Rapana thomasiana grosse (gastropoda) haemocyanin: spectroscopic studies of the structure in solution and the conformational stability of the native protein and its structural subunits.
1. The stability towards pH changes, thermal and chemical (guanidine hydrochloride) denaturation of the oxy- and apo-forms of the native Rapana thomasiana haemocyanin and its structural subunits, RHSS1 and RHSS2, has been investigated using fluorescence and CD spectroscopy. The association of the subunits into haemocyanin aggregates increases considerably the melting temperature and the free energy of stabilization in water. The guanidine hydrochloride denaturation of the aggregated oxygen-transporting protein depends slightly on the protein concentration. The denaturation of the individual subunits is concentration-independent. Rapana haemocyanin is 5.9-7.5 kJ/mol more stable than the constituent polypeptide chains. 2. Upon excitation of the native haemocyanin and the subunits at 295 or 280 nm the fluorescence emission is determined by tryptophyl residues 'buried' deeply in the hydrophobic interior of the protein globules. This is confirmed by quenching experiments with acrylamide, caesium and iodide ions. The efficiency of the radiationless energy transfer between the phenol (donor) and indole (acceptor) fluorophores in the three species, native haemocyanin, RHSS1 and RHSS2, has been determined. An efficient 'interchain' energy transfer between tyrosyl and tryptophyl residues from different polypeptide chains occurs in the non-dissociated form of the haemocyanin. 3. The tryptophan emission of the oxyhaemocyanin, oxy-RHSS1 and oxy-RHSS 2 is strongly quenched by the copper-dioxygen complex at the active site and the respective quantum yields of fluorescence of the oxygenated species are 4-7 times lower than those of the apo-forms. Protonated imidazole groups quench the fluorescence of neighbouring exited indole rings, probably by charge-transfer complex formation.
Dolashka
P
P
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria.
Genov
N
N
Parvanova
K
K
Voelter
W
W
Geiger
M
M
Stoeva
S
S
D000818:Animals; D002942:Circular Dichroism; D005453:Fluorescence; D006361:Heating; D006433:Hemocyanins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D011487:Protein Conformation; D011489:Protein Denaturation; D012908:Snails / Q000737:chemistry*; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D046911:Macromolecular Substances; D012996:Solutions; D014364:Tryptophan; D006433:Hemocyanins