STUDIES OF THE RECONSTITUTION BEHAVIOR OF RHUS VERNICIFERA LACCASE
Abstract
The problem of copper binding in the blue copper oxidase Rhus vernicifera laccase has been investigated in a study of the reconstitution properties of protein derived from Japanese and Chinese sources. Chinese protein has been obtained from several provincial sources. The general trend in the results indicates that reconstitution varies with protein age and protein source. One important facet of the protein reconstitution experiments is the development of a rapid dialysis technique based on the use of hollow fibers. This biofiber dialysis technique enables experiments to be performed in less than half the time required for the conventional dialysis technqiue. Preparation of biofiber dialysis devices is described in detail. A method has been developed to deplete Japanese Rhus laccase of copper. By dialysis against cyanide and ascorbate at pH = 7, complete removal of protein-bound metal is achieved. Addition of copper(I) by dialysis or incubation results in copper binding and activity, but the blue color characteristic of the native protein is missing. A model is proposed to explain the results. Chinese Rhus laccase shows variation in its reconstitution behavior. In general, three types of protein reconstitution are observed: (1) Native protein with A(,280)/A(,614) = 21; provincial origin unknown. Activity, copper content, and blue color are all returned to native levels when ascorbate/cyanide apoprotein is treated with copper(I). The fluorescence spectrum is sensitive to the rate of copper addition. (2) Native protein with A(,280)/A(,614) = 16-17; provincial origin uncertain. A partially active reconstituted protein results when apoprotein is treated with copper(I). No blue color is seen in the reconstituted protein although copper uptake approaches native levels. The EPR spectrum of the native protein is missing part of the signal attributed to Type 2 copper. (3) Native protein with A(,280)/A(,614) = 16-17; Cheng Kou province. Blue, active reconstituted protein results from addition of copper(I) to apoprotein. The fluorescence spectrum is not sensitive to the rate of copper addition. Two fractions--one blue and resembling native protein, the other colorless with low copper content--are separated by cation--exchange chromatography. The blue fraction has unusual EPR properties for a blue copper oxidase, while the EPR of the non-blue fraction shows evidence for more than one form of copper (II). Protein age and storage also affect the reconstitution process. The native protein isolated from Cheng Kou province slowly deteriorates on aging, with activity and spectral ratios decaying in a generally monotonic fashion. The aging of the native protein also affects the properties of its reconstituted protein. As the native protein ages, the properties of its reconstituted protein are less likely to resemble the native protein. Even the length of the protein isolation procedures affects reconstitution. Long isolations are detrimental to the recovery of native properties. Difference in protein storage causes differences in protein reconstitution. Of the three methods tested, shell-freezing in liquid nitrogen and subsequent storage below 0(DEGREES)C is preferred to lyophilization or storage at 10(DEGREES)C in phosphate buffer at pH 6.0. The best reconstitution results for Cheng Kou province Chinese Rhus laccase are obtained with freshly thawed protein which had been shell-frozen after a very short isolation. Several other items of interest are presented. The method of protein isolation is fully described. A method of copper analysis based on the color of a copper(I)--biquinoline or bathocuproine disulfonate complex has been developed, and the method of protein activity measurement is explained. Also, a method of protein preparation for EPR (concentration and freezing) has been developed.
Degree
Ph.D.
Subject Area
Chemistry
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