Structure and stability of arthropodan hemocyanin Limulus polyphemus.
In the hemolymph of many arthropodan species, respiratory copper proteins of high molecular weight, termed hemocyanins (Hcs) are dissolved. In this communication, we report on the protein stability of different hemocyanin species (Crustacea and Chelicerata) using fluorescence spectroscopy. Five to seven major electrophoretically separable protein chains (structural subunits) were purified by fast protein liquid chromatography (FPLC) ion exchange chromatography from different hemocyanins with very high sequence homology of the active site regions binding copper ions (CuA and CuB), and especially the relative sequence positions of histidine (His) and tryptophan (Trp) residues of these protein segments are in all cases identical. The conformational stabilities of the native dodecameric aggregates and their isolated structural subunits towards various denaturants (pH and guanidine hydrochloride (Gdn.HCl)) indicate that the quaternary structure is stabilized by hydrophilic and polar forces, whereby both, the oxy- and apo-forms of the protein are considered. These two classes of Crustacea and Chelicerata Hcs have the similar Trp-fluorescence quantum yields, but different values of lambda(max) emission (about 325 and 337 nm, respectively). Differences in the quantum yields are observed of the oxy- and apo-forms, which must be attributed to the fluorescence quenching effect of the two copper ions (CuA and CuB) in the active site. The position of emission maximum indicates tryptophan side chains are situated in a non-polar environment. Denaturation studies of Hcs by Gdn.HCl indicate that the denaturation process consists of two steps: dissociation of the native molecule into its structural subunits and denaturation of the subunits at concentrations >1.5M Gdn.HCl. Two steps of denaturation are also observed after keeping the protein in buffer solutions at different pH values with different pH-stability for holo-oxy and apo-Hc forms.
Dolashka-Angelova
Pavlina
P
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev, street, bldg. 9, 1113 Sofia, Bulgaria. pda54@yahoo.com
Dolashki
Alexander
A
Stevanovic
Stefan
S
Hristova
Rumijana
R
Atanasov
Boris
B
Nikolov
Peter
P
Voelter
Wolfgang
W
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry*; D001665:Binding Sites; D002853:Chromatography, Liquid; D003300:Copper / Q000378:metabolism; D003445:Crustacea / Q000737:chemistry*; D019791:Guanidine; D006433:Hemocyanins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006737:Horseshoe Crabs / Q000737:chemistry*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D021122:Protein Subunits; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D001059:Apoproteins; D021122:Protein Subunits; D003300:Copper; D014364:Tryptophan; D006433:Hemocyanins; D019791:Guanidine