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Immunoaffinity purification of β-amylase from mustard (Sinapis alba L.) cotyledons
Journal of Chromatography, 40X (lYX7) 385-3X-1 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands CHROM. 19 881
Note
lmmunoaffinity L.) cotyledons
purification
K. SUBBARAMAIAH
and R. SHARMA*
of P-amylase
from
mustard
(Sinapis
a/be
School of Life Sciences, Universify of Hyderabad, Hyderabad-SO0 134 (india)
(Received July 17th, 1987)
Immunoaffinity chromatography is a powerful technique for purification of proteins using corresponding antibody molecules immobilized on an insoluble matrixlp4. In this communication we report the purification of mustard P-amylase to homogeneity by immunoaffinity chromatography. EXPERIMENTAL
Immune serum
fi-Amylase was purified from mustard (Sinapis alba L.) cotyledon to homogeneity by a procedure described earlier 5,6. The purified enzyme was injected into rabbits to obtain fi-amylase specific antiserum. The immunoglobulin G (IgG) fraction from the antiserum was obtained by ammonium sulphate precipitation followed by ion-exchange chromatography on DEAE-cellulose7. Preparation of immunoafinity column
The immunoaffinity column was prepared by coupling 8.0 mg IgG to 1.5 g of cyanogen bromide-activated Sepharose (Pharmacia, Uppsala, Sweden) gel in coupling buffer (0.1 M borate buffer pH 8.0, 0.5 M sodium chloride) at 4°C overnight. Thereafter the gel wwas washed with coupling buffer and residual reactive groups were blocked by stirring with 0.1 M ethanolamine (pH 8.0) for 2 h. The gel was then equilibrated with 50 mM Verona1 buffer pH 8.6, containing 0.5 M sodium chloride and poured into a column. Extraction of P-amylase
Cotyledons from 6-day-old light-grown mustard seedlings were harvested for /?-amylase extraction. The cotyledons were homogenized in the extraction buffer (50 mM Verona1 buffer, pH 8.0, 0.2 M sodium chloride; 1 g cotyledons per 4 ml buffer) and the homogenate was centrifuged at 16 000 g at 4°C. The supernatant was dialyzed against Verona1 buffer and concentrated by lyophilization. The protein and /?-amylase activity was estimated by a procedure described earlier5. ImmunoaSJinity chromatography8
The supernatant 0021-9673/87/$03.50
0
containing /3-amylase was applied to the immunoaffinity 1987 Elsevier Science Publishers B.V.
col-
386
_
NOTES
2.0
I g 1.5 : .c f 1.0 n 0.5
0 o
2
4
6
8
Fig. 1. Immunoaffinity chromatography see Experimental.
0
lo
FRACTION
2
4
6
NUMBER
of mustard fi-amylase on an IgG Sepharose column. For details
umn at a flow-rate of 5 ml/h and the effluent was recycled through the column. Thereafter, the column was washed with extraction buffer to remove unbound proteins. Then the immunoaffinity column was eluted with about 8 ml of distilled water and the elution was interrupted. The column was allowed to stand at 20°C overnight and elution was then resumed at a flow-rate of 10 ml/h. The fractions were analysed for /I-amylase and protein as described earlier 5. The homogeneity of the purified @amylase was ascertained by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) by the procedure of Laemmli9. RESULTS AND DISCUSSION
Since antigen-antibody reactions are highly specific and have low dissociation constants, the dissociation of the immuno complex after immunosorption is generally a difficult task. Usually, a drastic pH or chaotropic reagents have been employed to dissociate which heads to a massive denaturation of the enzymelvs. In the present study, to circumvent this problem, a bland desorption procedure using distilled water was used to elute the bound /I-amylase molecules from the columnlo. The j?-amylase TABLE I PURIFICATION
OF P-AMYLASE FROM MUSTARD COTYLEDONS
Step
Volume (ml)
Activity (nkat)
Protein (w)
Specific activity
PuriJcation fold
Yield (%)
(nkatlmd
(1) Crude supernatant (2) Immunoaffinity chromatography
10 4.5
880 362
18.9 0.06
46.6 6033
1 130
100 41
in the supernatant on application to the immunoaffinity column is bound strongly to the IgG coupled gel. The bound enzyme was eluted with water in fOUr fraCtiOnS with a sharp peak (Fig. 1). The immunoaffinity purification of mustard b-amylase on an IgG Sepharose column resulted in a single step 130-fold purification of the enzyme (Table I). The SDS-PAGE of purified /3-amylase revealed a single P-amylase polypeptide having a molecular weight of 58 000 (data not shown) corresponding to the value for pure fi-amylase protein 6. There was no apparent modification in the physicochemical properties of /3-amylase purified by immunoaffinity chromatography. The purified enzyme revealed three isozymes identical to the isozymes present in the crude supernatant‘j (data not shown). The above results shows that mustard fi-amylase can be purified to homogeneity by immunoaffinity chromatography without any apparent modification of its molecular properties, in a single step directly from supernatant. ACKNOWLEDGEMENTS
The authors thank the Dean, School of Life Sciences, for providing facilities. This work was supported by grant 9 (159)/83/EMR-II from Council for Scientific and Industrial Research, New Delhi. REFERENCES 1 M. Wilchek, T. Miron and J. Kohn, Methods Enzymol., 104 (1984) 1. 2 J. Daussant and D. Bureau, in V. Neuhoff (Editor), Electrophoresis 84, Verlag Chemie, Weinheim, 1984. 3 R. E. Hunt and L. H. Pratt, Plant Physiol, 64 (1979) 332. 4 W. Liedgens, R. Grutzman and H. A. W. Schieder, Z. Naturforsch., 35 (1980) 958. 5 K. Subbaramaiah and R. Sharma, J. Biochem. Biophys. Methods, 10 (1985) 315. 6 K. Subbaramaiah, Ph.D. Dissertation, University of Hyderabad, Hyderabad, 1987. 7 C. A. Williams and M. W. Chase, Methoak in Immunology and Immunochemistry, Vol. I, Pergamon, New York, 1967, p. 321. 8 C. R. Lowe and P. D. F. Dean, A&ity Chromatography, Wiley, London, 1974. 9 U. K. Laemmli, Nature (London), 227 (1970) 680. 10 D. Bureau and J. Daussant, J. Zmmunol. Methods, 57 (1983) 205.
Note
lmmunoaffinity L.) cotyledons
purification
K. SUBBARAMAIAH
and R. SHARMA*
of P-amylase
from
mustard
(Sinapis
a/be
School of Life Sciences, Universify of Hyderabad, Hyderabad-SO0 134 (india)
(Received July 17th, 1987)
Immunoaffinity chromatography is a powerful technique for purification of proteins using corresponding antibody molecules immobilized on an insoluble matrixlp4. In this communication we report the purification of mustard P-amylase to homogeneity by immunoaffinity chromatography. EXPERIMENTAL
Immune serum
fi-Amylase was purified from mustard (Sinapis alba L.) cotyledon to homogeneity by a procedure described earlier 5,6. The purified enzyme was injected into rabbits to obtain fi-amylase specific antiserum. The immunoglobulin G (IgG) fraction from the antiserum was obtained by ammonium sulphate precipitation followed by ion-exchange chromatography on DEAE-cellulose7. Preparation of immunoafinity column
The immunoaffinity column was prepared by coupling 8.0 mg IgG to 1.5 g of cyanogen bromide-activated Sepharose (Pharmacia, Uppsala, Sweden) gel in coupling buffer (0.1 M borate buffer pH 8.0, 0.5 M sodium chloride) at 4°C overnight. Thereafter the gel wwas washed with coupling buffer and residual reactive groups were blocked by stirring with 0.1 M ethanolamine (pH 8.0) for 2 h. The gel was then equilibrated with 50 mM Verona1 buffer pH 8.6, containing 0.5 M sodium chloride and poured into a column. Extraction of P-amylase
Cotyledons from 6-day-old light-grown mustard seedlings were harvested for /?-amylase extraction. The cotyledons were homogenized in the extraction buffer (50 mM Verona1 buffer, pH 8.0, 0.2 M sodium chloride; 1 g cotyledons per 4 ml buffer) and the homogenate was centrifuged at 16 000 g at 4°C. The supernatant was dialyzed against Verona1 buffer and concentrated by lyophilization. The protein and /?-amylase activity was estimated by a procedure described earlier5. ImmunoaSJinity chromatography8
The supernatant 0021-9673/87/$03.50
0
containing /3-amylase was applied to the immunoaffinity 1987 Elsevier Science Publishers B.V.
col-
386
_
NOTES
2.0
I g 1.5 : .c f 1.0 n 0.5
0 o
2
4
6
8
Fig. 1. Immunoaffinity chromatography see Experimental.
0
lo
FRACTION
2
4
6
NUMBER
of mustard fi-amylase on an IgG Sepharose column. For details
umn at a flow-rate of 5 ml/h and the effluent was recycled through the column. Thereafter, the column was washed with extraction buffer to remove unbound proteins. Then the immunoaffinity column was eluted with about 8 ml of distilled water and the elution was interrupted. The column was allowed to stand at 20°C overnight and elution was then resumed at a flow-rate of 10 ml/h. The fractions were analysed for /I-amylase and protein as described earlier 5. The homogeneity of the purified @amylase was ascertained by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) by the procedure of Laemmli9. RESULTS AND DISCUSSION
Since antigen-antibody reactions are highly specific and have low dissociation constants, the dissociation of the immuno complex after immunosorption is generally a difficult task. Usually, a drastic pH or chaotropic reagents have been employed to dissociate which heads to a massive denaturation of the enzymelvs. In the present study, to circumvent this problem, a bland desorption procedure using distilled water was used to elute the bound /I-amylase molecules from the columnlo. The j?-amylase TABLE I PURIFICATION
OF P-AMYLASE FROM MUSTARD COTYLEDONS
Step
Volume (ml)
Activity (nkat)
Protein (w)
Specific activity
PuriJcation fold
Yield (%)
(nkatlmd
(1) Crude supernatant (2) Immunoaffinity chromatography
10 4.5
880 362
18.9 0.06
46.6 6033
1 130
100 41
in the supernatant on application to the immunoaffinity column is bound strongly to the IgG coupled gel. The bound enzyme was eluted with water in fOUr fraCtiOnS with a sharp peak (Fig. 1). The immunoaffinity purification of mustard b-amylase on an IgG Sepharose column resulted in a single step 130-fold purification of the enzyme (Table I). The SDS-PAGE of purified /3-amylase revealed a single P-amylase polypeptide having a molecular weight of 58 000 (data not shown) corresponding to the value for pure fi-amylase protein 6. There was no apparent modification in the physicochemical properties of /3-amylase purified by immunoaffinity chromatography. The purified enzyme revealed three isozymes identical to the isozymes present in the crude supernatant‘j (data not shown). The above results shows that mustard fi-amylase can be purified to homogeneity by immunoaffinity chromatography without any apparent modification of its molecular properties, in a single step directly from supernatant. ACKNOWLEDGEMENTS
The authors thank the Dean, School of Life Sciences, for providing facilities. This work was supported by grant 9 (159)/83/EMR-II from Council for Scientific and Industrial Research, New Delhi. REFERENCES 1 M. Wilchek, T. Miron and J. Kohn, Methods Enzymol., 104 (1984) 1. 2 J. Daussant and D. Bureau, in V. Neuhoff (Editor), Electrophoresis 84, Verlag Chemie, Weinheim, 1984. 3 R. E. Hunt and L. H. Pratt, Plant Physiol, 64 (1979) 332. 4 W. Liedgens, R. Grutzman and H. A. W. Schieder, Z. Naturforsch., 35 (1980) 958. 5 K. Subbaramaiah and R. Sharma, J. Biochem. Biophys. Methods, 10 (1985) 315. 6 K. Subbaramaiah, Ph.D. Dissertation, University of Hyderabad, Hyderabad, 1987. 7 C. A. Williams and M. W. Chase, Methoak in Immunology and Immunochemistry, Vol. I, Pergamon, New York, 1967, p. 321. 8 C. R. Lowe and P. D. F. Dean, A&ity Chromatography, Wiley, London, 1974. 9 U. K. Laemmli, Nature (London), 227 (1970) 680. 10 D. Bureau and J. Daussant, J. Zmmunol. Methods, 57 (1983) 205.