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Identification of the adenine binding site in the ricin toxin A-chain by fluorescence, CD, and electron spin resonance spectroscopy

Ramalingam, TS and Das, Puspendu K and Podder, Sunil K (1993) Identification of the adenine binding site in the ricin toxin A-chain by fluorescence, CD, and electron spin resonance spectroscopy. In: Biopolymers, 33 (11). pp. 1687-1694.

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Abstract

CD, electron spin resonance, and fluorescence spectroscopy have been utilized to study the adenine binding site of ricin and its toxic A-subunit. At acidic (4.5) and physiological (7.3) pH, adenine or a spin-labeled analogue of adenine, N6-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl) adenine, alters the near uv CD spectra of the ricin A-chain as well as intact ricin, whereas the far uv CD spectra of all proteins remain unchanged. Electron spin resonance data show that the adenine spin-labeled analogue interacts strongly with the A-chain both at pH 4.5 and 7.3, but no or very weak binding is observed for the intact ricin or the isolated B-chain. The adenine spin label gets highly immobilized (2AII = 65.5G) by the A-chain. The apparent dissociation constant Kd for the toxic A-chain ligand complex is 1.55 × 10-4 M and 5.6 × 10-5 M at pH 7.3 and 4.5, respectively. Fluorescence intensity of ricin A-chain bound 1,8-anilinonaphthalenesulfonic acid (ANS) decreases by 55% at pH 4.5 with the addition of the spin-labeled analogue of adenine, implying that both the ANS and adenine spin label (ADSL) bind to the hydrophobic domain of the A-chain. Fluorescence of the only intrinsic tryptophan probe of the A-chain is also efficiently quenched by ADSL, indicating that the tryptophan residue and the hydrophobic adenine binding site are closely located. All spectroscopic measurements indicate that adenine or its spin-labeled analogue has a single binding site adjacent to the TRP211 residue in the A-chain. Expansion of the A-chain globule and subsequent exposure of the hydrophobic binding site seem to be responsible for the increased binding of adenine at pH 4.5.

Item Type: Journal Article
Additional Information: Copyright of this article belongs to John Wiley & Sons, Inc.
Keywords: adenine binding;ricin toxin;spectroscopy
Department/Centre: Division of Chemical Sciences > Inorganic & Physical Chemistry
Division of Biological Sciences > Biochemistry
Date Deposited: 11 Jun 2004
Last Modified: 19 Sep 2010 04:12
URI: http://eprints.iisc.ernet.in/id/eprint/382

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