- Email: [email protected]
[23] Chicken pepsinogens
358
THE ACIDIC PROTEASES
[23]
Inactivation appears to be due to the intermolecular linking of sulfhydryl groups, since no activity is lost on reacting the SH groups with N-ethylmaleimide, p-mercurybenzoate, and 5,5'-dithiobis-(2-nitrobenzoic acid). p-Bromophenacylbromide and a-diazo, p-bromoacetophenone, reported to react with the carboxyl groups at the active site of swine pepsin, 12 also inactivate chicken pepsin at pH 6. Reaction of the free amino groups of chicken pepsinogen with trinitrobenzenesulfonate or with benzyl acetamidate, but not with methyl acetimidate, leads to complete loss of potential peptic activity. =B. F. Erlanger, S. M. Vratsanos, N. Wasserman, and A. G. Cooper, Biochem. Biophys. Res. Commun. 28, 203 (1967).
[23] C h i c k e n P e p s i n o g e n s B y SAM T. DONTA and HELEN VAN VUNAKIS
The purification of the chicken pepsinogens 1 is accomplished by ionexchange and molecular sieve chromatographic procedures similar to those used for the swine2 and dogfish3 systems. Most of the purification of the combined pepsinogens takes place on the first DE-11 cellulose column, with subsequent columns mainly used to achieve a separation of the zymogens from each other. The pepsins were generated from the purified precursors and no attempt was made to isolate the various enzymes from acidified extracts of the mucosae or from gastric juice. The preparation of chicken pepsinogens and pepsin has been reported in other laboratories. *-e Assay procedures for proteolytic activities utilize hemoglobin,7 milk,8 and synthetic peptides as substrates. Modifications of the original assay methods have been described2 Purification Procedures T h e proventriculus ("stomach equivalent" of the chick) is the source of the chicken pepsinogens and pepsins and is that area of the 1S. T. Donta and H. Van Vunakis, Biochemistry 9, 2791 (1970). 2A. P, Ryle, this volume [20]. s E. Bar-Eli and T. G. Merrett, this volume [24]. "R. M. Herriott, Q. R. Bartz, and J. H. Northrop, J. Gen. Physiol. 21, 575 (1938). T. P. Levchuk and V. N. Orekhovieh, Biokhimiya 28, 738 (1963). eZ. Bohak, J. Biol. Chem. 9,44, 4638 (1969); this volume [22]. M. L. Anson, J. Gen. Physiol. 22, 79 (1938). s R. M. Herriott, J. Gen. Physiol. 21, 501 (1938).
[23]
CHICKEN PEPSINOGENS
359
gastrointestinal tract lust proximal to the gizzard. The whole stomach is used as the starting material, as the mucosal layer containing the enzymes is too delicate and thin to be dissected away easily. Approximately 40-50 stomachs should be used as a minimum to obtain all three pepsinogens with relative ease; a lesser number decreases the chances of obtaining adequate amounts of pepsinogen C. Phosphate buffers at pH 6.9 are used unless otherwise stated and all operations are carried out in the cold. The stomachs from freshly killed chickens are immediately placed in 0.02M phosphate buffer, the fat removed from the outer layer of stomach, the organ opened, and the internal contents washed out with buffer. The stomachs, minced by one coarse and one fine grind with a meat grinder are then placed in 1 liter of buffer and stirred for 1 hour. The tissue is removed by centrifugation at 10,000 rpm for 1/2 hour. The supernatant is then applied to an ion-exchange column (35 X 6 cm) containing 750 ml of packed DE-11 cellulose equilibrated with 0.02M phosphate buffer, and the resin is washed with sufficient buffer (2-3 liters) to elute the inactive protein. This wash can be performed quickly and is continued until readings at OD 280 nm indicate that the peak has come off. The effluent should contain no potential proteolytic activity. The column is eluted with a linear gradient of 2 liters 0.02 M phosphate buffer connected to 2 liters of 0.02M phosphate, 0.8M NaC1, pH 6.9. The pepsinogens, located by suitable assay procedures, emerge in a pattern similar to the dogfish proteins a with pepsinogen C sometimes appearing as a slightly later shoulder (Fig. 1A). Pepsinogen B, the fraction active against N-Cbz-L-Glu-L-Tyr, is pooled and purified further by DE-11 and DE-52 cellulose chromatography using a gradient of 0.02 M phosphate, to 0.02 M phosphate containing 0.18 M NaC1, pH 6.9, followed by Sephadex chromatography. No attempt to separate the pepsinogens with potential enzyme activity toward hemoglobin is made here and they are pooled together. After dialysis against buffer (0.02M phosphate), this pooled fraction is reapplied to a smaller DE-11 column (30 X 3 cm), washed with 500 ml of buffer, and eluted with a shallower salt gradient of 2 liters 0.02 M phosphate, to 2 liters 0.02 M phosphate, 0.55 M NaC1, pH 6.9. A separation of two fractions with potential proteolytic activity may be observed, the smaller and later emerging peak being pepsinogen C. If pepsinogen C is not readily separated from the pepsinogen A-D peak on this column, the pepsinogen peaks are pooled, dialyzed against phosphate buffer, then adsorbed onto a DE-52 cellulose column and eluted with a similar salt gradient (Fig. 1B). Even though a separation of zymogens is not always achieved on the second DE-11 cellulose column, this chromatographic step greatly improves the flow properties of the solutions through the
360
THE ACIDIC PROTEASES I
80
[23]
t
60
o
r-, 4 0 o
2.0
E c o I0
20
50
I00
150
Fraction
3o.-.
lit
~~
B
250
number
'.4t
--~
20. ~,.~. ,o
200
::
:"/ ": ~
'-~r
c-'
/~\
......... 0
20
40
60
I~~
~"°r N~ \ ~o.~[ p, \ °~[1 \\ o.~r)/ k
,
80
o~ <~
I00
40
60
Fraction
number
80
100
120 1,~ 1'~o 1°B ,.io
120
140
160
Fro. 1 (A). Chromatography of original crude stomach extract on DE-11 ionexchange cellulose. The extract from 72 chicken stomachs was applied to a column (35 X 6 cm) and eluted with a gradient of 2.0 liters 0.02 M phosphate buffer, pH 6.9, to 2.0 liters 0.02 M phosphate -~- 0.9 M NaCl, pH 0.9, with 20-ml fractions being collected. Arrows indicate fractions pooled. (B). Separation of pepsinogen C from main pepsinogen fraction with DE~2 column (8 X 3 cm) and gradient of 2.0 liters, 0.02 M phosphate, pH 6.9, to 2.0 liters, 0.02M phosphate, 0.6M NaCI, pH 6.9; 15-ml fractions were collected. (C). Separation of pepsinogens D and A; DE-52 column (8 X 3 cm) eluted with gradient of 1.O liter, 0.02 M phosphate, pit 6.9 to 1.O
[23]
CHICKEN PEPSINOGENS
361
DE-52 cellulose columns used in subsequent steps of the purification procedure (Figs. 1B and 1C). On DF_,-52 cellulose columns, the major fraction consisting of pepsinogens D and A should separate easily at salt concentrations of 0.15 and 0.20 M NaC1, respectively, while pepsinogen C is eluted at 0.35 M NaC1. Acrylamide electrophoresis should be used to follow the purification, especially the final steps, as sometimes pepsinogen A can be found in the early eluted portion of an apparently homogeneous (protein concentration paralleling potential proteolytic activity) pepsinogen C preparation. • The purified DE-52 pepsinogens are concentrated by ultrafiltration (Amicon Corp. Diaflo--Membrane with 10,000 MW cutoff) and filtered through a Sephadex G-100 column (100 X 1 cm) equilibrated with 0.02 M phosphate, 0.15 M NaC1, pH 6.9 as the final purification step for each pepsinogen. Only with pepsinogen D was any further slight purification noticed. As an approximation, there are 30 mg of total pepsinogen in each stomach, in roughly a ratio of 10:15:5 between D, A, and C. In the chromatographic separations, only the midareas of protein peaks were pooled to obtain homogeneous preparations of the individual pepsinogens; thus the final yields of the purified precursors were between 10-20%. During acrylamide electrophoresis at pH 8.5, the zymogens migrate toward the anode. Pepsinogen C moves well in front of pepsinogens A and D and right behind or coincident with the bromphenol blue dye marker (Fig. 2). Pepsinogen D has a slightly slower mobility than pepsinogen A; this difference is detectable when the zymogens are electrophoresed in a single tube. The pepsins are derived from their respective purified pepsinogens by first incubating the pepsinogens at 37 ° and pH 2.0 for 4 minutes (conditions of maximal activation), then dialyzing at 0 ° against 0.1 N acetate, pH 4.0 for 8 hours followed by dialysis against 0.02 M phosphate, 0.15 M NaC1, pH 6.9, for 8 hours. The activation mixtures are then chromatographed on Sephadex under conditions used for the pepsinogens. Properties The amino acid compositions of chicken pepsinogens A, D, and C and the pepsins derived from these purified precursors are given in the table and are based on the molecular weights obtained from high-speed sedimentation to equilibrium determinations. Amide, tryptophan, phosphate, and polysaccharide analyses have not yet been done. liter, 0.02 M P0, + 0.45 M NaCl, pH 6.9; 10 ml fractions were collected. (O) Optical density at 280 nm; (A) activity against hemoglobin at pYcI2 expressed as increase of optical density at 280 nm of digested trichloroacetic acid-soluble material; (l-q) optical density at 570 nm used to determine hydrolysis of synthetic substrate N-Cbz-L-GIu-L-Tyr by ninhydrin assay.
362
THE ACIDIC PROTEASES
II
[23]
I
--!
FIe. 2. Schematic representation of polyacrylamide gel electrophoresis of the pepsinogens in Tris-HCl buffer system, pH 8~5. Arrow indicates direction of electrophoresis; bottom line in each case represents dye front.
I D
A
C
Pepsinogens
AMINO ACID COMPOSITIONS OF CHICKEN PEPSINOGENS AND PEPSINS
Pepsinogens
Lys His Arg Asp Thr Set Glu Pro Gly Ala Ha~-Cys Val Met Ile Leu Tyr Phe MW
Pepsins
A
D
C
A
D
C
17 7 7 44 28 39 32 19 33 21 (6) 27 10 24 30 24 25 42,000
15 7 7 44 28 40 29 20 34 20 (6) 28 10 24 29 27 22 42,000
5 3 3 41 33 40 48 22 40 22 (6) 22 9 24 30 24 27 42,000
11 5 6 44 31 44 31 19 35 20 (6) 27 10 25 29 24 23 42,000
10 5 6 44 32 46 29 19 34 19 (6) 28 10 26 29 24 22 42,000
3 1 2 35 32 39 43 17 39 18 (6) 21 10 23 28 20 24 38,500
F r o m this d a t a a n d t h a t o b t a i n e d from i m m u n o c h e m i c a l a n a l y s i s , 9 p e p s i n o g e n s A a n d D a p p e a r to be closely r e l a t e d to each o t h e r a n d different from p e p s i n o g e n C. P e p s i n s A a n d D closely resemble t h e i r precursors, b e i n g s m a l l e r b y some 15 a m i n o acid residues. U s i n g i m m u n o 9S. T. Donta and H. Van Vunakis, Biochemistry 9, 2798 (1970).
[23]
C~ICKEN PEPSINOGENS
363
chemical techniques which can detect small conformational differences in macromolecules,1° pepsins A and D were shown to be very similar to their precursors and to each other. Pepsin C (but not its precursor, pepsinogen C) also reacted with antipepsinogen A. The pepsinogens were homogeneous by a number of criteria, i.e., superposition of activity and optical density curves in the elution patterns obtained from DEAE-cellulose and Sephadex chromatography, ultracentrifugal analysis, the formation of single bands with the homologous antibodies on immunodiffusion and immunoelectrophoresis and migration of each protein as a single band on disc-gel electrophoresis. The pepsins generated from these purified precursors also meet these criteria for homogeneity except that several specific bands are observed for each pepsin on acrylamide electrophoresis and two bands are observed for pepsin C on immunoelectrophoresis. Pepsinogen B has been obtained in small yields only and is still heterogeneous. Stabilities. Chicken pepsins are much more stable than swine pepsin,l, 4-6,9 they maintain their activities throughout the purification procedure which is carried out at neutral pH and are stored in phosphate buffer, pH 6.9, either refrigerated or frozen. Pepsinogen preparations are stable if stored in 0.03 M Tris buffer, pH 7.5, either refrigerated or frozen. Preparations frozen in phosphate buffer at pH 6.9 were converted into pepsin'. It is strongly suggested that the pepsinogen samples be assayed routinely by the milk clot assay to assure that conversion has not taken place. Neither the pepsinogens nor the pepsins should be lyophilized since they tend to lose activity in this process. Specificities. All the enzymes are active at acid pH. The comparative proteolytic activities of swine pepsin and chick pepsins D, A, and C toward hemoglobin are 1.0, 3.4, 2.3, and 1.7, respectively, and 1.0, 0.29, 0.33, and 0.18 in the milk clot assay. The greater reactivity of swine pepsin compared to chick pepsin in the milk clot assay has already been noted? Even at concentrations of 1 mg/ml and incubation times of 1 hour, chick pepsins D, A, and C did not hydrolyze N-Cbz-L-Glu-L-Tyr, either at pH 2 or pH 4, the latter being the pH optimum of the chick peptidase (pepsinogen B). Conversely, on exposure to acid pH, the protein corresponding to pepsinogen B showed no enzymatic activity in the hemoglobin or milk clot assays, even at concentrations (100-fold) and incubation times (10-fold) greater than those used for the other pepsins. Under standard assay conditions, each milligram of peptidase B contains 0.5 units of enzyme activity using N-Cbz-L-Glu-L-Tyr as substrate. 1oL. Levine and H. Van Vunakis, Vol. XI, p. 928.
THE ACIDIC PROTEASES
[23]
Inactivation appears to be due to the intermolecular linking of sulfhydryl groups, since no activity is lost on reacting the SH groups with N-ethylmaleimide, p-mercurybenzoate, and 5,5'-dithiobis-(2-nitrobenzoic acid). p-Bromophenacylbromide and a-diazo, p-bromoacetophenone, reported to react with the carboxyl groups at the active site of swine pepsin, 12 also inactivate chicken pepsin at pH 6. Reaction of the free amino groups of chicken pepsinogen with trinitrobenzenesulfonate or with benzyl acetamidate, but not with methyl acetimidate, leads to complete loss of potential peptic activity. =B. F. Erlanger, S. M. Vratsanos, N. Wasserman, and A. G. Cooper, Biochem. Biophys. Res. Commun. 28, 203 (1967).
[23] C h i c k e n P e p s i n o g e n s B y SAM T. DONTA and HELEN VAN VUNAKIS
The purification of the chicken pepsinogens 1 is accomplished by ionexchange and molecular sieve chromatographic procedures similar to those used for the swine2 and dogfish3 systems. Most of the purification of the combined pepsinogens takes place on the first DE-11 cellulose column, with subsequent columns mainly used to achieve a separation of the zymogens from each other. The pepsins were generated from the purified precursors and no attempt was made to isolate the various enzymes from acidified extracts of the mucosae or from gastric juice. The preparation of chicken pepsinogens and pepsin has been reported in other laboratories. *-e Assay procedures for proteolytic activities utilize hemoglobin,7 milk,8 and synthetic peptides as substrates. Modifications of the original assay methods have been described2 Purification Procedures T h e proventriculus ("stomach equivalent" of the chick) is the source of the chicken pepsinogens and pepsins and is that area of the 1S. T. Donta and H. Van Vunakis, Biochemistry 9, 2791 (1970). 2A. P, Ryle, this volume [20]. s E. Bar-Eli and T. G. Merrett, this volume [24]. "R. M. Herriott, Q. R. Bartz, and J. H. Northrop, J. Gen. Physiol. 21, 575 (1938). T. P. Levchuk and V. N. Orekhovieh, Biokhimiya 28, 738 (1963). eZ. Bohak, J. Biol. Chem. 9,44, 4638 (1969); this volume [22]. M. L. Anson, J. Gen. Physiol. 22, 79 (1938). s R. M. Herriott, J. Gen. Physiol. 21, 501 (1938).
[23]
CHICKEN PEPSINOGENS
359
gastrointestinal tract lust proximal to the gizzard. The whole stomach is used as the starting material, as the mucosal layer containing the enzymes is too delicate and thin to be dissected away easily. Approximately 40-50 stomachs should be used as a minimum to obtain all three pepsinogens with relative ease; a lesser number decreases the chances of obtaining adequate amounts of pepsinogen C. Phosphate buffers at pH 6.9 are used unless otherwise stated and all operations are carried out in the cold. The stomachs from freshly killed chickens are immediately placed in 0.02M phosphate buffer, the fat removed from the outer layer of stomach, the organ opened, and the internal contents washed out with buffer. The stomachs, minced by one coarse and one fine grind with a meat grinder are then placed in 1 liter of buffer and stirred for 1 hour. The tissue is removed by centrifugation at 10,000 rpm for 1/2 hour. The supernatant is then applied to an ion-exchange column (35 X 6 cm) containing 750 ml of packed DE-11 cellulose equilibrated with 0.02M phosphate buffer, and the resin is washed with sufficient buffer (2-3 liters) to elute the inactive protein. This wash can be performed quickly and is continued until readings at OD 280 nm indicate that the peak has come off. The effluent should contain no potential proteolytic activity. The column is eluted with a linear gradient of 2 liters 0.02 M phosphate buffer connected to 2 liters of 0.02M phosphate, 0.8M NaC1, pH 6.9. The pepsinogens, located by suitable assay procedures, emerge in a pattern similar to the dogfish proteins a with pepsinogen C sometimes appearing as a slightly later shoulder (Fig. 1A). Pepsinogen B, the fraction active against N-Cbz-L-Glu-L-Tyr, is pooled and purified further by DE-11 and DE-52 cellulose chromatography using a gradient of 0.02 M phosphate, to 0.02 M phosphate containing 0.18 M NaC1, pH 6.9, followed by Sephadex chromatography. No attempt to separate the pepsinogens with potential enzyme activity toward hemoglobin is made here and they are pooled together. After dialysis against buffer (0.02M phosphate), this pooled fraction is reapplied to a smaller DE-11 column (30 X 3 cm), washed with 500 ml of buffer, and eluted with a shallower salt gradient of 2 liters 0.02 M phosphate, to 2 liters 0.02 M phosphate, 0.55 M NaC1, pH 6.9. A separation of two fractions with potential proteolytic activity may be observed, the smaller and later emerging peak being pepsinogen C. If pepsinogen C is not readily separated from the pepsinogen A-D peak on this column, the pepsinogen peaks are pooled, dialyzed against phosphate buffer, then adsorbed onto a DE-52 cellulose column and eluted with a similar salt gradient (Fig. 1B). Even though a separation of zymogens is not always achieved on the second DE-11 cellulose column, this chromatographic step greatly improves the flow properties of the solutions through the
360
THE ACIDIC PROTEASES I
80
[23]
t
60
o
r-, 4 0 o
2.0
E c o I0
20
50
I00
150
Fraction
3o.-.
lit
~~
B
250
number
'.4t
--~
20. ~,.~. ,o
200
::
:"/ ": ~
'-~r
c-'
/~\
......... 0
20
40
60
I~~
~"°r N~ \ ~o.~[ p, \ °~[1 \\ o.~r)/ k
,
80
o~ <~
I00
40
60
Fraction
number
80
100
120 1,~ 1'~o 1°B ,.io
120
140
160
Fro. 1 (A). Chromatography of original crude stomach extract on DE-11 ionexchange cellulose. The extract from 72 chicken stomachs was applied to a column (35 X 6 cm) and eluted with a gradient of 2.0 liters 0.02 M phosphate buffer, pH 6.9, to 2.0 liters 0.02 M phosphate -~- 0.9 M NaCl, pH 0.9, with 20-ml fractions being collected. Arrows indicate fractions pooled. (B). Separation of pepsinogen C from main pepsinogen fraction with DE~2 column (8 X 3 cm) and gradient of 2.0 liters, 0.02 M phosphate, pH 6.9, to 2.0 liters, 0.02M phosphate, 0.6M NaCI, pH 6.9; 15-ml fractions were collected. (C). Separation of pepsinogens D and A; DE-52 column (8 X 3 cm) eluted with gradient of 1.O liter, 0.02 M phosphate, pit 6.9 to 1.O
[23]
CHICKEN PEPSINOGENS
361
DE-52 cellulose columns used in subsequent steps of the purification procedure (Figs. 1B and 1C). On DF_,-52 cellulose columns, the major fraction consisting of pepsinogens D and A should separate easily at salt concentrations of 0.15 and 0.20 M NaC1, respectively, while pepsinogen C is eluted at 0.35 M NaC1. Acrylamide electrophoresis should be used to follow the purification, especially the final steps, as sometimes pepsinogen A can be found in the early eluted portion of an apparently homogeneous (protein concentration paralleling potential proteolytic activity) pepsinogen C preparation. • The purified DE-52 pepsinogens are concentrated by ultrafiltration (Amicon Corp. Diaflo--Membrane with 10,000 MW cutoff) and filtered through a Sephadex G-100 column (100 X 1 cm) equilibrated with 0.02 M phosphate, 0.15 M NaC1, pH 6.9 as the final purification step for each pepsinogen. Only with pepsinogen D was any further slight purification noticed. As an approximation, there are 30 mg of total pepsinogen in each stomach, in roughly a ratio of 10:15:5 between D, A, and C. In the chromatographic separations, only the midareas of protein peaks were pooled to obtain homogeneous preparations of the individual pepsinogens; thus the final yields of the purified precursors were between 10-20%. During acrylamide electrophoresis at pH 8.5, the zymogens migrate toward the anode. Pepsinogen C moves well in front of pepsinogens A and D and right behind or coincident with the bromphenol blue dye marker (Fig. 2). Pepsinogen D has a slightly slower mobility than pepsinogen A; this difference is detectable when the zymogens are electrophoresed in a single tube. The pepsins are derived from their respective purified pepsinogens by first incubating the pepsinogens at 37 ° and pH 2.0 for 4 minutes (conditions of maximal activation), then dialyzing at 0 ° against 0.1 N acetate, pH 4.0 for 8 hours followed by dialysis against 0.02 M phosphate, 0.15 M NaC1, pH 6.9, for 8 hours. The activation mixtures are then chromatographed on Sephadex under conditions used for the pepsinogens. Properties The amino acid compositions of chicken pepsinogens A, D, and C and the pepsins derived from these purified precursors are given in the table and are based on the molecular weights obtained from high-speed sedimentation to equilibrium determinations. Amide, tryptophan, phosphate, and polysaccharide analyses have not yet been done. liter, 0.02 M P0, + 0.45 M NaCl, pH 6.9; 10 ml fractions were collected. (O) Optical density at 280 nm; (A) activity against hemoglobin at pYcI2 expressed as increase of optical density at 280 nm of digested trichloroacetic acid-soluble material; (l-q) optical density at 570 nm used to determine hydrolysis of synthetic substrate N-Cbz-L-GIu-L-Tyr by ninhydrin assay.
362
THE ACIDIC PROTEASES
II
[23]
I
--!
FIe. 2. Schematic representation of polyacrylamide gel electrophoresis of the pepsinogens in Tris-HCl buffer system, pH 8~5. Arrow indicates direction of electrophoresis; bottom line in each case represents dye front.
I D
A
C
Pepsinogens
AMINO ACID COMPOSITIONS OF CHICKEN PEPSINOGENS AND PEPSINS
Pepsinogens
Lys His Arg Asp Thr Set Glu Pro Gly Ala Ha~-Cys Val Met Ile Leu Tyr Phe MW
Pepsins
A
D
C
A
D
C
17 7 7 44 28 39 32 19 33 21 (6) 27 10 24 30 24 25 42,000
15 7 7 44 28 40 29 20 34 20 (6) 28 10 24 29 27 22 42,000
5 3 3 41 33 40 48 22 40 22 (6) 22 9 24 30 24 27 42,000
11 5 6 44 31 44 31 19 35 20 (6) 27 10 25 29 24 23 42,000
10 5 6 44 32 46 29 19 34 19 (6) 28 10 26 29 24 22 42,000
3 1 2 35 32 39 43 17 39 18 (6) 21 10 23 28 20 24 38,500
F r o m this d a t a a n d t h a t o b t a i n e d from i m m u n o c h e m i c a l a n a l y s i s , 9 p e p s i n o g e n s A a n d D a p p e a r to be closely r e l a t e d to each o t h e r a n d different from p e p s i n o g e n C. P e p s i n s A a n d D closely resemble t h e i r precursors, b e i n g s m a l l e r b y some 15 a m i n o acid residues. U s i n g i m m u n o 9S. T. Donta and H. Van Vunakis, Biochemistry 9, 2798 (1970).
[23]
C~ICKEN PEPSINOGENS
363
chemical techniques which can detect small conformational differences in macromolecules,1° pepsins A and D were shown to be very similar to their precursors and to each other. Pepsin C (but not its precursor, pepsinogen C) also reacted with antipepsinogen A. The pepsinogens were homogeneous by a number of criteria, i.e., superposition of activity and optical density curves in the elution patterns obtained from DEAE-cellulose and Sephadex chromatography, ultracentrifugal analysis, the formation of single bands with the homologous antibodies on immunodiffusion and immunoelectrophoresis and migration of each protein as a single band on disc-gel electrophoresis. The pepsins generated from these purified precursors also meet these criteria for homogeneity except that several specific bands are observed for each pepsin on acrylamide electrophoresis and two bands are observed for pepsin C on immunoelectrophoresis. Pepsinogen B has been obtained in small yields only and is still heterogeneous. Stabilities. Chicken pepsins are much more stable than swine pepsin,l, 4-6,9 they maintain their activities throughout the purification procedure which is carried out at neutral pH and are stored in phosphate buffer, pH 6.9, either refrigerated or frozen. Pepsinogen preparations are stable if stored in 0.03 M Tris buffer, pH 7.5, either refrigerated or frozen. Preparations frozen in phosphate buffer at pH 6.9 were converted into pepsin'. It is strongly suggested that the pepsinogen samples be assayed routinely by the milk clot assay to assure that conversion has not taken place. Neither the pepsinogens nor the pepsins should be lyophilized since they tend to lose activity in this process. Specificities. All the enzymes are active at acid pH. The comparative proteolytic activities of swine pepsin and chick pepsins D, A, and C toward hemoglobin are 1.0, 3.4, 2.3, and 1.7, respectively, and 1.0, 0.29, 0.33, and 0.18 in the milk clot assay. The greater reactivity of swine pepsin compared to chick pepsin in the milk clot assay has already been noted? Even at concentrations of 1 mg/ml and incubation times of 1 hour, chick pepsins D, A, and C did not hydrolyze N-Cbz-L-Glu-L-Tyr, either at pH 2 or pH 4, the latter being the pH optimum of the chick peptidase (pepsinogen B). Conversely, on exposure to acid pH, the protein corresponding to pepsinogen B showed no enzymatic activity in the hemoglobin or milk clot assays, even at concentrations (100-fold) and incubation times (10-fold) greater than those used for the other pepsins. Under standard assay conditions, each milligram of peptidase B contains 0.5 units of enzyme activity using N-Cbz-L-Glu-L-Tyr as substrate. 1oL. Levine and H. Van Vunakis, Vol. XI, p. 928.