Photochemical derivatization of agarose: synthesis of an affinity agarose using a photoaffinity ligand specific for the peripheral-type benzodiazepine receptor.
PK 14105, a photoaffinity ligand specific for the peripheral-type benzodiazepine receptor (PBZR), was photochemically coupled to omega-aminobutyl agarose (ABAg) to yield PK 14105 agarose (PKAg). 19F and 1H NMR spectroscopy were consistent with the proposed site of coupling at the 2'-fluorine of PK 14105 by the primary amine moiety of ABAg. Quantitation of the affinity gel using two different colorimetric assays for primary amines suggests approximately 50% of the available primary amine groups of ABAg were bound by PK 14105. The estimated concentration of PK 14105 bound to ABAg was 2.3 mumols/ml of settled gel (2.3 mM effective ligand concentration). PKAg specifically binds the bovine PBZR solubilized by digitonin. The affinity of PKAg for the soluble PBZR was estimated by varying the concentration of PKAg. PBZR binding to PKAg was saturable and the apparent affinity of PKAg for the bovine receptor was estimated from the saturation data. A PKAg affinity column bound 85% of the solubilized PBZR from rat adrenals partially purified by anion exchange chromatography. These results indicate PKAg is a receptor-specific affinity media which may be useful in the purification of the native PBZR from various species.
Parola
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Department of Pharmacology & Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
MacKenzie
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NE
Laird
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HE
D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D002082:Butylamines / Q000737:chemistry; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D007546:Isoquinolines / Q000138:chemical synthesis* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010777:Photochemistry; D051381:Rats; D011963:Receptors, GABA-A / Q000032:analysis / Q000378:metabolism*; D012685:Sepharose / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D000345:Affinity Labels; D002082:Butylamines; D007546:Isoquinolines; C067040:PK 14105 agarose; D011963:Receptors, GABA-A; C067039:4-aminobutylagarose; D012685:Sepharose; C051238:PK 14105