Iminobiotin binding induces large fluorescent enhancements in avidin and streptavidin fluorescent conjugates and exhibits diverging pH-dependent binding affinities.
The pH-dependent binding affinity of either avidin or streptavidin for iminobiotin has been utilized in studies ranging from affinity binding chromatography to dynamic force spectroscopy. Regardless of which protein is used, the logarithmic dependence of the equilibrium dissociation constant (K(d)) on pH is assumed conserved. However a discrepancy has emerged from a number of studies which have shown the binding affinity of streptavidin for iminobiotin in solution to be unexpectedly low, with the K(d) at values usually associated with non-specific binding even at strongly basic pH levels. In this work we have utilized a Bodipy fluorescent conjugate of avidin and an Oregon Green fluorescent conjugate of streptavidin to determine the K(d) of the complexes in solution in the pH range of 7.0 to 10.7. The study was made possible by the remarkable fluorescent enhancement of the two fluorescent conjugates (greater than 10 fold) upon saturation with iminobiotin. The streptavidin-iminobiotin interaction exhibited almost no pH dependence over the range studied, with K(d) consistently on the order of 10(-5) M. In contrast, under identical experimental conditions the avidin-iminobiotin interaction exhibited the expected logarithmic dependence on pH. We discuss the possible origins for why these two closely related proteins would diverge in their binding affinities for iminobiotin as a function of pH.
Materials and Sensors Branch, Naval Research Laboratory, 4555 Overlook Ave SW, Washington, DC 20375-5320, USA. raphael@anvil.nrl.navy.mil
Marc P
MP
Raphael
Catherine A
CA
Rappole
Lynn K
LK
Kurihara
Joseph A
JA
Christodoulides
Syed N
SN
Qadri
Jeff M
JM
Byers
D001360:Avidin / Q000737:chemistry* / Q000378:metabolism*; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D019809:Streptavidin / Q000737:chemistry* / Q000378:metabolism*
D008024:Ligands; C518163:iminobiotin; D001360:Avidin; D001710:Biotin; D019809:Streptavidin