ABSTRACT The dodecameric hemocyanin of the arthropod Homarus americanus dissociates at high pH and in the absence of alkaline earth cations into six major and 2 minor electrophoretically separable polypeptide chains (structural subunits). All subunits can be purified by FPLC ion exchange chromatography. N-terminal amino acid sequences of six subunits from H. americanus were determined after additional purification of the subunits on a HPLC column Nucleosil RP 18, and compared with already known N-terminal sequences. The various protein forms have been characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, cesium chloride and potassium iodide as tryptophan quenchers. The results show that the tryptophyl side chains of dodecameric Hc are deeply buried in hydrophobic regions of the hemocyanin aggregates and the quenching efficiency values for the native He in comparison with those from the constituent subunits are 7-10 times less. We have also studied the conformational stability of the native dodecameric aggregate and its isolated structural subunits as a function of pH and temperature, as well as in the presence of guanidinium hydrochloride. Both, the oxy- and apo-forms of the protein have been considered. The conformational changes, induced by the various treatments, were monitored by far UV, CD and fluorescence spectroscopy. The dodecameric form of H. americanus hemocyanin shows a markedly higher thermostability in comparison to other arthropod hemocyanins as well as to its constitutent polypeptide chains. The free energy of stabilization in water, ΔGDH2O, toward guanidinium hydrochloride is about twofold higher for the dodecamer as compared to isolated subunits, and this difference can be accounted for the stabilizing effects of intrasubunit interactions exerted within the oligomer.
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