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Further studies on the purification of the blood-clotting enzyme from the venom of Bothrops jararaca
IRCHIVES
OF
BIOCHEMISTRY
Further
ANI)
Studies
Enzyme
BIOPHYSICS
on the
from
98,
‘35-08
(1962)
Purification
the Venom
of the
Blood-Clotting
of Bothrops
jararaca
The blood-clotting enzyme from the venom of no2h/,o/~s Justin \vas purified fold by chromatography on diethylaminoethylcellulose. Some propert,ies of the enzyme have been dcterminrd and nrc reported. INTRODUCTIOS
Partial purificat,ion of the blood-clotting enzyme from the wnom of Bothrops jurarata has been achieved by acid treatment followed by fract’ionation with acetone and paper electrophoresis (1) and by fractional precipitation wit,11 ammonium sulfate followed by electrophoresis on a st’arch column (2). It should be noted that both tcchniques (1, 2) are time consuming. In this paper we report a method of purification of the blood-clotting enzyme from the venom of Bothrops jaw-aca by chromatography on (DEAE-celludicthylaminoethylccllulose low), which has the advantage of being simpler and giving a bett’er yield. Some addit,ional properties of the clotting cnsyme of the venom are also reported here. EXPERIMEKTAL
ninc-
Fibriirogcn was prepared by thr method of Sturtevant et al. (3), using as starting material fibrinogen I prepared as described by Laki (4) or by the technique of Blombiirk and Blomb%ck (5). The start,ing material for the fibrinogen I or the preparation by t,hc method of Blombiick and Blombiick (5) was commercial fibrinogen supplied by thr Xut ritionrd Biochcmicnls Corporation. The fibrinogc,n was dissolved in 0.3 $1 SaCl or KCI and kc,pt frozen until used. 7’1lrwtr/bin cmplo~etl WW “Thrombin Topico” from Parke, Davis & Co.. Detroit, Mivh. Stclrctltri,ti 2’hi~ov,I)irr from the National Innt itutf>s of Hcnlth, Lot So. B3, iaaucd Jurlc 1961, l~otc~nc~ of 21.7 unit*:/mg., was uacd :I$ sln~~d:~rtl for the ijotcncy test of the clotting cnzymr from the j~cnom of Botl1i’O/‘S ;fcl~~/Nlc.o. NETIIOIJS
Protein
Es tima tion
Protcins were estimated by the method of Lowry et c/l. (6), using bovine albumin from Bios Laboratories, Inc. as standard, or in terms of ultra\-iolet absorption at 280 m+
MATERIALS TTcnom. The venom of Bothrops jarurncn used in the experiments here reported was collected from young snakes (a few months to l-year old), and dried in VUCI~O over CaCl~ at room temperature. It was dissolved in saline (0.9% NaCl) or the indicated buffer, and the insoluble residnr was centrifuged off, DEAE-cellulose, Ccllex D, capacity 0.7 meq./g., W:~S purchased from Bio-Rnd Laboratories, Richmond, Calif.
Proteolytic activity on casein was assayed bj the method of kiunitz (7) as drscribcd by Henriqucs et al. (8). When benzoyl-L-arginine amide teas used as substrate (amidnse activity), the method of Schm-crt et crl. (9) was applied, as modified by Hrnriques el al. (8).
1 One of us (M.F.) is indebted to the Brazilian Research Council for a personal grant, We are also indebted to the Instituto Butantan Research Funds:.
Clotting activity was determined by the same method as previously dcscrihctl (2), at, least three
Proteolytic
Activity
different concentrations of protein bring used. In all cases these different concentrations were obtained by independent xld direct dilution of the material to be tested, since some experimental results included in this paper in the section Znhibition by Dilution under Elc.sults have shown that there is loss of blood-clotting activity when the purified preparation is submitkd to successive dilution. The determinations were always performed immediately after dilution. The specific clotting act,ivity was calculated as prrviously describrd by Henriques ct crl. (2). The potency of t,he purified clotting enzyme from the venom of Botkrops jararaca was compared to that of standard thrombin using the method described by the U. S. Department of Health, Edttcation, and Relfare (10). To verify the effect of ions or buffers on the clotting enzymes, Botkrops jaramx venom or thrombin were used as a 2% (w/v) solution in 0.9% NaCl. These enzymes were diluted 20-fold with the corresponding solution of ion or Imffcr, immediately before being used in the clotting test. Fibrinogen was used in a concentration of 0.2% foi the estimation of both enzymes.
Chromatography
and Rechromatography
This was made on DEAE-cellulose columns folloxing the procedure of Peterson and Sober (11). A 12% solution of the venom (5 g.), in 0.05 111 sodium cacodylate buffer, pH 6.5, was chromatographed on a 2.6 X 42 cm. DEAE-cellulose column equilibrated with the same buffer; stepwise elution was employed, reepectirely with 0.05, 0.1, 0.2, and 0.4 M sodium cacodylate buffers, pH 6.5. Fractions were collected in 24-ml. aliquots at the average rate of 124 ml./hr. All experiments were performed at room temperature. For rechromatography, the fractions with higher clotting specific act,ivity were mixed, diluted four times with distilled water, and passed on a 1 X 20 cm. DEAE-cellulose column, equilibrated with 0.05 ill sotlium cacodylate buffer, pH 6.5. The column was then warhcd with 0.05 M cacodylate buffer, pH 6.5, and the active material was eluted with 0.4 211 ,sodium cacodylate buffer, pH 6.5. RESULTS
SND
DISCUSSlOP;
PROPERTIESOF THE EMYME Innctivation,
by Dilution
It was observed that gradual dilution of the enzyme is accompanied by inactivation of its clotting potency. Figure 1 shows comparatively the clotting t,imes obtained with different concentrations of crude venom and
FIG. 1. Comparison between gradual and direct dilution as regards the clotting activity of Botkrops jararaca venom or purified enzyme obtained from the same venom. A and B: crude venom. C and D : purified enzyme. O--O, successive dilution ; a---e, direct dilution. The numbers indicate the specific clotting activity of the sample.
purified enzyme after direct and successive dilution, respectively. It can be noted that t,he inactivation of the purified preparation is more marked than that of the crude venom. The clot,ting specific activity of the crude venom was found to bc 900 when the dilutions were made directly from the concentrated solution, and 690 when each point corresponding t’o a lower concentration was obtained by diluting the solution pertinent to the immediately preceding point in the graph. The corresponding figures for the clotting specific activity of t,hc purified preparation were respectively 3600 and 1740. The use of siliconized or plastic (Lusheroid,l tubes did not prevent, this inactivation. Effect of Buffer
Cowentration.
Sodium racodylate buffer, pH 6.8, was used when the clotting activity of the venom was determined, in a final concentration which varied bet,ween 0.0125 and 0.2 M. Table I shows that the coagulation times were the same in all the buffer conccntrations used. Table I also shotvs that sodium phosphate buffer strongly inhibited t,he rlot-
BLOOD-CLOTTING
ting enzyme or t,he venom, and this inhibition increased with the rise of concent’ration of the buffer.
',-.IRLE
as rompwed Tlzro~~ bin
OF BOTHROPSJARARACA Reaction mist,llre for the drterminxtion of clotting activity: 0.1 ml. of rmom buffer solution and 0.2 ml. of fibrinogen sollltion.
to
Table II shows that sodium chloride and pot,assium chloride used in increasing concentrat’ions inhibit the fibrinogen clotting by the venom or by thrombin to the same degree in each case, t,he inhibition being, however, much stronger in the case of t’he venom. Sodium acetat’e and potassium acct’ate had a very slight effect, on both enzymes, showing that in the first case the inhibition was probably due to the chloride ion. Calcium acetate inhibited considerably the activity of thrombin and almost completely the venom enzyme activity, suggcsting that the inhibitory effect in this case is dependent on t,he calcium ion. Calcium chloride used in the same molar concentrations as the other salts caused t,he highest inhibition of fibrinogen clotting by the venom or by thrombin. With lower calcium chloride concentrations, tile inhibition of the venom enzyme was much stronger than t’he inhibition of thrombin. This finding suggcsts that such a high inhibition might be due t,o the ~111 of effects of calcium and chloride ions. ‘I’ABLR EFFECT
OF SEVERIIL ROTHROPS
10x3
OS THE
5.4rt~~~c.4,
1
EFFECT OF THE COSCE~XTRATIO~ OF Pi 6.8 Ponrr:a~ CACODE-LATE ASD SODIUM PHOSPHATE RVFFERX ON THE CLOTTING ACTIVITY OB THEVEXOM
Effect of Seve,ral Ions on the Clotting dctiait~~ of the T*enom of Bothrops ,jararacn
EXZYMIC
Clotting time Sodium Sodium phosphate cacodylate
Final molarity
~
(CC.
sec.
0.0125 0.025 0.05
10 10 10
0.10
10
0.20
10
11 12.5 15.5 30.5 > 15 min.
.lf
OF TIN EXZYME
PURIFICATIOK
Chromatoyraphic
Studies
As can be seen from Fig. 2, four peaks of protein were obtained in the chromatographic run. The enzymic activity appeared in one peak (see clott,ing activity in Fig. 2), and accounted for 34.5% of the total activity applied to the column (Table III 1. The specific blood-clotting ac&ity of the two fractions of t,he peak, which had litt’le caseinasc or benzoylarginine amidase activity, was 7.6 t,imes higher than t,hat of the crude venom. The amidase act,ivity could be due to contamination with Bothrops protcase A II
CLOTTING ACTIVITY OF TIIE .4s COMPARELI TO THROMBIX
Reaction mixture for the determination of the clotting activity: tjhromhin ion solution and 0.2 ml. of 0.2C,;,fibrinogen solution.
VENOM
OF
0.1 ml. of venom ion solrltion or
Clotting time, ~rlin. Final molarity -___ .v
0.020 0.025 0.05 0.1 0.2
-__~Sodium chloride
Potassiumchloride 1 Calcium chloride / Sodium acetate
Potassiumacetate
Calcium acetate
I
V” -ILI
0.26 i 0.28 0.1T 0.5 0.48 >60 0.71 :>60 -l I (1Venom of Bothrops h Thrombin.
0.10 0.12 0.13 0.16 0.26 jararaca.
-
0.15 0.14 0.18 0.17
0.25 1 0.21 , >60
0.2 0.23 0.67 2.5
98
5 1
CACODYLATE ph 65
04M
0.05M
FIG. 2. Chromatography of venom of Bothrops O-0, blood-clotting specific activity; l -0, TABLE Br,oou
(h.OTTING,
CASEIN~SE
III AND
BJG:NZOYLARG-
N~NEAMIL)ASESPECIF~CACTIVITIESOFSAMPLE~~ OBTAINED ON THE COL-RSE OF PURIFI~ATION OF THE CLOTTING ENZYME OF THE VENOM OF HOTHROPSJARARACA I
Specilic
activities
I
Yield
step
Crude venom DEAE-cellulose chromatogr:tph> I)EAI’:-cellulose lechrom:ttograph~
PK. tein
Clotting actiw ity
7
!;
100 100 1.6 31.5 2.9
26.5
(81, or to the blood-clotting enzyme itself. This can only be settled by further work,
jararaca
optical
snake on DEAE-cellulose density at 280 rnb.
column
but, a decision in this aspect should have some bearing on t,he mechanism of blood clotting induced by snake venom. If the last hypot,hesis proved to be true, this activity should be similar to that of thrombin, which is known to have benzoylarginine amidase activity (12). In the rechromatography of the clot’ting fractions, t,he peak of the clot’ting activity was eluted with 0.4 M sodium cacodylate buffer, pH 6.5, bctwcen the 17th and 22nd ml. of effluent. The preparation obtained was nine times more active than the crude venom in its blood-clott’ing activity, its potency corresponded to approximately 153 X.I.H. thrombin units/mg. lirotcin (lo), had no caseinase activity, its amidase activity was about t,hree times greater than that of the init’ial venom (Table III), and it was slightlv contaminntcd with n-amino acid oxidase. The blood-clotting activitv of this
BLOOD-CT,OTTISG
preparation is preserved if it is stored freeze-dried in z’c~uo at 10”. REFEREKCES E., Ad. m7)tl. Prcthol. I’hwmnkol. 234, 291 (1958).
1. HABI%R~~.~~,
2. HESI~IQLXS, 0. B., FICHM.~~. M., ASD IIFSRIQCER,S. B., Niochm. J. 75, 551 (1960). 3. f+l’URTEVATT, J. &I., L.4SKo\VSIiI, ?vl. JR., 110xSELLY, T. H., ASD SCHER.4G.4, H. Ai., J. Am Chcm. Sm. 77, 6168 (1955). 4. LAHI, Ii., AK/~. Biochcna. Hiophgs. 32, 317 (1951). 5. BLOMB~K, B., AKD BI.o~RcK, M., Arkiv Semi 10,415 (1956). 6. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.,
EX%TRIE:
00
OF
BIOCHEMISTRY
Further
ANI)
Studies
Enzyme
BIOPHYSICS
on the
from
98,
‘35-08
(1962)
Purification
the Venom
of the
Blood-Clotting
of Bothrops
jararaca
The blood-clotting enzyme from the venom of no2h/,o/~s Justin \vas purified fold by chromatography on diethylaminoethylcellulose. Some propert,ies of the enzyme have been dcterminrd and nrc reported. INTRODUCTIOS
Partial purificat,ion of the blood-clotting enzyme from the wnom of Bothrops jurarata has been achieved by acid treatment followed by fract’ionation with acetone and paper electrophoresis (1) and by fractional precipitation wit,11 ammonium sulfate followed by electrophoresis on a st’arch column (2). It should be noted that both tcchniques (1, 2) are time consuming. In this paper we report a method of purification of the blood-clotting enzyme from the venom of Bothrops jaw-aca by chromatography on (DEAE-celludicthylaminoethylccllulose low), which has the advantage of being simpler and giving a bett’er yield. Some addit,ional properties of the clotting cnsyme of the venom are also reported here. EXPERIMEKTAL
ninc-
Fibriirogcn was prepared by thr method of Sturtevant et al. (3), using as starting material fibrinogen I prepared as described by Laki (4) or by the technique of Blombiirk and Blomb%ck (5). The start,ing material for the fibrinogen I or the preparation by t,hc method of Blombiick and Blombiick (5) was commercial fibrinogen supplied by thr Xut ritionrd Biochcmicnls Corporation. The fibrinogc,n was dissolved in 0.3 $1 SaCl or KCI and kc,pt frozen until used. 7’1lrwtr/bin cmplo~etl WW “Thrombin Topico” from Parke, Davis & Co.. Detroit, Mivh. Stclrctltri,ti 2’hi~ov,I)irr from the National Innt itutf>s of Hcnlth, Lot So. B3, iaaucd Jurlc 1961, l~otc~nc~ of 21.7 unit*:/mg., was uacd :I$ sln~~d:~rtl for the ijotcncy test of the clotting cnzymr from the j~cnom of Botl1i’O/‘S ;fcl~~/Nlc.o. NETIIOIJS
Protein
Es tima tion
Protcins were estimated by the method of Lowry et c/l. (6), using bovine albumin from Bios Laboratories, Inc. as standard, or in terms of ultra\-iolet absorption at 280 m+
MATERIALS TTcnom. The venom of Bothrops jarurncn used in the experiments here reported was collected from young snakes (a few months to l-year old), and dried in VUCI~O over CaCl~ at room temperature. It was dissolved in saline (0.9% NaCl) or the indicated buffer, and the insoluble residnr was centrifuged off, DEAE-cellulose, Ccllex D, capacity 0.7 meq./g., W:~S purchased from Bio-Rnd Laboratories, Richmond, Calif.
Proteolytic activity on casein was assayed bj the method of kiunitz (7) as drscribcd by Henriqucs et al. (8). When benzoyl-L-arginine amide teas used as substrate (amidnse activity), the method of Schm-crt et crl. (9) was applied, as modified by Hrnriques el al. (8).
1 One of us (M.F.) is indebted to the Brazilian Research Council for a personal grant, We are also indebted to the Instituto Butantan Research Funds:.
Clotting activity was determined by the same method as previously dcscrihctl (2), at, least three
Proteolytic
Activity
different concentrations of protein bring used. In all cases these different concentrations were obtained by independent xld direct dilution of the material to be tested, since some experimental results included in this paper in the section Znhibition by Dilution under Elc.sults have shown that there is loss of blood-clotting activity when the purified preparation is submitkd to successive dilution. The determinations were always performed immediately after dilution. The specific clotting act,ivity was calculated as prrviously describrd by Henriques ct crl. (2). The potency of t,he purified clotting enzyme from the venom of Botkrops jararaca was compared to that of standard thrombin using the method described by the U. S. Department of Health, Edttcation, and Relfare (10). To verify the effect of ions or buffers on the clotting enzymes, Botkrops jaramx venom or thrombin were used as a 2% (w/v) solution in 0.9% NaCl. These enzymes were diluted 20-fold with the corresponding solution of ion or Imffcr, immediately before being used in the clotting test. Fibrinogen was used in a concentration of 0.2% foi the estimation of both enzymes.
Chromatography
and Rechromatography
This was made on DEAE-cellulose columns folloxing the procedure of Peterson and Sober (11). A 12% solution of the venom (5 g.), in 0.05 111 sodium cacodylate buffer, pH 6.5, was chromatographed on a 2.6 X 42 cm. DEAE-cellulose column equilibrated with the same buffer; stepwise elution was employed, reepectirely with 0.05, 0.1, 0.2, and 0.4 M sodium cacodylate buffers, pH 6.5. Fractions were collected in 24-ml. aliquots at the average rate of 124 ml./hr. All experiments were performed at room temperature. For rechromatography, the fractions with higher clotting specific act,ivity were mixed, diluted four times with distilled water, and passed on a 1 X 20 cm. DEAE-cellulose column, equilibrated with 0.05 ill sotlium cacodylate buffer, pH 6.5. The column was then warhcd with 0.05 M cacodylate buffer, pH 6.5, and the active material was eluted with 0.4 211 ,sodium cacodylate buffer, pH 6.5. RESULTS
SND
DISCUSSlOP;
PROPERTIESOF THE EMYME Innctivation,
by Dilution
It was observed that gradual dilution of the enzyme is accompanied by inactivation of its clotting potency. Figure 1 shows comparatively the clotting t,imes obtained with different concentrations of crude venom and
FIG. 1. Comparison between gradual and direct dilution as regards the clotting activity of Botkrops jararaca venom or purified enzyme obtained from the same venom. A and B: crude venom. C and D : purified enzyme. O--O, successive dilution ; a---e, direct dilution. The numbers indicate the specific clotting activity of the sample.
purified enzyme after direct and successive dilution, respectively. It can be noted that t,he inactivation of the purified preparation is more marked than that of the crude venom. The clot,ting specific activity of the crude venom was found to bc 900 when the dilutions were made directly from the concentrated solution, and 690 when each point corresponding t’o a lower concentration was obtained by diluting the solution pertinent to the immediately preceding point in the graph. The corresponding figures for the clotting specific activity of t,hc purified preparation were respectively 3600 and 1740. The use of siliconized or plastic (Lusheroid,l tubes did not prevent, this inactivation. Effect of Buffer
Cowentration.
Sodium racodylate buffer, pH 6.8, was used when the clotting activity of the venom was determined, in a final concentration which varied bet,ween 0.0125 and 0.2 M. Table I shows that the coagulation times were the same in all the buffer conccntrations used. Table I also shotvs that sodium phosphate buffer strongly inhibited t,he rlot-
BLOOD-CLOTTING
ting enzyme or t,he venom, and this inhibition increased with the rise of concent’ration of the buffer.
',-.IRLE
as rompwed Tlzro~~ bin
OF BOTHROPSJARARACA Reaction mist,llre for the drterminxtion of clotting activity: 0.1 ml. of rmom buffer solution and 0.2 ml. of fibrinogen sollltion.
to
Table II shows that sodium chloride and pot,assium chloride used in increasing concentrat’ions inhibit the fibrinogen clotting by the venom or by thrombin to the same degree in each case, t,he inhibition being, however, much stronger in the case of t’he venom. Sodium acetat’e and potassium acct’ate had a very slight effect, on both enzymes, showing that in the first case the inhibition was probably due to the chloride ion. Calcium acetate inhibited considerably the activity of thrombin and almost completely the venom enzyme activity, suggcsting that the inhibitory effect in this case is dependent on t,he calcium ion. Calcium chloride used in the same molar concentrations as the other salts caused t,he highest inhibition of fibrinogen clotting by the venom or by thrombin. With lower calcium chloride concentrations, tile inhibition of the venom enzyme was much stronger than t’he inhibition of thrombin. This finding suggcsts that such a high inhibition might be due t,o the ~111 of effects of calcium and chloride ions. ‘I’ABLR EFFECT
OF SEVERIIL ROTHROPS
10x3
OS THE
5.4rt~~~c.4,
1
EFFECT OF THE COSCE~XTRATIO~ OF Pi 6.8 Ponrr:a~ CACODE-LATE ASD SODIUM PHOSPHATE RVFFERX ON THE CLOTTING ACTIVITY OB THEVEXOM
Effect of Seve,ral Ions on the Clotting dctiait~~ of the T*enom of Bothrops ,jararacn
EXZYMIC
Clotting time Sodium Sodium phosphate cacodylate
Final molarity
~
(CC.
sec.
0.0125 0.025 0.05
10 10 10
0.10
10
0.20
10
11 12.5 15.5 30.5 > 15 min.
.lf
OF TIN EXZYME
PURIFICATIOK
Chromatoyraphic
Studies
As can be seen from Fig. 2, four peaks of protein were obtained in the chromatographic run. The enzymic activity appeared in one peak (see clott,ing activity in Fig. 2), and accounted for 34.5% of the total activity applied to the column (Table III 1. The specific blood-clotting ac&ity of the two fractions of t,he peak, which had litt’le caseinasc or benzoylarginine amidase activity, was 7.6 t,imes higher than t,hat of the crude venom. The amidase act,ivity could be due to contamination with Bothrops protcase A II
CLOTTING ACTIVITY OF TIIE .4s COMPARELI TO THROMBIX
Reaction mixture for the determination of the clotting activity: tjhromhin ion solution and 0.2 ml. of 0.2C,;,fibrinogen solution.
VENOM
OF
0.1 ml. of venom ion solrltion or
Clotting time, ~rlin. Final molarity -___ .v
0.020 0.025 0.05 0.1 0.2
-__~Sodium chloride
Potassiumchloride 1 Calcium chloride / Sodium acetate
Potassiumacetate
Calcium acetate
I
V” -ILI
0.26 i 0.28 0.1T 0.5 0.48 >60 0.71 :>60 -l I (1Venom of Bothrops h Thrombin.
0.10 0.12 0.13 0.16 0.26 jararaca.
-
0.15 0.14 0.18 0.17
0.25 1 0.21 , >60
0.2 0.23 0.67 2.5
98
5 1
CACODYLATE ph 65
04M
0.05M
FIG. 2. Chromatography of venom of Bothrops O-0, blood-clotting specific activity; l -0, TABLE Br,oou
(h.OTTING,
CASEIN~SE
III AND
BJG:NZOYLARG-
N~NEAMIL)ASESPECIF~CACTIVITIESOFSAMPLE~~ OBTAINED ON THE COL-RSE OF PURIFI~ATION OF THE CLOTTING ENZYME OF THE VENOM OF HOTHROPSJARARACA I
Specilic
activities
I
Yield
step
Crude venom DEAE-cellulose chromatogr:tph> I)EAI’:-cellulose lechrom:ttograph~
PK. tein
Clotting actiw ity
7
!;
100 100 1.6 31.5 2.9
26.5
(81, or to the blood-clotting enzyme itself. This can only be settled by further work,
jararaca
optical
snake on DEAE-cellulose density at 280 rnb.
column
but, a decision in this aspect should have some bearing on t,he mechanism of blood clotting induced by snake venom. If the last hypot,hesis proved to be true, this activity should be similar to that of thrombin, which is known to have benzoylarginine amidase activity (12). In the rechromatography of the clot’ting fractions, t,he peak of the clot’ting activity was eluted with 0.4 M sodium cacodylate buffer, pH 6.5, bctwcen the 17th and 22nd ml. of effluent. The preparation obtained was nine times more active than the crude venom in its blood-clott’ing activity, its potency corresponded to approximately 153 X.I.H. thrombin units/mg. lirotcin (lo), had no caseinase activity, its amidase activity was about t,hree times greater than that of the init’ial venom (Table III), and it was slightlv contaminntcd with n-amino acid oxidase. The blood-clotting activitv of this
BLOOD-CT,OTTISG
preparation is preserved if it is stored freeze-dried in z’c~uo at 10”. REFEREKCES E., Ad. m7)tl. Prcthol. I’hwmnkol. 234, 291 (1958).
1. HABI%R~~.~~,
2. HESI~IQLXS, 0. B., FICHM.~~. M., ASD IIFSRIQCER,S. B., Niochm. J. 75, 551 (1960). 3. f+l’URTEVATT, J. &I., L.4SKo\VSIiI, ?vl. JR., 110xSELLY, T. H., ASD SCHER.4G.4, H. Ai., J. Am Chcm. Sm. 77, 6168 (1955). 4. LAHI, Ii., AK/~. Biochcna. Hiophgs. 32, 317 (1951). 5. BLOMB~K, B., AKD BI.o~RcK, M., Arkiv Semi 10,415 (1956). 6. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.,
EX%TRIE:
00