Catalogo Articoli (Spogli Riviste)

OPAC HELP

Titolo:
BIOTINYLATION OF PROTEINS VIA AMINO-GROUPS CAN INDUCE BINDING TO U937CELLS, HL-60 CELLS, MONOCYTES AND GRANULOCYTES
Autore:
STORM D; LOOS M; KAUL M;
Indirizzi:
INST MED MICROBIOL & HYG,AUGUSTUSPL,HOCHHAUS D-55101 MAINZ GERMANY INST MED MICROBIOL & HYG D-55101 MAINZ GERMANY
Titolo Testata:
Journal of immunological methods
fascicolo: 1, volume: 199, anno: 1996,
pagine: 87 - 99
SICI:
0022-1759(1996)199:1<87:BOPVAC>2.0.ZU;2-E
Fonte:
ISI
Lingua:
ENG
Soggetto:
GUINEA-PIG MACROPHAGES; MONOCLONAL-ANTIBODIES; COMPLEMENT COMPONENT; RECEPTOR EXPRESSION; MEMBRANE-PROTEIN; FLOW-CYTOMETRY; 1ST COMPONENT; CLQ RECEPTOR; C1Q RECEPTOR; LINE;
Keywords:
BIOTINYLATION; C1Q; FLOW CYTOMETRY; MONOCYTE; GRANULOCYTE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
40
Recensione:
Indirizzi per estratti:
Citazione:
D. Storm et al., "BIOTINYLATION OF PROTEINS VIA AMINO-GROUPS CAN INDUCE BINDING TO U937CELLS, HL-60 CELLS, MONOCYTES AND GRANULOCYTES", Journal of immunological methods, 199(1), 1996, pp. 87-99

Abstract

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 after 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 andactivated 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-FlTC 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 withtheir 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.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 15/07/20 alle ore 05:37:40