the weak fluorescence emission by EITC–strep) fluctuations generated by protein diffusion. The associated autocorrelation curve for EITC–streptavidin (orange trace in Figure 4 B) is best described by a single diffusing species with diffusion coefficient D ∼50 μm 2 /s, in agreement with literature values, 32 and a triplet decay with lifetime around 10 μs. Upon addition of increasing concentrations of biotinylated IgG, the cross-correlation curve becomes progressively slower due to binding between IgG and EITC–strep, as shown in Figure 4 B for [IgG] = 32 nM (green circles).…”

“…The curve is best described by a single diffusing species with diffusion coefficient D = 49 μm 2 /s plus a triplet-state contribution with τ T ∼ 10 μs. The diffusion coefficient is consistent with literature values for the streptavidin tetramer, confirming conjugation of EITC to the protein . The low fluorescence emission by the conjugate (see Figure C) results in poor signal-to-noise ratio and prevents lowering the concentration below 50 nM.…”

“…The diffusion coefficient is consistent with literature values for the streptavidin tetramer, confirming conjugation of EITC to the protein. 32 The low fluorescence emission by the conjugate (see Figure 2 C) results in poor signal-to-noise ratio and prevents lowering the concentration below 50 nM. The triplet contribution shows that intersystem crossing occurs to an appreciable extent in the conjugate (vide infra).…”