Biotinylation of proteins via amino groups can induce binding to U937 cells, HL-60 cells, monocytes and granulocytes.

The use of biotinylated ligands for the flow cytometric detection of cell surface receptors has become a popular alternative to radioreceptor assays. Although the biotinylation of a protein is a relatively mild chemical reaction several reports have mentioned the fact that the number and location of biotin moieties coupled to amino groups of a protein can alter its physicochemical properties and impair biological activity. In the present study we show for a variety of biotinylated functionally unaltered ligands that biotinylation by N-hydroxysuccinimide (NHS) esters of biotin can induce a binding to cell surfaces, which is not specific for the respective unlabelled ligand. C1q, C1 inhibitor (C1-INH), alpha 1-antitrypsin (AT), ovalbumin (OV), transferrin and soybean trypsin inhibitor (STI) were labelled with S-NHS-LC-biotin and activated C1s (C1s) with NHS-biotin. Biotinylation of C1q, C1s and C1-INH exerted negligible effects on biological function, antigenicity or electrophoretic mobility but when labelled and unlabelled proteins were assayed for binding to monocytic U937 cells, promyelocytic HL-60 cells, monocytes and granulocytes, a remarkable binding was observed for biotinylated C1q, C1-INH and C1s. In contrast, no binding was observed when we used unlabelled C1q, C1s and C1-INH and employed specific antibodies, alpha-mouse-FITC or alpha-rabbit-FITC for detection. Increasing molar ratios of biotin-to-protein (B : P) for biotinylated AT, OV and STI evoked increased fluorescence intensities of the cells. Most importantly the unlabelled ligands did not compete for cell binding with their biotinylated derivatives, with the exception of transferrin. Preincubation of the cells with an excess of free d-biotin did not reduce binding of biotinylated proteins, thus excluding a potential involvement of biotin receptors. Hydrophobic interaction chromatography revealed a remarkable increase in hydrophobicity of the biotinylated proteins compared to their unlabelled counterparts, suggesting that the biotinylation-induced binding is due to increased hydrophobicity. Our findings indicate that biotinylation by the common amino acid esterification method may be critical for proteins if they are to be used as ligands for receptor binding studies.