Studies of two blue copper proteins: Photophysics of azurin and isolation, purification, and characterization of porcine ceruloplasmin

Frank Karl Klemens, Purdue University

Abstract

The tryptophan phosphorescence from a series of derivatives of Pseudomonas aeruginosa azurin is monitored at 30$\sp\circ$C in pH 8.5 buffer solution. The phosphorescence lifetimes fall in the range of 230-270 ms for deoxygenated solutions of derivatives containing Cd(II), Cu(I), Co(II), Ni(II), Hg(II) or apoazurin. A weak signal with a lifetime of about 130 ms is observed from solutions of oxidized native azurin. This component arises from the protein heterogeneity and is ascribed to a modified form of azurin in solution. This modified form is observed in the phosphorescence emission and phosphorescence lifetime, and has been assigned on the basis of the unique sensitivity to quenching by dioxygen. Aside from this minor component, the tryptophan phosphorescence in the Cu(II) protein appears to be fully quenched. The quenching is assigned an electron-transfer mechanism involving transient reduction of the metal center. The same mechanism is deemed to be responsible for fluorescence quenching in oxidized native azurin as well. These observations are of interest because aromatic groups like tryptophan may be conduits for physiological electron-transfer processes involving the copper center. Isolation and purifiction of porcine ceruloplasmin (Cp) is accomplished with the use of a derivatized Sepharose CL-4B-primary amine resin in conjunction with a chloroform/ethanol precipitation. The spectral ratio, A$\sb{610}$/A$\sb{280}$, after the extraction is in the range of 0.042-0.046. Characterization of native Cp revealed that 5.9-6.3 coppers are present and that 2.4-3.3 of these coppers are EPR-active. ApoCp was prepared via 3.5 hr room temperature anaerobic dialysis against 0.1 M cyanide. Reconstitution of the apoCp was accomplished at 25$\sp\circ$C as well as at 4$\sp\circ$C. The product from the 25$\sp\circ$C reconstitution is identical to native Cp in 75-85% yield. However, the major product from the 4$\sp\circ$C reconstitution contains no Type 1 coppers based on the EPR signal which is primarily a Type 2 signal. Mercury(II) is added to apoCp at pH 5.5 and 6.25. From UV absorption at 250 nm, the binding of Hg(II) is at the Type 1 site since the difference spectrum (Hg(II)Cp $-$ apoCp) reveals an absorbance peak at 245-255 nm. This is indicative of mercury(II) binding at the Type 1 site. Attempts to increase the yield (30-33%) of the Hg(II)-bound protein are not successful.

Degree

Ph.D.

Advisors

McMillin, Purdue University.

Subject Area

Chemistry

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