ISSN:
0006-3525
Keywords:
Chemistry
;
Polymer and Materials Science
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Chemistry and Pharmacology
Notes:
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. © 1993 John Wiley & Sons, Inc.
Additional Material:
7 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/bip.360331106
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