CONFORMATIONAL-CHANGES OF TARANTULA (EURYPELMA-CALIFORNICUM) HEMOCYANIN DETECTED WITH A FLUORESCENT-PROBE, 7-CHLORO-4-NITROBENZO-2-OXA-1,3-DIAZOLE

Different fluorescent labels were tested in order to monitor conformational transitions of the four‐hexamer haemocyanin from the tarantula Eurypelma californicum during the oxygenation process. When the four‐hexamer was labelled with 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole, the maximum wavelength λmax of the fluorescence emission spectrum was significantly shifted up to 5 nm, depending on pH and the degree of oxygenation. The values for λmax of the fully oxygenated haemocyanin were 531.5 nm (pH < 7.8) and 530.0 nm (pH > 7.8). For deoxygenated haemocyanin the values were 533.5 nm (pH < 7.2) and 535.2 nm (pH > 7.2). The occurrence of four distinct emission maxima supports the hypothesis of four conformational species for the tarantula haemocyanin, which have been predicted by the nesting model [Robert, C. H., Decker, H., Richey, B., Gill, S. J. & Wyman, J. (1987) Proc. Natl Acad. Sci. USA 84, 1891–1895]. Only four amino acids of the four‐hexamer were labelled with 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole. They were identified as lysine 484 on the purified peptide Leu‐Arg‐Lys‐Phe‐His‐Arg. This amino acid is located on the surface of the four copies of subunit d. The sharp shift of the maxima of the emission wavelengths during oxygenation indicates that the four copies of subunits d synchronously take part in the conformational switch. This points to a concerted mechanism for the conformational transitions of the tarantula haemocyanin. Copyright © 1990, Wiley Blackwell. All rights reserved