Fluorescence properties and conformational stability of the beta-hemocyanin of Helix pomatia.
The beta-hemocyanin (beta-HpH) is one of the three dioxygen-binding proteins found freely dissolved in the hemolymph of the gastropodan mollusc Helix pomatia. The didecameric molecule (molecular mass 9 MDa) is built up of only one type of subunits. The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Upon excitation of the hemocyanins at 295 or 280 nm, tryptophyl residues buried in the hydrophobic interior of the protein determine the fluorescence emission. This is confirmed by quenching experiments with acrylamide, cesium chloride and potassium iodide. The copper-dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-beta-HpH. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophyl residues in the apo-form. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the beta-HpH and its subunits exist in different conformations. The thermal stability of the oxy- and apo-beta-HpH is characterized by a transition temperature (Tm) of 84 degrees C and 63 degrees C, respectively, obtained by differential scanning calorimetry. Increase of the temperature influences the active site at lower temperatures than the environments of tryptophans and tyrosines causing a loss of oxygen bound to the copper atoms. This process is, at least partially, reversible as after cooling of the protein samples, around 60% reinstatement of the copper-peroxide band has been observed. The results confirm the role of the copper-dioxygen complex for the stabilization of the hemocyanin structure in solution. The other important stabilizing factor is oligomerization of the hemocyanin molecule.
Idakieva
Krassimira
K
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Akad. G. Bonchev-Str. Bl. 9, Sofia 1113, Bulgaria. idakieva@orgchm.bas.bg
Siddiqui
Nurul I
NI
Parvanova
Katja
K
Nikolov
Peter
P
Gielens
Constant
C
D000818:Animals; D002152:Calorimetry, Differential Scanning; D006372:Helix, Snails / Q000737:chemistry; D006433:Hemocyanins / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
C021724:oxy-beta-hemocyanin; D014364:Tryptophan; D006433:Hemocyanins