ACHATINA-FULICA HEMOCYANIN AND ITS INTERACTIONS WITH IMIDAZOLE, POTASSIUM CYANIDE, AND FLUORIDE AS STUDIED BY SPECTROPHOTOMETRY AND NUCLEAR MAGNETIC-RESONANCE AND RESONANCE RAMAN SPECTROMETRY

Hemocyanin from Taiwan snails, A. fulica, has not been studied previously. UV spectrophotometric, PMR, 19F-NMR, and resonance Raman methods were used to study the interaction of several small ligands with this hemocyanin species. Addition of imidazole (up to 0.5 M) and KCN (up to 0.001 M) to oxyhemocyanin causes a relatively rapid drop in absorbance at 345 nm, a primary reaction, followed by a much slower secondary process. The primary reaction between imidazole and hemocyanin takes about 2 h, while that between cyanide and hemocyanin takes only a fraction of a second. The 1H-NMR signal of 0.20 M imidazole (4,5-H) and the 19F-NMR signal of 0.20 M KF at pH 9.0 are broadened by oxyhemocyanin, and the extent of broadening is linearly dependent on hemocyanin concentration. Addition of KCN (up to 0.001 M) results in reduction of paramagnetic Cu(II) to diamagnetic Cu(I), with the copper still bound to the protein. Above 0.001 M, Cu begins to be removed from the protein, forming aqueous cuprous-cyanide complexes. The rate constant of the primary reaction of KCN (0.0001-0.001 M) with hemocyanin decreases with pH increase in the pH range 8.5-9.5. The data suggest that the neutral HCN, or its kinetic equivalent, is the reactive species in the primary reaction. A resonance Raman spectrum of the oxyhemocyanin, by excitation with the 514.4-nm band, shows the O22- vibration at 752 cm-1. Literature values give 744 cm-1 in Cancer magister (arthropod) and 749 cm-1 in Busycon canaliculatum (mollusk) hemocyanins for the O22- vibration. The position of the O22- vibration together with the NMR and spectrophotometric results suggests that A. fulica and B. canaliculatum hemocyanins have similar structures and properties.