BIOTECHNOLOGY TECHNIQUES Volume 8 No.9 (Sept.1 994) pp.655458 Received as revised 22~ui July

COPPER AFFINITY PRECIPITATION AS AN INITIAL STEP IN PROTEIN PURIFICATION

Ritu Agarwal & M.N.Gupta*

Department of Chemistry Indian Institute of Technology, Delhi-110 016, India

SUMMARY

The affinity of copper ions for histidine residues can be utilized to carry out fractional precipitation of proteins. Using this approach it is possible to purify concanavalin A by a simple two step procedure. The fold purification obtained was 4.5 fold.

INTRODUCTION

Selective precipitation of proteins by affinity reagents has shown considerable promise in recent years (Gupta et al., 1993). Recently it has been shown that the concept of Immobilized Metal Ion Affinity Chromatography can be extended to separate proteins by affinity precipitation (van Dam et al., 1989). This suggested the possibility that precipitation by CuSO4 can be used as an initial step in separation of proteins. This limited fractionation of proteins may be possible on the basis of varying number of histidines (and other groups capable of chelating with

copper 1 available for complexing with copper. This work explores these possibilities and shows that metal affinity precipitation may indeed be an useful step in protein/enzyme separation. It is also shown that a higher purity concanavalin A can be obtained from crude extract simply by ammonium sulphate precipitation followed by affinity precipitation with copper ions.

MATERIALSAND HIWHODS

Materials: o(-Amylase (from bovine pancreas) and latex beads (polystyrene, 0.46 urn diameter, 10% solid content) were from Sigma, USA, Alkaline phosphatase (from chicken intestine), chymotrypsin and peroxidase were from Sisco Research Laboratories (India). Jack bean meal was from Difco, USA-B- Amylase was from Merck (India). Concanavalin A was obtained from Centre for Biochemical Technology, India.

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Methods: Agglutination assay: Lectin activity was measured using latex beads to which ovalbumin was physically adsorbed (Kaul et al., 1991). The minimum amount of protein still giving positive agglutination was determined by this method. Protein was measured using the dye binding method of Bradford (1976). Polyacrylamide gel electrophoresis: SDS-PAGE with 10% gel was performed according to Laemmli (1970). Precipitation of proteins by CuSO4: Parameters, such as pH, CuSO4 concentration and protein concentration were varied to optimize conditions for maximum precipitation of protein with CuSO4. Purification of concanavalin A: Jack bean meal extract was prepared and (NH4)2S04 precipitation was done by the method of Agrawal and Goldstein (1967). To approximately 2 mg per ml of protein in (NH4)2SO4 precipitate dissolved in distilled water, 1 ml of 0.1 M NaCl, 1 ml CuSO4 (2 ma/ml), 1 ml acetate buffer (0.04 M, pH 6.0) was added. After incubation for two hours, the precipitate was collected by centrifugation at 12,000 g for 12 min. The precipitate was dissolved in minimum amount of Tris-HCl buffer (0.5 M, pH 7.0) and dialysed against the same buffer for 24 h and finally against 0.1 M NaCl.

RESULTS AND DISCUSSION van Dam et al, (1989) has shown that in free solution

Cu2+ crosslinks protein molecules and leads to precipitation in case of haemoglobin. This work also showed that myoglobin with fewer surface histidine residues did not precipitate under these conditions.

In this work with larger number of enzymes/protein

(Fig-l), it was found that Cu 2+ from 0.2 to 0.4 ml4 gave

optimum precipitation and the extent of precipitation was different for different proteins.

The extent of precipitation, was also found to be dependent upon protein concentration (Fig.2). It was further found that precipitation in all these cases was optimum at pH 6.0 and in some cases marginally improved at pH 6.5. While all the precipitation experiments were done in duplicates, which agreed within 5%, it was found that variation between different batches of experiments was up to 12%. Encouraged by above results, we used ammonium sulphate precipitation followed by precipitation with copper ions to purify concanavalin A from the crude extract of jackbean

meal (Table 1). These two simple precipitation Steps were

found to give concanavalin A, which gave as good a titre value as a commercially available concanavalin A (3.9 ng L

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rk”“““6”““’ &ntratio~ (mM)*

~-~‘“‘~.G’~“-F--~““& ProteinaCance~tration ~mc~/ml 3

Figure 2: Effect of protein concentration on precipitation by CuSO4. To 1 ml of protein (0.05 mg/ml to 3 mg/ml) in dis- tilled water, one ml of 0.1 W NaCl, 1 ml acetate buffer (0.04 M, pH 6.0), and 1 ml CuSO4 (2 mM) was added. Rest of the details were as in legend to Fig.1.

Figure 1:Effect of CuSO4 concentration on precipi- tation of various proteins. To 2 mg/ml of protein in distilled water, 1 ml of 0.1 M NaCl, 1 ml acetate buffer (0.04 M, pH 6.0) and 1 ml CuSO4 (0.2 mM to 4.0 mM) was added. After incubation for 2 hrs the mixture was centrifuged for 12 min at 12,000 g and protein was read in the supernatant. Total protein precipitated was calculated by substracting the protein in the super- natant from the total protein added. (D) &-amylase,(x) alkaline phosphatase,(+) chymotryp- sin, (0)B -amylase, (a) peroxidase, and (9) Con A.

12 3 4

Figure 3: SDS-PAGE Elect- rophoresis. Lane 1: commercial Concanavalin A; lane 2: (NH4)2SO4 precipi- tate;lane 3:CuSO4 precipi- tate; lane 4: sequential precipitate of (NH4)2S04 and CuSO4.

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Table 1 Purification of Concanavalin A ----------------------------------------------------------- Protein Acti- Fold

(mg) vity* purifi- cation

----------------------------------------------------------- Crude 45 17.5 1.0 0.28 extract Ammonium 20 7.8 2.25 0.12 sulphate precipitate Co per

B 10 3.9 4.5 0.06

EFeE$?ate ----------------------------------------------------------- * (minimum protein in ng showing positive agglutination)

3.6 ng respectively). The fold purification was of the same order as observed by Olson and Liener (1967), (4.5 and 2.2 respectively) and yield was also the same (0.06 g/100 g of jackbean meal). The SDS-PAGE pattern of concanavalin A at different stages of purification along with pure concanavalin A is shown in Fig.3

These results show that precipitation with copper ions at early stages may yield effective fractionation in some cases. It could be a useful method for large scale

purification of concanavalin A.

RA was supported by Council of Scientific and Industrial Research, India in the form of senior research fellowship. This work was also supported by Department of Biotechnology, Govt. of India and Swedish Agency for Research Cooperation with developing countries (SARKC).

REFERENCES

Agrawal, B.B.L. and Goldstein, 1-J. (1967). Biochim. Biophys. Acta, 147, 262-271.

Bradford, M.M., (1976). Anal. Biochem. 72, 248. Gupta H.N., Guoqiang, D., Mattiasson B. (1993). Biotechnol.

APPl l Biochem. 18, 321-327,

Kaul, R., Read, J. and Mattiasson, B. (1991). Phytochemistry 30, 4005-4009.

Laemmli, U.K. (1970). Nature, 227, 680. Olson, M.O.J. and Liener, I.E. (1967). Biochemistry, 6, 105-

111. van Dam, M.E., Wuenscheli, G.F. and Arnold, F.H. (1989).

Biotechnol. Appl. Biochem. 11, 492-502.

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