Structural and spectroscopic studies of the native hemocyanin from Maia squinado and its structural subunits

The dodecameric hemocyanin of the crab Maia squinado contains five major el ectrophoretically separable polypeptide chains (structural subunits) which have been purified by FPLC ion exchange chromatography. The various protein s have been characterized by fluorescence spectroscopy, combined with fluor escence quenching studies, using acrylamide, caesium chloride and potassium iodide as tryptophan quenchers. The results show that the tryptophyl side chains of dodecameric He are deeply buried in hydrophobic regions of the he mocyanin aggregates and the quenching efficiency values for the native He i n comparison with those from the constituent subunits are two to four times less. The conformational stabilities of the native dodecameric aggregate a nd its isolated structural subunits towards various denaturants (pH, temper ature, guanidinium hydrochloride) indicate that the quaternary structure is stabilized by hydrophilic and polar forces, whereby, both, the oxy- and ap e-forms of the protein have been considered. The critical temperatures for the structural subunits, T-c, determined by fluorescence spectroscopy, are in the region of 50-60 degrees C, coinciding with the melting temperatures, T-m, determined by CD spectroscopy. The free energy of stabilization in wa ter, Delta G(D)(H2O), toward guanidinium hydrochloride is about two times h igher for the dodecamer as compared to the isolated subunits. These studies reveal that oligomerization between functional subunits has a stabilizing effect on the whole molecule and differences in the primary structures resu lt in different stabilities of the subunits. (C) 2000 Elsevier Science B.V. All rights reserved.