Isolation and spectroscopic characterization of the structural subunits of keyhole limpet hemocyanin.
Keyhole limpet hemocyanin is a respiratory glycoprotein of high molecular weight from the gastropod mollusc Megathura crenulata. Two subunits, KLH1 and KLH2, were isolated using ion exchange chromatography and their physical properties are compared with the parent molecule. The various proteins are characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, cesium chloride and potassium iodide as tryptophan quenchers. The conformational stability of the native aggregate and its isolated structural subunits are also studied by circular dichroism and fluorescence spectroscopy as a function of temperature, as well as in the presence of guanidinium hydrochloride and urea. The associated subunits in the hemocyanin aggregates increase considerably the melting temperature to 67 degrees C and the free energy of stabilization in water, DeltaG(H(2)O)(D), towards guanidinium hydrochloride is higher for the decamer as compared to the isolated subunits; this difference can be accounted for by the stabilizing effects of intra-subunit interactions exerted within the oligomer. The copper-dioxygen complex at the active site additionally stabilizes the molecule, and removing of the copper ions increases the tryptophan emission and the quantum yield of the fluorescence.
Schütz
J
J
abteilung für Physikalische Biochemie des Physiologisch-chemischen Instituts der Universität Tübingen, Hoppe-Seyler-Strasse 4, D-72076 Tübingen, Germany.
Dolashka-Angelova
P
P
Abrashev
R
R
Nicolov
P
P
Voelter
W
W
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D019791:Guanidine / Q000494:pharmacology; D006433:Hemocyanins / Q000737:chemistry*; D008974:Mollusca; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects / Q000502:physiology; D021122:Protein Subunits; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D021122:Protein Subunits; D014508:Urea; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin; D019791:Guanidine