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Simultaneous isolation of trypsin inhibitor and anti-A phytohemagglutinin from Vicia cracca by means of biospecific adsorption
394
SHORT COMMUNICATIONS
BBA 3 3 2 5 5
Simultaneous isolation of trypsin inhibitor and anti-A phytohemagglutinin from Vicia cracca by means of biospecific adsorption The purification of enzymes and enzyme inhibitors by means of specific adsorption has been reported by several authors I 7. In order to simplify the purification step, a method is introduced that should be of general applicability in handling suspensions and emulsions, making removal of insoluble material unnecessaryL The adsorbent is enclosed in a bag which is freely permeable to liquid but impermeable to suspended solids and adsorbent beads. This report describes the use of trypsin coupled to Sepharose 6B, and blood group substance A coupled to Sepharose 2B, as specific adsorbents for trypsin inhibitor and anti-A phytohemagglutinin from Ficia cracca, respectively. The coupling was performed according to the cyanogen bromide methodS, 9. Preparation of the A-substance agarose has been reported as well as its use in the purification of human anti-A serum isoantibody 1°,11. A detailed report on tile purification and characterization of the agglutinin is in preparation by Dr. K. Aspberg and will appear separately. The preparation of suitable containers has recently been reported 5. The trypsin agarose (dry weight, I8o mg) and the A-substance-agarose (dry weight, 6o mg), 3 ml of each, were enclosed in separate bags made by welding together circular sheets (diameter, 3.2 cm) of nylon net (mesh size, 5o/,). The two bags, together with a control bag filled with untreated agarose, were placed in I5o ml of a suspension of ground seeds of V. cracca (I g) in o.o 5 M Tris-HC1 buffer containing o.5 M NaC1 of pH 7.6. After 3o min stirring, the bags were removed and introduced into short, close-fitting columns (internal diameter, 3.2 cm). The bag was sandwiched in each colunm between porous plastic membranes. In this manner they could be conveniently washed with buffer (o.o5 M Tris-HC1, pH 7.6, containing o.5 .~I NaC1) until no ultraviolet-absorbing material was detectable. Material adsorbed to the trypsin gel was then eluted with 0.o 5 M glycine HC1 buffer, pH 3.o, containing o.5 M NaC1. Fractions of 2.5 ml were collected. The ultraviolet-absorbing (A280 nrn) fractions inhibited trypsin (substrate: benzoyl-DL-arginine-p-nitroanilide) as well as chymotrypsin (substrate: acetvltyrosine ethyl ester) but showed no agglutinating activity against A erythrocytes. After dialysis against distilled water and subsequent freeze-drying, about I m g of protein was obtained. No inhibition activity against trypsin remained in tile suspension. Material adsorbed to the A-substance gel was removed with o.o5 M acetate buffer, pH 4.5, containing o. 5 M NaC1. The pH of the fractions was immediately adjusted to 7-4 with o.5 M NaOH. A 2 % solution ofA erythrocytes in o.o2 M phosphate buffer, pH 7.4, containing o. 9 % NaC1 was used to determine the agglutinating activity. Active fractions were pooled and dialysed against distilled water. About 3 mg of freezedried protein was obtained. Not all A erythrocyte-agglutinating activity in the suspension was extracted in the above experiment, but could be obtained by repeating the adsorption step. No material inhibiting trypsin or chymotrypsin was displaced from the A-substance agarose in the desorption step. The control bag with untreated agarose adsorbed neither agglutinin nor any material inhibiting trypsin or chym~trypsin. J3iochim. Hiopllys..4cta, -,2~ ([(~7o) 384.3(~5
395
SHORT COMMUNICATIONS
TrypsinSepharose 6 B
Blood group Substance A Sepharose 2 B
Fig. 1. S i m u l t a n e o u s a d s o r p t i o n of t r y p s i n inhibitor a n d a n t i - A p h y t o h e m a g g l u t i n i n f r o m a suspension.
In the above experiment the inhibitor and the agglutinin were isolated within 9° min after the seeds were ground. The example shows how the trypsin inhibitor and the agglutinin from V. cracca can be isolated simultaneously. Fig. I shows the adsorption step schematically. Bags containing inhibitors specifically directed to different enzymes can be used for the simultaneous separation of many enzymes. Enzymatic degradation can be controlled by use of bags filled with various enzyme gels. A particular reaction is stopped simply by removing the bag. The technique described is useful in cases where the dissociation constant of a complex (e.g. enzyme-inhibitor, antigen-antibody) is sufficiently small to allow virtually complete adsorption.
Group of Applied Biochemistry in Uppsala, (Swedish Board for Technical Development), Institute of Biochemistry, Box. 53I, S - 7 5 I 21 Uppsala I (Sweden) i 2 3 4 5 6 7 8 9 io II
LARS SUNDBERG J E R K E R PORATH K~RE ASPBERG
P. CUATRECASAS, M. WILCHEK AND C. B. ANFINSEN, Proc. Natl. Acad. Sci. U.S., 61 (1968) 636. M. WILCHEK AND M. GORECKI, European J. Biochem., II (1969) 49. F. FRITZ, B. BREY, S. SCHMAL AND E. WERLE, Z. Physiol. Chem., 350 (1969) 617. T. KRISTIANSEN, M. EINARSSON, L. SUNDBERG AND J, PORATH, F E B S Letters, 7 (1969) 294. J. PORATH AND L. SUNDBERG, in H. PEETERS, Protides of the Biological Fluids, 197 ° , in t h e press. G. FEINSTEIN, F E B S Letters, 7 (1969) 353. H. FRITZ, I. TRAUTSCHOLD, H. HAENDLE AND E. WERLE, Ann. N . Y . Acad. Sci., 146 (1968) 40o. R. AXI~N, J. PORATH AND S. ERNBACK, Nature, 214 (1967) 13o2. J. PORATH, R. AX#~N AND S. ERNBACK, Nature, 215 (1967) 1491. T. KRISTIANSEN, L. SUNDBERG AND J. PORATH, Biochim. Biophys. Acta, 184 (1969) 93. J. PORATH, Nature, 218 (1968) 834.
Biochim. Biophys. Acta, 221 (197 o) 394-395
SHORT COMMUNICATIONS
BBA 3 3 2 5 5
Simultaneous isolation of trypsin inhibitor and anti-A phytohemagglutinin from Vicia cracca by means of biospecific adsorption The purification of enzymes and enzyme inhibitors by means of specific adsorption has been reported by several authors I 7. In order to simplify the purification step, a method is introduced that should be of general applicability in handling suspensions and emulsions, making removal of insoluble material unnecessaryL The adsorbent is enclosed in a bag which is freely permeable to liquid but impermeable to suspended solids and adsorbent beads. This report describes the use of trypsin coupled to Sepharose 6B, and blood group substance A coupled to Sepharose 2B, as specific adsorbents for trypsin inhibitor and anti-A phytohemagglutinin from Ficia cracca, respectively. The coupling was performed according to the cyanogen bromide methodS, 9. Preparation of the A-substance agarose has been reported as well as its use in the purification of human anti-A serum isoantibody 1°,11. A detailed report on tile purification and characterization of the agglutinin is in preparation by Dr. K. Aspberg and will appear separately. The preparation of suitable containers has recently been reported 5. The trypsin agarose (dry weight, I8o mg) and the A-substance-agarose (dry weight, 6o mg), 3 ml of each, were enclosed in separate bags made by welding together circular sheets (diameter, 3.2 cm) of nylon net (mesh size, 5o/,). The two bags, together with a control bag filled with untreated agarose, were placed in I5o ml of a suspension of ground seeds of V. cracca (I g) in o.o 5 M Tris-HC1 buffer containing o.5 M NaC1 of pH 7.6. After 3o min stirring, the bags were removed and introduced into short, close-fitting columns (internal diameter, 3.2 cm). The bag was sandwiched in each colunm between porous plastic membranes. In this manner they could be conveniently washed with buffer (o.o5 M Tris-HC1, pH 7.6, containing o.5 .~I NaC1) until no ultraviolet-absorbing material was detectable. Material adsorbed to the trypsin gel was then eluted with 0.o 5 M glycine HC1 buffer, pH 3.o, containing o.5 M NaC1. Fractions of 2.5 ml were collected. The ultraviolet-absorbing (A280 nrn) fractions inhibited trypsin (substrate: benzoyl-DL-arginine-p-nitroanilide) as well as chymotrypsin (substrate: acetvltyrosine ethyl ester) but showed no agglutinating activity against A erythrocytes. After dialysis against distilled water and subsequent freeze-drying, about I m g of protein was obtained. No inhibition activity against trypsin remained in tile suspension. Material adsorbed to the A-substance gel was removed with o.o5 M acetate buffer, pH 4.5, containing o. 5 M NaC1. The pH of the fractions was immediately adjusted to 7-4 with o.5 M NaOH. A 2 % solution ofA erythrocytes in o.o2 M phosphate buffer, pH 7.4, containing o. 9 % NaC1 was used to determine the agglutinating activity. Active fractions were pooled and dialysed against distilled water. About 3 mg of freezedried protein was obtained. Not all A erythrocyte-agglutinating activity in the suspension was extracted in the above experiment, but could be obtained by repeating the adsorption step. No material inhibiting trypsin or chymotrypsin was displaced from the A-substance agarose in the desorption step. The control bag with untreated agarose adsorbed neither agglutinin nor any material inhibiting trypsin or chym~trypsin. J3iochim. Hiopllys..4cta, -,2~ ([(~7o) 384.3(~5
395
SHORT COMMUNICATIONS
TrypsinSepharose 6 B
Blood group Substance A Sepharose 2 B
Fig. 1. S i m u l t a n e o u s a d s o r p t i o n of t r y p s i n inhibitor a n d a n t i - A p h y t o h e m a g g l u t i n i n f r o m a suspension.
In the above experiment the inhibitor and the agglutinin were isolated within 9° min after the seeds were ground. The example shows how the trypsin inhibitor and the agglutinin from V. cracca can be isolated simultaneously. Fig. I shows the adsorption step schematically. Bags containing inhibitors specifically directed to different enzymes can be used for the simultaneous separation of many enzymes. Enzymatic degradation can be controlled by use of bags filled with various enzyme gels. A particular reaction is stopped simply by removing the bag. The technique described is useful in cases where the dissociation constant of a complex (e.g. enzyme-inhibitor, antigen-antibody) is sufficiently small to allow virtually complete adsorption.
Group of Applied Biochemistry in Uppsala, (Swedish Board for Technical Development), Institute of Biochemistry, Box. 53I, S - 7 5 I 21 Uppsala I (Sweden) i 2 3 4 5 6 7 8 9 io II
LARS SUNDBERG J E R K E R PORATH K~RE ASPBERG
P. CUATRECASAS, M. WILCHEK AND C. B. ANFINSEN, Proc. Natl. Acad. Sci. U.S., 61 (1968) 636. M. WILCHEK AND M. GORECKI, European J. Biochem., II (1969) 49. F. FRITZ, B. BREY, S. SCHMAL AND E. WERLE, Z. Physiol. Chem., 350 (1969) 617. T. KRISTIANSEN, M. EINARSSON, L. SUNDBERG AND J, PORATH, F E B S Letters, 7 (1969) 294. J. PORATH AND L. SUNDBERG, in H. PEETERS, Protides of the Biological Fluids, 197 ° , in t h e press. G. FEINSTEIN, F E B S Letters, 7 (1969) 353. H. FRITZ, I. TRAUTSCHOLD, H. HAENDLE AND E. WERLE, Ann. N . Y . Acad. Sci., 146 (1968) 40o. R. AXI~N, J. PORATH AND S. ERNBACK, Nature, 214 (1967) 13o2. J. PORATH, R. AX#~N AND S. ERNBACK, Nature, 215 (1967) 1491. T. KRISTIANSEN, L. SUNDBERG AND J. PORATH, Biochim. Biophys. Acta, 184 (1969) 93. J. PORATH, Nature, 218 (1968) 834.
Biochim. Biophys. Acta, 221 (197 o) 394-395