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Studies on the biotin-binding site of avidin. Minimized fragments that bind biotin.

ABSTRACT: The object of this study was to define minimized biotin-binding fragments, or 'prorecognition sites', of either the egg-white glycoprotein avidin or its bacterial analogue streptavidin. Because of the extreme stability to enzymic hydrolysis, fragments of avidin were prepared by chemical means and examined for their individual biotin-binding capacity. Treatment of avidin with hydroxylamine was shown to result in new cleavage sites in addition to the known Asn-Gly cleavage site (position 88-89 in avidin). Notably, the Asn-Glu and Asp-Lys peptide bonds (positions 42-43 and 57-58 respectively) were readily cleaved; in addition, lesser levels of hydrolysis of the Gln-Pro (61-62) and Asn-Asp (12-13 and 104-105) bonds could be detected. The smallest biotin-binding peptide fragment, derived from hydroxylamine cleavage of either native or non-glycosylated avidin, was identified to comprise residues 1-42. CNBr cleavage resulted in a 78-amino acid-residue fragment (residues 19-96) that still retained activity. The data ascribe an important biotin-binding function to the overlapping region (residues 19-42) of avidin, which bears the single tyrosine moiety. This contention was corroborated by synthesizing a tridecapeptide corresponding to residues 26-38 of avidin; this peptide was shown to recognize biotin. Streptavidin was not susceptible to either enzymic or chemical cleavage methods used in this work. The approach taken in this study enabled the experimental distinction between the chemical and structural elements of the binding site. The capacity to assign biotin-binding activity to the tyrosine-containing domain of avidin underscores its primary chemical contribution to the binding of biotin by avidin.

SUBMITTER: Hiller Y

PROVIDER: S-EPMC1151383 | biostudies-other | 1991 Sep

REPOSITORIES: biostudies-other

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Project description:Affinity interactions between the small molecule biotin and the protein avidin have been used extensively to functionalize biomaterials. More recently, researchers have leveraged the changes in biotin-avidin affinity that occur upon biotin conjugation to larger molecules to control the release of biotinylated drugs and proteins. However, the effects of biotin-avidin interactions on hydrogel properties have not been thoroughly investigated. The objective of this study was to evaluate the effect of increasing biotin and avidin concentrations on hydrogel swelling properties, as an indicator of crosslinking. Gelatin, selected as a model hydrogel material, was biotinylated at increasing fold molar excesses of biotin with a PEG linker using N-hydroxysuccinimide chemistry. Afterwards, biotinylated gelatin was formed into hydrogels and stabilized with glutaraldehyde. Swelling properties of the biotinylated hydrogels were investigated by conducting swelling studies in different avidin solutions. Increasing the degree of biotinylation caused significant decreases in swelling ratios of the hydrogels in a dose-dependent manner, suggesting increases in crosslinking of the hydrogels. However, increasing avidin concentrations in excess of biotin content did not significantly affect swelling ratios. Moving hydrogels to phosphate-buffered saline following avidin incorporation resulted in increased swelling ratios for hydrogels prepared with a lower concentration of biotin. However, hydrogels prepared with the highest concentration of biotin did not experience increased swelling ratios, implying that the stability of biotin-avidin-mediated crosslinking depends on the number of biotin molecules available for binding. Collectively, these results demonstrate that biotin-avidin interactions control hydrogel swelling properties, and that the magnitude and stability of the effects depend on the biotin concentration. These results have important implications for affinity-based controlled release of biotinylated drugs or proteins from biotin-avidin-crosslinked hydrogels.

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Studies on the biotin-binding site of avidin. Minimized fragments that bind biotin.

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