A simple procedure for regeneration of an organomercurial agarose column

ANALYTICAL

BIOCHEMISTRY

91, 367-369 (1978)

A Simple Procedure Organomercurial

for Regeneration of an Agarose Column

~rganomer~urial agarose has been used in the purification of various thiol compounds including enzymes (1). Thiol compounds are first adsorbed on a column of organomercurial agarose, and then eluted with a second thiol compound. e.g., 2-mercaptoethanol(2-ME)’ and cysteine. Although this column can be used repeatedly, a usual method for regeneration of the column is to remove the second thiol by HgCl,. It would be desirable to regenerate the column without using HgC&, since it is biohazardous. In the study of the purification of a thiol-containing enzyme, we found that organomer~urial agarose, which had previously been treated with 2-ME, could adsorb the enzyme and that the enzyme was eluted with 2-ME. This finding led us to examine whether the column can be used repeatedly without the regeneration using HgCl,. MATERIALS

AND METHODS

Sepharose 4B was the product of Pharmacia Fine Chemicals, Uppsala. ~-Aminophenylmercuri~ acetate, 5,5’-dithiobis-(Z-nitrobenzoi~ acid), 2-ME, and L-cysteine were obtained from Nakarai Chemicals, Kyoto, and bovine serum albumin from Sigma Chemical Co., St. Louis. Other chemicals were of analytical grade. Rabbit hemoglobin was prepared as described by Geraci et al. (2). p-Chloromercurianiline-agarose (MAA) was prepared as described by Sluyterman and Wijdenes (l), and its capacity for 2-nitro-5mercaptobenzoic acid was estimated to be 6.5 pmoli ml by the method of the same authors (1). The column of a thiol-treated MAA was prepared as follows. Ten bed volumes of 50 mM potassium phosphate buffer, pH 7.0, containing 0.2 M NaCl, 1 mM EDTA, and 10 mM 2-ME (buffer A) was passed through a column of MAA, and then the column was washed with 10 bed volumes of 50 mM potassium phosphate buffer, pH 7.0, containing 0.2 M NaCl and 1 mM EDTA (buffer B). The capacity of MAA and 2-ME- or cysteine-treated MAA for albumin and hemoglobin was determined as follows. A solution of each protein (I m&ml) was passed through a column of MAA or thiol-treated MAA (0.55 x 1 cm) until the concentration of the effluent became the same as that of the initial solution. The column was washed with buffer B until the absorbance at 280 nm of the effluent became nearly zero, and the protein I Abbreviations

used: 2-ME, 2-mercaptoethanol; 367

MAA. p-chloromercurianiline-agarose. 0003-2697178~09~ l-0367$02.00/0 Copyright :a 1918 by Academic Press, Inc. All righta of reproduction tn any form reserved.

368

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COMMUNICATIONS

was eluted with buffer A. The amount of the eluted protein was determined by the method of Lowry ef al. (3) after removing 2-ME or cysteine by dialysis. RESULTS AND DISCUSSION

The binding of thiol compounds such as glutathione, cysteine, and reduced CoA to 2-ME-treated p-acetoxymercurianiline-agarose was reported by Matsuo et al. (4). To our knowledge, however, the utility of 2ME-treated MAA in the purification of thiol-containing proteins has not been tested. In the present study, it was found that bovine serum albumin and rabbit hemoglobin, thiol-containing proteins, were adsorbed on a column of MAA previously treated with 2-ME or cysteine. The capacities of 2-ME- and cysteine-treated MAA for bovine serum albumin were 12 and 7.4 m&ml, respectively, while the capacity of the original MAA was 18 mgiml. This result indicates that the capacity of MAA was decreased considerably by the treatment with a thiol compound. However, since the capacities of the thiol-treated MAAs are still high, the decrease in capacity does not injure their utility. Then, we tested whether the MAA column could be used repeatedly. Hemoglobin (OS mg) was applied onto a column of 2-ME-treated MAA (0.55 x 1 cm) and the bound hemoglobin was eluted with the buffer containing 10 mM 2-ME (buffer A). The column was washed with the buffer for equilibration (buffer B). The second application of hemoglobin was made on the column, and the bound hemoglobin was eluted with buffer A. It was noted that the elution profile for the second chromatography was almost the same as that of the first one. A similar elution pattern was observed in the chromatography on the same MAA at least six times. Even after these processes, the capacity of the column for hemoglobin decreased only by 20%. These results indicate that the present method which does not require HgC& for the recycling process of a MAA column is useful. The HgCl, treatment is not desirable especially in the puri~cation of enzymes containing a thiol group essential for activity, in that HgCl, should be completely washed out from the column in the recycling process. In the present method, the washing of the column with 10 bed volumes of the buffer for equilibration regenerates the column ready for next use. In our laboratory, column chromatography on 2-ME-treated MAA has been successfully used in the purification of L-galactonolactone oxidase from yeast mitochondria (Nishikimi, Noguchi, and Yagi, unpublished result). In the chromatography described above, thiol-containing proteins are bound to the mercury of the column through their exchange with the thiol compound previously bound to the mercury. The present chromatography, therefore, may be termed “mercaptide-tool ~terch~ge” chromato~phy.

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369

REFERENCES 1. Stuyterman, L. A. &., and Wijdenes. J. f 1974) irr Methods in Enzymology (Jakoby. W. B.. and Wilchek, M.. eds.), Vol. 34, Part B. pp. 546547, Academic Press. New York. 2. Geraci. G., Parkhurst. L. J., and Gibson. Q. H. ( 1969) J. Bio/. CkPJR. 244, 4664-4667. 3. Lowry, 0. H.. Rosebrough. N. J., Parr, A. L., and Randall. R. J. (1951)5. Bio(. Chrm. 193, 265-275. 4. Matsuo, Y., Sano, R., Tosa. T., and Chibata, 1. (1975) Anal. Bidzem. 68, 349-357. ETSUKO NOGUCHI MORIMITSU NISHIKIMI KLJNIO YAGI