Allosteric Oxygen-Binding Properties of Reassembled Tarantula(Eurypelma californicum)Hemocyanin with Incorporated Apo- or Met-Subunits

Abstract

4.times.6-meric hemocyanin of the tarantula Eurypelma Californicum was dissociated into subunits; one type of subunit was removed by immunoaffinity chromatography and replaced by its apo-or met-form. The mixture was reassembled and reconstituted 4.times.6-mers were isolated. This was performed for subunits a, bc, d, e, f and g, respectively. It was varified by crossed immunoelectrophoresis that each type of subunit including the modified one, was incorporated in the reassembled 4.times.6-mers. Oxygen binding curves of the purified reconstituted 4.times.6-mers were recorded at different pH values (pH 7.0-9.0; 20 .degree. C). Half-saturation pressures (p50) and the cooperativities were calculated using unmodified, reassembled 4.times.6 as reference. In all cases, incorporation of a met-subunit increased oxygen affinity. In contrast, incorporation of an apo-subunit either slightly decreased oxygen affinity (bc, f and) or had no detected influence (others). The Bohr effect remained more or less unchanged in every case. Cooperativity was generally decreased. The met-modification of subunit d had the strongest effect. No significant differences could be observed between the respective met-and apomodification, except for experiments with subunit d. Generally, the value of hmax exceeded h50 by a factor of 1.3 to 1.5. pH-sensitivity of cooperativity was distinctly influenced depending on the modified subunit. The strongest effect was observed for subunit bc. Our results demonstrate, for the first time, that each subunit of tarantula hemocyanin is involved in the allosteric processes. Apparently, they uniformly contribute to oxygen affinity and Bohr effect, but distinctly to cooperativity and pH senstitvity of the latter.