950
Biochimica et Biophysica Acta, 429 (1976) 950--953 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 67780 C H A R A C T E R I Z A T I O N OF AGAROSE-BOUND TRYPSIN
BERT WALTER Department of Biochemistry, Brandeis University, Waltham, Mass. 02154 (U.S.A.)
(Received October 16th, 1975)
Summary Agarose-bound trypsin (EC 3.4.21.4) was prepared and its properties were compared with those of soluble trypsin. The b o u n d form of the enzyme was found to be equally available to large and small molecular weight substrates as the soluble form. In addition, the b o u n d form of the enzyme showed the same specificity towards protein substrates as the soluble enzyme. However, the agarose-bound trypsin showed greater stability than the soluble trypsin to denaturing conditions for prolonged period of time.
Introduction There have been conflicting reports on the comparative properties of free and matrix-bound enzymes [1--3] and this work was undertaken with the purpose of establishing some comparative data on one well characterized enzyme, namely trypsin (EC 3.4.21.4), in the soluble form and bound to agarose. The specific properties investigated were (1) relative activity towards large and small substrates; (2) specificity of cleavage in protein substrates, and (3) relative activity under denaturing conditions. Materials and Methods
Bovine pancreatic trypsin (Worthington) was covalently b o u n d to agarose A-15 M, 50--100 mesh (Bio-Rad) by the CNBr procedure of Ax~n et al. [4]; the binding was conducted in the presence of 10 mM CaC12 to reduce trypsin autolysis. The a m o u n t of trypsin covalently attached to agarose was determined by amino acid analysis [5,6] on Beckman Model 120C amino acid analyzer after hydrolysis of 1 ml of settled agarose-trypsin in 6 M HCI for 24 h at l l 0 ° C . Soluble and matrix-bound trypsin activity was assayed titrimetrically with a Radiometer pH-stat system using 0.01 M KOH as a titrant; the temperature was
951 maintained at 25°C with a Haake temperature control bath. A typical assay consisted of 10 ml of 15 mMp-tosylarginine methyl ester (TosArgOMe) (Sigma), pH 8.0, and 0.006--0.009 pmol of soluble trypsin or agarose-trypsin having a comparable a m o u n t of active trypsin. The total a m o u n t of active trypsin per given quantity of agarose was determined from a standard curve constructed using the rate of TosArgOMe hydrolysis by soluble trypsin as a function of enzyme concentration. In addition, proteolytic action of trypsin and agarosetrypsin on proteins was also monitored titrimetrically. In all cases, the slope of the tangent to the initial part of the titration curve was taken as the rate of substrate hydrolysis. Results and Discussion
Specific activity and relative activity towards p-tosyl arginine methyl ester, casein and insoluble protein substrates A total of 0.01 #mol of enzymatically active trypsin was b o u n d per 1 ml of settled agarose based on the rate of TosArgOMe hydrolysis; this activity accounted for 20% of the total a m o u n t of b o u n d enzyme. In the subsequent discussion, reference to a given quantity of agarose-trypsin refers only to the a m o u n t of active trypsin as determined by the rate of TosArgOMe hydrolysis. When agarose-bound trypsin and soluble trypsin of comparable activity levels were allowed to act on TosArgOMe and bovine casein in parallel experiments, the ratio of the rate of TosArgOMe hydrolysis to casein hydrolysis (TosArgOMe/ casein) was found to be 13.9 for both the matrix-bound and soluble form of the enzyme. This clearly shows that the agarose-bound enzyme is equally available to the large protein substrate (casein approx. 25 000 mol.wt.) as it is to the small substrate (TosArgOMe approx. 379 mol.wt.). On the other hand, when soluble and agarose-bound trypsin were allowed to act on insoluble denatured proteins, a marked difference in activity was observed. Whereas no insoluble substrate was found to be resistant to soluble trypsin, the agarose-bound enzyme showed little or no activity towards insoluble substrates.
Specificity of free and agarose-bound trypsin Using several protein substrates (bovine casein, performic acid oxidized RNAase A and horse hemoglobin), the following survey w a s conducted. After exhaustive exposure of substrate to soluble trypsin (where proton production subsided as determined by pH-stat assay), the solution was heated to denature the trypsin, and agarose-bound trypsin was introduced at 25°C. The opposite experiment was carried o u t too; after exposure to agarose-bound trypsin, the b o u n d enzyme was removed, the solution heated and soluble trypsin was introduced at 25°C. A typical set of results, where casein was used as a substrate, is illustrated in Fig. 1. In no case was significant further hydrolysis of substrate observed after the second introduction of enzyme. This suggests that the same bonds must be cleaved by both enzyme preparations. In the case of oxidized ribonuclease A, the peptides produced by the action of agarose-trypsin were compared to those produced by soluble trypsin; the resulting peptide profiles upon fractionation on phosphocellulose were found to be identical for both forms of the enzyme. Amino acid analysis showed that agarose-trypsin cleaved
952 o,~D-
2.0 o 1.5-
x
I 0
1.0-
0.5-
20
40
60 mln 80
100
I20
140
Fig. 1. E x h a u s t i v e digestion o f b o v i n e casein by soluble trypsin (o) and agarose-trypsin (e). 10 m l of 0.2% casein s o l u t i o n w a s digested at 2 5 ° C w i t h 0 . 0 1 ~ m o l o f soluble or aga~ose-bound trypsin f o l l o w e d b y heating the s o l u t i o n to 7 0 ° C for 15 m i n , c o o l i n g to 2 5 ° C and i n t r o d u c i n g 0 . 0 1 / ~ m o i o f agarose-trypsin or soluble trypsin, respectively. C o n t r o l e x p e r i m e n t s c o n t a i n e d 0 . 0 1 ~ m o l o f either soluble trypsin or agarose-trypsin in t h e absence o f t h e substrate casein. D i g e s t i o n w a s m o n i t o r e d at pH 8.0 b y a R a d i o m e t e r pH-stat.
only at lysine and arginine residues as reported by Chin and Wold [ 7 ] where eight lysine- and four arginine-containing peptides were found in each case with the expected amino acid content [8]. A comparison of the rate and extent of activation of chymotrypsinogen and trypsinogen by the two forms of the enzyme also gave identical results. 1.0. >,
>_ 0.8. v_
E (3>, o',,
•~ 0 , 4 -
x
c. L 0.2-
r ...... BO
~Urea ] ( M )
, , 40 50 Temperature
'
r 60
7()
(°C)
Fig. 2 . ( A ) A c t i v i t y profile o f soluble trypsin (o) a n d ags.rose-trypsin (o) as a f u n c t i o n o f urea concentration. E n z y m a t i c activity w a s d e t e r m i n e d b y introducillg 0 , 0 0 6 - - 0 . 0 0 9 ~ m o l o f soluble or agarose-bound trypsin into I 0 m l o f 15 mM T o s A r g O M e s o l u t i o n , w i t h t h e appropriate urea c o n c e n t r a t i o n , and m o n i toring t h e initial h y d r o l y s i s rate at 2 5 ° C , p H 8 . 0 , w i t h a R a d i o m e t e r pH-stat s y s t e m . (B) A c t i v i t y profile o f soluble t r y p s i n (o) and agarosc-trypsin ( e ) as a f u n c t i o n o f t e m p e r a t u r e . A 0 . 0 0 6 - - 0 , 0 0 9 ~mol of soluble or agarose-bound trypsin w a s p r e i n c u b a t e d for 3 rain in 5.0 m l o f w a t e r at a given t e m p e r a t u r e and 5 . 0 m l o f substrate solution~ malntalnml at t h e same t e m p e r a t u r e was i n t r o d u c e d i n t o t h e p r e i n c u b a t c d trypsin s o l u t i o n to give a 1 0 m M T o s A r g O M e c o n c e n t r a t i o n . T h e rate o f substrate h y d r o l y s i s w a s determ i n e d as described in A. All activities are expressed relative to m a x i m a l activity at 62~C.
953
Effect of temperature and urea on free and agarose-bound trypsin The urea denaturation profiles for agarose-trypsin and soluble trypsin are illustrated in Fig. 2A. Both species show identical profiles where enzymatic activity decreases nearly linearly with increasing urea concentration and total inactivation is observed in 8 M urea. The temperature profiles for both forms of the enzyme in the presence of 10 mM CaC12 were also examined and the results are summarized in Fig. 2B. Both species show similar temperature profiles where specific activity increases with increasing temperature and o p t i m u m activity is observed at 62°C. At temperatures above 62°C inactivation due to heat denaturation predominates and loss of activity is observed; these results are in good agreement with the observations made by Sipos and Merkel [9] for trypsin activity in the presence of calcium as a function of temperature.
Stability of agarose-bound trypsin All the observations on the stability of agarose-trypsin u p o n storage under different conditions or under exposure to severe conditions for extended periods of time are constant with an increased stability over soluble trypsin. Agarose-trypsin maintained constant specific activity after repeated use over a 9-month period when stored at 5°C at neutral pH. In 4 M urea, agarose-trypsin maintained constant activity for 4 h at 25°C where activity was 50% of that observed in the absence of urea; upon removal of urea, full activity was recovered. In addition, the b o u n d enzyme maintained a constant specific activity at 50°C for a duration of 5 h, making it feasible to digest trypsin-resistant proteins at elevated temperatures. The high stability of agarose-trypsin is in agreement with earlier observations [1,3] and is most likely due to protection against autolysis which is prevented when individual trypsin molecules are kept apart on the agarose matrix. It is concluded that active trypsin attached to agarose beads behaves like soluble trypsin in terms of specificity, availability to soluble substrates and behavior to denaturing conditions for short periods of time when autolysis is minimized. The agarose-bound trypsin is distinctly different from soluble trypsin in its inability to interact with insoluble substrates and this property is undoubtedly reflected in the increased stability of the agarose-bound enzyme. Acknowledgement This work was supported in part by a U.S.P.H. research grant (GM 15053) ~ Dr. F. Wold, Department of Biochemistry, at the University of Minnesota in whose laboratory these experiments were conducted. References 1 2 3 4 5 6 7 8 9
K n i g h t , R . J . a n d L i g h t , A. ( 1 9 7 4 ) A r c h . B i o c h e m . B i o p h y s . 1 6 0 , 3 7 7 - - 3 8 6 A x ~ n , R . and Ernbach, S. ( 1 9 7 1 ) E u r . J. B i o c h e r n . 1 8 , 3 5 1 - - 3 6 0 G a b l e , D., V r e t b a l d , P., A x ~ n , It. and Porath, J. ( 1 9 7 0 ) B i o c h i m . B i o p h y s . A c t a 2 1 4 , 5 6 1 - - 5 6 3 A x ~ n , R., P o r a t h , J. and Ernbach, S. ( 1 9 6 7 ) N a t u r e 2 1 4 , 1 3 0 2 - - 1 3 0 4 M o o r e , S., S p a c k m a n , D . H . a n d S t e i n , W.H. ( 1 9 5 8 ) A n a l . C h e m . 3 0 , 1 1 8 5 - - 1 2 0 6 B e n s o n , J r . , J . V . and Patterson, J . A . ( 1 9 6 5 ) A n a l . C h e m . 3 7 , 1 1 0 8 - - 1 1 1 0 C h i n , C . Q . a n d W o l d , F. ( 1 9 7 2 ) A n a l . B i o c h e m . 4 6 , 5 8 5 - - 5 9 3 Hits, C . H . W . , M o o r e , S. a n d S t e i n , W.H. ( 1 9 5 6 ) J. Biol. C h e m . 2 1 9 , 6 2 3 - - 6 4 2 Sipos, T. a n d M e r k e l , J . A . ( 1 9 7 0 ) B i o c h e m i s t r y 9, 2 7 6 6 - - 2 7 7 5
Biochimica et Biophysica Acta, 429 (1976) 950--953 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 67780 C H A R A C T E R I Z A T I O N OF AGAROSE-BOUND TRYPSIN
BERT WALTER Department of Biochemistry, Brandeis University, Waltham, Mass. 02154 (U.S.A.)
(Received October 16th, 1975)
Summary Agarose-bound trypsin (EC 3.4.21.4) was prepared and its properties were compared with those of soluble trypsin. The b o u n d form of the enzyme was found to be equally available to large and small molecular weight substrates as the soluble form. In addition, the b o u n d form of the enzyme showed the same specificity towards protein substrates as the soluble enzyme. However, the agarose-bound trypsin showed greater stability than the soluble trypsin to denaturing conditions for prolonged period of time.
Introduction There have been conflicting reports on the comparative properties of free and matrix-bound enzymes [1--3] and this work was undertaken with the purpose of establishing some comparative data on one well characterized enzyme, namely trypsin (EC 3.4.21.4), in the soluble form and bound to agarose. The specific properties investigated were (1) relative activity towards large and small substrates; (2) specificity of cleavage in protein substrates, and (3) relative activity under denaturing conditions. Materials and Methods
Bovine pancreatic trypsin (Worthington) was covalently b o u n d to agarose A-15 M, 50--100 mesh (Bio-Rad) by the CNBr procedure of Ax~n et al. [4]; the binding was conducted in the presence of 10 mM CaC12 to reduce trypsin autolysis. The a m o u n t of trypsin covalently attached to agarose was determined by amino acid analysis [5,6] on Beckman Model 120C amino acid analyzer after hydrolysis of 1 ml of settled agarose-trypsin in 6 M HCI for 24 h at l l 0 ° C . Soluble and matrix-bound trypsin activity was assayed titrimetrically with a Radiometer pH-stat system using 0.01 M KOH as a titrant; the temperature was
951 maintained at 25°C with a Haake temperature control bath. A typical assay consisted of 10 ml of 15 mMp-tosylarginine methyl ester (TosArgOMe) (Sigma), pH 8.0, and 0.006--0.009 pmol of soluble trypsin or agarose-trypsin having a comparable a m o u n t of active trypsin. The total a m o u n t of active trypsin per given quantity of agarose was determined from a standard curve constructed using the rate of TosArgOMe hydrolysis by soluble trypsin as a function of enzyme concentration. In addition, proteolytic action of trypsin and agarosetrypsin on proteins was also monitored titrimetrically. In all cases, the slope of the tangent to the initial part of the titration curve was taken as the rate of substrate hydrolysis. Results and Discussion
Specific activity and relative activity towards p-tosyl arginine methyl ester, casein and insoluble protein substrates A total of 0.01 #mol of enzymatically active trypsin was b o u n d per 1 ml of settled agarose based on the rate of TosArgOMe hydrolysis; this activity accounted for 20% of the total a m o u n t of b o u n d enzyme. In the subsequent discussion, reference to a given quantity of agarose-trypsin refers only to the a m o u n t of active trypsin as determined by the rate of TosArgOMe hydrolysis. When agarose-bound trypsin and soluble trypsin of comparable activity levels were allowed to act on TosArgOMe and bovine casein in parallel experiments, the ratio of the rate of TosArgOMe hydrolysis to casein hydrolysis (TosArgOMe/ casein) was found to be 13.9 for both the matrix-bound and soluble form of the enzyme. This clearly shows that the agarose-bound enzyme is equally available to the large protein substrate (casein approx. 25 000 mol.wt.) as it is to the small substrate (TosArgOMe approx. 379 mol.wt.). On the other hand, when soluble and agarose-bound trypsin were allowed to act on insoluble denatured proteins, a marked difference in activity was observed. Whereas no insoluble substrate was found to be resistant to soluble trypsin, the agarose-bound enzyme showed little or no activity towards insoluble substrates.
Specificity of free and agarose-bound trypsin Using several protein substrates (bovine casein, performic acid oxidized RNAase A and horse hemoglobin), the following survey w a s conducted. After exhaustive exposure of substrate to soluble trypsin (where proton production subsided as determined by pH-stat assay), the solution was heated to denature the trypsin, and agarose-bound trypsin was introduced at 25°C. The opposite experiment was carried o u t too; after exposure to agarose-bound trypsin, the b o u n d enzyme was removed, the solution heated and soluble trypsin was introduced at 25°C. A typical set of results, where casein was used as a substrate, is illustrated in Fig. 1. In no case was significant further hydrolysis of substrate observed after the second introduction of enzyme. This suggests that the same bonds must be cleaved by both enzyme preparations. In the case of oxidized ribonuclease A, the peptides produced by the action of agarose-trypsin were compared to those produced by soluble trypsin; the resulting peptide profiles upon fractionation on phosphocellulose were found to be identical for both forms of the enzyme. Amino acid analysis showed that agarose-trypsin cleaved
952 o,~D-
2.0 o 1.5-
x
I 0
1.0-
0.5-
20
40
60 mln 80
100
I20
140
Fig. 1. E x h a u s t i v e digestion o f b o v i n e casein by soluble trypsin (o) and agarose-trypsin (e). 10 m l of 0.2% casein s o l u t i o n w a s digested at 2 5 ° C w i t h 0 . 0 1 ~ m o l o f soluble or aga~ose-bound trypsin f o l l o w e d b y heating the s o l u t i o n to 7 0 ° C for 15 m i n , c o o l i n g to 2 5 ° C and i n t r o d u c i n g 0 . 0 1 / ~ m o i o f agarose-trypsin or soluble trypsin, respectively. C o n t r o l e x p e r i m e n t s c o n t a i n e d 0 . 0 1 ~ m o l o f either soluble trypsin or agarose-trypsin in t h e absence o f t h e substrate casein. D i g e s t i o n w a s m o n i t o r e d at pH 8.0 b y a R a d i o m e t e r pH-stat.
only at lysine and arginine residues as reported by Chin and Wold [ 7 ] where eight lysine- and four arginine-containing peptides were found in each case with the expected amino acid content [8]. A comparison of the rate and extent of activation of chymotrypsinogen and trypsinogen by the two forms of the enzyme also gave identical results. 1.0. >,
>_ 0.8. v_
E (3>, o',,
•~ 0 , 4 -
x
c. L 0.2-
r ...... BO
~Urea ] ( M )
, , 40 50 Temperature
'
r 60
7()
(°C)
Fig. 2 . ( A ) A c t i v i t y profile o f soluble trypsin (o) a n d ags.rose-trypsin (o) as a f u n c t i o n o f urea concentration. E n z y m a t i c activity w a s d e t e r m i n e d b y introducillg 0 , 0 0 6 - - 0 . 0 0 9 ~ m o l o f soluble or agarose-bound trypsin into I 0 m l o f 15 mM T o s A r g O M e s o l u t i o n , w i t h t h e appropriate urea c o n c e n t r a t i o n , and m o n i toring t h e initial h y d r o l y s i s rate at 2 5 ° C , p H 8 . 0 , w i t h a R a d i o m e t e r pH-stat s y s t e m . (B) A c t i v i t y profile o f soluble t r y p s i n (o) and agarosc-trypsin ( e ) as a f u n c t i o n o f t e m p e r a t u r e . A 0 . 0 0 6 - - 0 , 0 0 9 ~mol of soluble or agarose-bound trypsin w a s p r e i n c u b a t e d for 3 rain in 5.0 m l o f w a t e r at a given t e m p e r a t u r e and 5 . 0 m l o f substrate solution~ malntalnml at t h e same t e m p e r a t u r e was i n t r o d u c e d i n t o t h e p r e i n c u b a t c d trypsin s o l u t i o n to give a 1 0 m M T o s A r g O M e c o n c e n t r a t i o n . T h e rate o f substrate h y d r o l y s i s w a s determ i n e d as described in A. All activities are expressed relative to m a x i m a l activity at 62~C.
953
Effect of temperature and urea on free and agarose-bound trypsin The urea denaturation profiles for agarose-trypsin and soluble trypsin are illustrated in Fig. 2A. Both species show identical profiles where enzymatic activity decreases nearly linearly with increasing urea concentration and total inactivation is observed in 8 M urea. The temperature profiles for both forms of the enzyme in the presence of 10 mM CaC12 were also examined and the results are summarized in Fig. 2B. Both species show similar temperature profiles where specific activity increases with increasing temperature and o p t i m u m activity is observed at 62°C. At temperatures above 62°C inactivation due to heat denaturation predominates and loss of activity is observed; these results are in good agreement with the observations made by Sipos and Merkel [9] for trypsin activity in the presence of calcium as a function of temperature.
Stability of agarose-bound trypsin All the observations on the stability of agarose-trypsin u p o n storage under different conditions or under exposure to severe conditions for extended periods of time are constant with an increased stability over soluble trypsin. Agarose-trypsin maintained constant specific activity after repeated use over a 9-month period when stored at 5°C at neutral pH. In 4 M urea, agarose-trypsin maintained constant activity for 4 h at 25°C where activity was 50% of that observed in the absence of urea; upon removal of urea, full activity was recovered. In addition, the b o u n d enzyme maintained a constant specific activity at 50°C for a duration of 5 h, making it feasible to digest trypsin-resistant proteins at elevated temperatures. The high stability of agarose-trypsin is in agreement with earlier observations [1,3] and is most likely due to protection against autolysis which is prevented when individual trypsin molecules are kept apart on the agarose matrix. It is concluded that active trypsin attached to agarose beads behaves like soluble trypsin in terms of specificity, availability to soluble substrates and behavior to denaturing conditions for short periods of time when autolysis is minimized. The agarose-bound trypsin is distinctly different from soluble trypsin in its inability to interact with insoluble substrates and this property is undoubtedly reflected in the increased stability of the agarose-bound enzyme. Acknowledgement This work was supported in part by a U.S.P.H. research grant (GM 15053) ~ Dr. F. Wold, Department of Biochemistry, at the University of Minnesota in whose laboratory these experiments were conducted. References 1 2 3 4 5 6 7 8 9
K n i g h t , R . J . a n d L i g h t , A. ( 1 9 7 4 ) A r c h . B i o c h e m . B i o p h y s . 1 6 0 , 3 7 7 - - 3 8 6 A x ~ n , R . and Ernbach, S. ( 1 9 7 1 ) E u r . J. B i o c h e r n . 1 8 , 3 5 1 - - 3 6 0 G a b l e , D., V r e t b a l d , P., A x ~ n , It. and Porath, J. ( 1 9 7 0 ) B i o c h i m . B i o p h y s . A c t a 2 1 4 , 5 6 1 - - 5 6 3 A x ~ n , R., P o r a t h , J. and Ernbach, S. ( 1 9 6 7 ) N a t u r e 2 1 4 , 1 3 0 2 - - 1 3 0 4 M o o r e , S., S p a c k m a n , D . H . a n d S t e i n , W.H. ( 1 9 5 8 ) A n a l . C h e m . 3 0 , 1 1 8 5 - - 1 2 0 6 B e n s o n , J r . , J . V . and Patterson, J . A . ( 1 9 6 5 ) A n a l . C h e m . 3 7 , 1 1 0 8 - - 1 1 1 0 C h i n , C . Q . a n d W o l d , F. ( 1 9 7 2 ) A n a l . B i o c h e m . 4 6 , 5 8 5 - - 5 9 3 Hits, C . H . W . , M o o r e , S. a n d S t e i n , W.H. ( 1 9 5 6 ) J. Biol. C h e m . 2 1 9 , 6 2 3 - - 6 4 2 Sipos, T. a n d M e r k e l , J . A . ( 1 9 7 0 ) B i o c h e m i s t r y 9, 2 7 6 6 - - 2 7 7 5
