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 electrophoretically separable polypeptide chains (structural subunits) which have been purified by FPLC ion exchange chromatography. The various proteins have been characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, caesium 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 Hc in comparison with those from the constituent subunits are two to four times less. The conformational stabilities of the native dodecameric aggregate and its isolated structural subunits towards various denaturants (pH, temperature, guanidinium hydrochloride) indicate that the quaternary structure is stabilized by hydrophilic and polar forces, whereby, both, the oxy- and apo-forms of the protein have been considered. The critical temperatures for the structural subunits, Tc, determined by fluorescence spectroscopy, are in the region of 50-60 degrees C, coinciding with the melting temperatures, Tm, determined by CD spectroscopy. The free energy of stabilization in water, deltaG(D)H2O, toward guanidinium hydrochloride is about two times higher 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 result in different stabilities of the subunits.
Dolashka-Angelova
P
P
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia. pda54@hotmail.com
Hristova
R
R
Schuetz
J
J
Stoeva
S
S
Schwarz
H
H
Voelter
W
W
D000595:Amino Acid Sequence; D000818:Animals; D003386:Brachyura / Q000737:chemistry*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006433:Hemocyanins / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature
D006433:Hemocyanins