Avidin

Abstract: The adhesion force between the tip of an atomic force microscope cantilever derivatized with avidin and agarose beads functionalized with biotin, desthiobiotin, or iminobiotin was measured Under conditions that allowed only a limited number of molecular pairs to interact, the force required to separate tip and bead was found to be quantized in integer multiples of 160 +/- 20 piconewtons for biotin and 85 +/- 15 piconewtons for iminobiotin The measured force quanta are interpreted as the unbinding forces of individual molecular pairs

Abstract: Avidin has an extraordinary affinity for the small-molecule vitamin biotin. Covalently coupling biotin or avidin to peroxidase molecules does not interfere with their normal biochemical functions. The avidin or biotin molecules, either peroxidase conjugated or unconjugated, can be brought to the antigen sites by means of an antiavidin antibody. Several immunohistochemical staining technics based on this principal have been described. The method utilizing an avidin-biotin-peroxidase complex was found to be more sensitive than the unlabeled antibody (PAP) method. This method involved four sequential staining procedures: (1) primary antibody (goat anti-human antigen); (2) secondary antibody (rabbit antigoat IgG) added in relative excess; (3) goat antiavidin antibody; (4) avidin-biotin-peroxidase complex. The applications of this technic are discussed.

Abstract: Publisher Summary This chapter discusses the fundamental molecular properties of avidin and streptavidin. Carbohydrate-free avidin can be obtained by use of deglycosylating enzymes and it has been shown to be present in significant amounts in some commercial preparations from which it may be separated by use of lectin columns. Improvement on the original use of biotinyl cellulose came with the introduction of iminobiotinyl derivatives of Sepharose that utilized the pH dependence of the binding 24 to achieve efficient elution. In iminobiotin, the ureido group becomes a guanidinium group; the form in which this is uncharged is strongly bound. The gene for streptavidin has been cloned and sequenced with the ultimate objective of using it in general expression systems for detecting and isolating fusion proteins. The stability is greatly enhanced by biotin binding, because the total free energy of binding is about 330 kJ/mol of tetramer. The dissociation constant for biotin is so low that it can be estimated only from the ratio of the rate constants for binding and exchange. The binding is accompanied by a red shift of the tryptophan spectrum and by a decrease in fluorescence, either of which can be used as the basis for quantitative assays.