The intrinsic tryptophan fluorescence of beta 1-bungarotoxin and the Ca2+-binding domains of the toxin as probed with Tb3+ luminescence.
beta 1-Bungarotoxin has only one tryptophan residue, namely Trp-19 in the phospholipase A2 subunit. The environment of Trp-19 was studied by intrinsic fluorescence and solute quenching. The native protein showed an emission peak at 330 nm. About 90% of the fluorescent tryptophan was accessible to quenching by either acrylamide or KI but not to CsCl. A red-shift in the emission peak occurred between 2.0 M- and 4.0 M-guanidinium chloride, and the helix-coil transition of the polypeptide backbone occurred between 4.0 M- and 6.0 M-guanidinium chloride. These results suggested that Trp-19 was in a less polar medium but near a positive charge. The local conformation around Trp-19 could be disturbed by binding of Tb3+ or Ca2+ or Sr2+ to the toxin molecule. Tb3+ a tervalent lanthanide ion, effectively substituted for Ca2+ in stimulating the phospholipase A2 activity of beta 1-bungarotoxin. Upon the binding of Tb3+ to the toxin, the Tb3+ fluorescence in the 450-650 nm region was enhanced. This resulted from the energy transfer from Trp-19 to Tb3+. The distance between the energy-transfer pair was estimated to be 0.376-0.473 nm at pH 7.6 and 0.486-0.609 nm at pH 6.3. Assuming that there were two Tb3+-binding sites on the toxin molecule, at pH 7.6 the association constants of the high-affinity and the low-affinity sites were determined to be 3.82 x 10(3) M-1 and 2.85 x 10(2) M-1 respectively. At between pH 6.0 and 7.0 Tb3+ bound to the high-affinity site decreased greatly but did not disappear entirely. Both Ca2+ and Sr2+ competed with Tb3+ at the high-affinity sites, but Sr2+ could not substitute for Ca2+ in stimulating the phospholipase A2 activity.
Chu
S T
ST
Institute of Biochemical Science, College of Science, National Taiwan University, Taipei, Republic of China.
Chen
Y H
YH
D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000032:analysis*; D004735:Energy Transfer; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010741:Phospholipases A / Q000032:analysis; D054467:Phospholipases A2; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013324:Strontium; D013725:Terbium; D014364:Tryptophan / Q000032:analysis
D002038:Bungarotoxins; D013725:Terbium; D014364:Tryptophan; D010741:Phospholipases A; D054467:Phospholipases A2; D013324:Strontium