[22] Chicken pepsinogen and chicken pepsin

[22]

347

CHICKEN PEPSINOGEN AND CHICKEN PEPSIN AMINO ACID COMPOSITION OF BOVINE PEPSINOGEN a

Amino acid

Residues per molecule b

Amino acid

Residues per molecule b

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamie acid Proline Glycine Alanine

8 2 6 40 27 50 32 15 35 16

Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Amide groups

6 25 4 32 25 18 15 6 37

Total

362 +_ 2

a R. B. Chow and B. Kassell, J. Biol. Chem. 243, 1718 (1968). a Molecular weight 38,943; total nitrogen 14.56%. n u m b e r of acidic and small n u m b e r of basic residues and in the size of the molecule. However, there are at least 22 amino acid substitutions. Distribution. The pepsinogen is found only in the fundic mucosa of the a b o m a s u m or fourth stomach. The total pepsinogen content of a single mueosa varies from 0.4 to 1 g. Terminal Groups. The bovine pepsinogens all have amino terminal Ser-Val- and carboxyl terminal -Val-Ala. The bovine pepsins have amino terminal valine and carboxyl terminal alanine.

[22] Chicken

Pepsinogen

and Chicken

Pepsin 1

B y Zvx BOHAK

Assay Methods Chicken pepsin catalyzes the hydrolysis of acid-denatured hemoglobin at p H 1.5-4.0, and the extent of digestion under standard conditions is a measure of the amount of enzyme present. The digestion of hemoglobin is followed either by determining the amount of split products released in a given time la, ~ or by determining the rate of hydrolysis of peptide bonds with the aid of a p H - s t a t ? 1This investigation was supported by Grant GM-12971 from the National Institutes of Health, U.S. Public Health Service. '~ M. L. Anson, J. Gen. Physiol. 20, 79 (1938). M. L. Anson, in "Crystalline Enzymes" (J. H. Northrop, M. Kunitz, and R. M. Herriott, eds.), 2nd ed., p. 305. Columbia Univ. Press, New York, 1948.

348

THE ACIDIC PROTEASES

[22]

Chicken pepsin possesses a marked milk-clotting activity at pH 5.06.5, and can therefore be assayed by the procedure of Berridge for the assay of rennin 4 or by following the splitting of casein 5 at pH 6.0. As these assays are carried out under conditions where negligible conversion of chicken pepsinogen to chicken pepsin takes place, they can be employed to assay the enzyme in the presence of the zymogen. The sensitivity of these assays is, however, much smaller than that of the assays based on the hydrolysis of hemoglobin. The potential peptic activity of chicken pepsinogen is determined by any of the above methods after conversion to pepsin at pH 1.8-2.0. METHOD I: THE ASSAY OF CHICKEN PEPSIN BY THE SPECTROPHOTOMETRIC DETERMINATION OF TeA-sOLUBLE HYDROLYSISPRODUCTSOF ACID-DENATURED HEMOGLOBIN Reagents

Substrate. Dissolve 25 g of substrate grade hemoglobin (Worthington) in 1 liter of distilled water by stirring for 30 minutes. Dialyze the solution (20/32 Visking tubes) for 3~4 days at 4 ° against 10 liters of distilled water changed twice daily. Divide the solution into 20-ml portions and keep frozen. For the assay thaw one 20-ml portion, add 5 ml 0.3 N He1, and keep at room temperature for 15-30 minutes. The pH of this solution is 1.8 ± 0.05. Enzyme. Prepare a solution containing 0.5-1 mg per milliliter of chicken pepsin in 0.03 N HC1 or of chicken pepsinogen in 0.005 M phosphate buffer, pH 7.5, and determine its absorbanee at 280 nm. Calculate protein concentration taking E 1 mg/ml----1.46 for chicken pepsin and E 1 mg/ml ---- 1.26 for chicken pepsinogen. Dilute the solution with 0.03N He1 to a final concentration of 5-25 #g ml. If the zymogen is assayed, leave this solution for about 5 minutes at room temperature to allow for complete activation. 5% Trichloroacetic acid solution. Use within 1 week of preparation. Procedure. Pipette 1.25-ml portions of the substrate solutions into three 16 X 150 mm test tubes and keep 10 minutes at 37 °. To one tube,

aC. F. Jacobsen, J. I~onis, K. LinderstrCm-Lang,and M. Ottesen, in "Methods of Biochemical Analysis" (D. Glick, ed.), Vol. IV, p. 171. Wiley (Interscienee), New York, 1957. N. J. Berridge, Vol. II, p. 69. M. Kunitz, in "Crystalline Enzymes" (J. H. Northrop, M. Kunitz, and R. M. l=Ierriott, eds.), 2rid ed., p. 30~. Columbia Univ. Press, New York, 1948.

[22]

CHICKEN PEPSINOGEN AND CHICKEN PEPSIN

349

the blank, add 2.5 ml 5% TCA, shake well, then add 250 ~l of the enzyme solution. To each of the other tubes add 250 ~1 enzyme solution, incubate for 10 minutes at 37 °, then add 2.5 ml 5% TCA and shake well. Filter the suspension through hardened filter paper (Whatman No. 50 or S & S No. 576), read the absorbance of the filtrate at 280 nm against distilled water, and subtract the blank reading from the readings obtained for the test reaction mixtures. The figure thus obtained (AA28o) represents the absorbance of the hydrolysis products. Calculations. (a) Pure chicken pepsin yields AA28o= 0.167 per /~g enzyme taken for assay and pure chicken pepsinogen yields AA2so = 0.140 per t~g zymogen taken for assay. ( b ) The pepsin unit defined by Anson 2 is the amount of enzyme which produces an increase in the absorbance at 280 nm of TCA-soluble hydrolysis products of 0.001 per minute when the assay is carried out in a reaction mixture whose volume is 4 times that used in the present procedure. Specific activities in Anson's units are calculated from AA2so values obtained as described above by employing the equation

AA2so

Units/mg = ~g enzyme in assay × 25 × 103 Notes. The assay is linear in time and in enzyme concentration up to AA2so z 0.8.

The duplicate test readings should agree within 5%. Blank values are 0.15-0.25, and blank readings higher than 0.3 indicate unsuitable hemoglobin solutions, which often yield completely nonlinear calibration curves. Turbidity of the TCA-filtrate is often due to the presence of traces of some detergents or to protein adhering to glassware repeatedly used for this assay. Glassware, in particular filter funnels, should be thoroughly rinsed with water immediately after the experiment before washing with detergent, and after repeated use funnels should be cleand by overnight immersion in a mixture of conc. HNO3 (3 vols) and conc. H2SO4 (1 vol). METHOD II: AUTOMATEDASSAY OF CHICKEN PEPSIN BY DETERMINATION OF DIALYZABLE HYDROLYSIS PRODUCTS OF HEMOGLOBIN, EMPLOYING FOLIN'S REAGENT This method employing the Technicon Autoanalyzer was originally developed for clinical investigations. 6 It was found very convenient for monitoring peptic activity in column effluents. The method is suitable for the assay of chicken pepsin concentrations of 10-250 ~g/ml. J. Vatier, A. M. Cheret, and S. Bonfils, in "Automation in Analytical Chemistry," Vol. II, p. 371. Mediad, New York, 1967.

350

[22]

THE ACIDIC PROTEASES

(~ 0.025 0.045

'T From ccolumn :)-120 ml/hr 20-120 AIR

( ~ 0.073 HEMOG. To (~) 0.046

AIR

Fraction collector

® ® ( ~ 0.090HCl 0.2 N

o.oi°

~

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0.045

AIR

( ~ 0.056 No0H 0.9/1/

( ~ 0.056 FOLIN

® @ ~) Drain

0.100

@

15 mm

660 nm

Fro. 1. Autoanalyzer flow diagram for the assay of peptic activity?

Reagents and Apparatus Hemoglobin. Dissolve 20 g substrate hemoglobin in 400 ml distilled water and add 100 ml 0.3 N HC1. The solution should be used within 2-3 days of preparation and stored at 4 ° when not in use. Folin Reagent. Mix 500 ml commercial Folin-Ciocalteu 2 N (Harleco) with 1500 ml distilled water. Store in amber bottles. HC1, 0.2 N, and NaOH, 0.9 N Equipment. The manifold arrangement is given in Fig. 1. METHOD III. pH-STATIC ASSAY OF CHICKEN PEPSIN

Reagents and Apparatus Substrate. Prepare a 2% solution of acid-denatured hemoglobin as described in Method I. For assay this solution is titrated with 2 N NaOH to pH 3.1-3.2, kept at 0 °, and used on the day prepared.

[22]

CHICKEN PEPSINOGEN AND CHICKEN PEPSIN

351

Enzyme. Bring the solution of the enzyme to pH 3.0, and dilute with 0.001 N HC1 to a final concentration of 0.5-2.5 mg/ml. HC1, 0.05 N, standardized. Equipment. Autotitrator operated as pH-stat (Radiometer) equipped with an 0.5 ml microburet and a 10-ml jacketed titration vessel thermostatted at 37 °. Procedure. Pipette 4 ml substrate solution into the titration vessel, and allow 5-10 minutes for temperature equilibration. Set end point to 3.0 and start titrator. A small acid uptake, required to bring the substrate solution to pH 3.0, is registered and a flat base line is then recorded for at least 1 minute. Add 50 ~1 of enzyme solution and record acid uptake for 2-3 minutes. Measure the initial slope of the curve and calculate from it proton uptake in micromoles per minute. Calculations. To obtain the rate of hydrolysis of peptide bonds, divide the rate of proton uptake by 0.832 a The number of moles of peptide bonds hydrolyzed per mole of pure chicken pepsin is 1.5 X 103 per minute. Isolation

Procedures

Avian pepsinogen is produced and stored in gastric glands located in the forestomach (proventriculus) which is the part of the alimentary canal leading into the stomach. In addition to zymogen granules, these glands produce acid-containing granules and are rich in mucinlike materials 7 To extract the pepsinogen without activation, an alkaline solution must be employed for homogenization of the tissue in order to neutralize the acid. To minimize losses of the zymogen during isolation, the mucinlike materials must be removed from the extract in an early step of purification. In the original method for the isolation of pepsinogen from chicken stomachs, 8 extraction was carried out with 0.1 M NaHCO3, mucopolysaccharides were adsorbed on basic cupric sulfate, and the zymogen was precipitated from the extract between 0.2 and 0.6 saturation with ammonium sulfate. In the procedure 9 described below, mucinel Calculated from LinderstrCm-Lang'sformula3 for the conversion factor fl, 1 = 1 + 10PH-pK

employing the experimentally determined value pK z 3.7 for the dissociation of the carboxylic groups liberated in the reaction. 7p. D. Sturkie, in "Avian Physiology," p. 152. Cornell Univ. Press, Ithaca, New York, 1954. s R. M. Herriott, Q. R. Bartz, and J. H. Northrop, J. Gen. Physiol. 21, 575 (1938). Z. Bohak, J. Biol. Chem. 244, 4638 (1969).

352

THE ACIDIC PROTEASES

[22]

like materials are rendered insoluble by treating the forestomaeh homogenate with acetone, and homogenization is therefore carried out with a solution of Tris-acetate buffer which is miscible with acetone. Acetone precipitation is also employed for the isolation of the zymogen from the tissue extract. Chicken pepsinogen is then purified by chromatography on DEAEcellulose followed by gel filtration on Sephadex G-100. The pepsinogen emerges from the DEAE-cellulose columns as a single peak, when the elution schedule described below is employed. Multiple peaks apparently containing chicken pepsinogen together with some contaminants can be obtained by employing other elution schedules, and rechromatography on DEAE-cellulose is then required? ,1° Pure chicken pepsin is obtained by activation of the pure zymogen and separation of the products by gel filtration on Sephadex G-100 in an acid solution. Chicken pepsin preparations were also obtained from extracts of chicken forestomachs by acidification to pH 2 and precipitation by the addition of solid NaC1 to a final concentration of 250 g per liter. 1~ The crude pepsin thus obtained was further purified by gel filtration to yield an enzyme preparation possessing 70-90% of the specific activity of the pure enzyme. Both procedures for the preparation of chicken pepsin are described below.

Preparation o] Chicken Pepsinogen Step 1. Chicken Forestomachs. The forestomachs were removed from fowl immediately after slaughter, sliced open, rinsed, and adhering fat was removed. The forestomachs were kept frozen. A single forestomach weighs 6-8 g. To determine the total potential activity in the tissue, small pieces were removed from the central part of several forestomachs and homogenized with 10 ml/g of 0.1 M NaHCO3. A 0.1 ml portion of the homogenate was diluted with 5 ml HC1 0.03 M and activity was determined as described in assay Method I. The activity found in 1 g of forestomachs corresponds to 5-12 mg of pure pepsinogen. Step 2. Homogenization and Acetone Treatment. One hundred grams of forestomachs were partly thawed to a semisolid state, and homogenized in a Sorvall Omnimix with 200 ml of ice-cold 0.4 M Tris-acetate buffer, pH 8.6 (0.4 M Tris solution adjusted to pH 8.6 with 50% acetic acid). Homogenization was carried out for 10 minutes at full speed while cooling the jar in an ice-salt mixture. The slurry was poured into 1 liter of acetone precooled to --15 °. The mixture was stirred for 5 minutes and filtered with suction through Whatman No. 3 MM paper moistened with I°T. P. Levchuck and V. N. Orekovich, Biokhimiya (Engl. Transl.) 28, 738 (1963). " D . Gabison, Y. Levin, E. Katchalski, and Z. Bohak, unpublished results.

[22]

CHICKEN P E P S I N O G E N AND CHICKEN P E P S I N

353

acetone. The filter cake was squeezed and kept on the pump until it started turning brown at the edges.

Step 3. Extraction o] Chicken Pepsinogen and Precipitation with Acetone. The wet filter cake was suspended in 400 ml of 0.4 M Trisacetate buffer, pH 8.6, and homogenized with cooling for about 5 minutes. The suspension was then stirred 30 minutes at room temperature, and filtered through a double layer of gauze. The cake was squeezed out by hand and kept at 0 °. The red-brown milky filtrate was mixed with 50 g of Celite 535 and centrifuged for 1 hour at the maximum speed (about 11,000 g) in the Sorval RC-2 centrifuge employing the large volume head, and the supernatant was carefully decanted. In most preparations a slightly opalescent solution was obtained, whereas in some runs the supernatant had a milky appearance and was mixed with 25 g Celite 535 and recentrifuged for 30 minutes. The precipitates, collected by gauze filtration and by centrifugation, were combined and reextracted with 200 ml 0.4 M Tris-acetate buffer, pH 8.6, employing the same procedure as for the first extraction, and the two extracts were combined. The crude extract was poured into 1.4 liter acetone precooled to --15 ° and 3 g cellulose powder (Whatman standard grade) were added. The mixture was allowed to stand at 0 ° for about 30 minutes, until most of the precipitate settled. It was then filtered through a layer 3-5 mm thick of cellulose powder spread on a sintered polyethylene filter (Bel Art, Labpor filter). The solution was transferred slowly to the filter bed starting with the upper layer and filtration was started with light suction which was slowly increased as filtration slowed down. The filter cake was kept on the pump until all the liquid was sucked out. The filter cake was homogenized for 10 minutes with 100 ml 0.4 M Tris-acetate, pH 8.6, buffer. The mixture was stirred for 15 minutes at room temperature then centrifuged in the small head on the Sorval centrifuge at maximum speed (27,000 g) for 15 minutes. The supernatant was kept at 0 °, and the precipitate was reextracted as above. The supernatants were combined to give about 190 ml of a clear pinkish brown solution. The solution was transferred to 18/32 Visking dialysis tubing and dialyzed against 4 liters of 0.005 M borate buffer, pH 7.6 (0.005 M boric acid adjusted to pH 7.6 with 4 N NaOH), at 4 °. The outer buffer was changed after 2-3 hours and then twice daily for 2 days. Step 4. Chromatography on DEAE-Cellulose. The dialyzed solution was run at a rate of 120 ml/hour, into a 2.5 X 30 cm column of DEAEcellulose preequilibrated with 0.05 M borate buffer, pH 7.6. The column was then eluted at a rate of 120 ml per hour with a solution 0.05 M in boric acid and 0.1 M in NaC1 adjusted to pH 7.6 with 4 N NaOH, the absorbance at 280 nm of the effluent was monitored, and the effluent was discarded. After about 1.5 liters of the wash solution passed through the

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Effluent, ml

Fie. 2. C h r o m a t o g r a p h y of crude chicken pepsinogen on DEAE-cellulose. T h e zymogen preparation contained 11.4 X 105 potential peptic units, was chromotographed on a 2.5 X 20 cm column employing the elution schedule described in step 3. The right ordinate represents hA~0 values obtained when 10 #1 aliquots of the effluent were mixed with 1.5 ml of HC1 0.03 N and the activity was determined using 250 ~1 of this solution employing assay M e t h o d I?

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Fie. 3. Gel filtration of chicken pepsinogen on a column (1.8 X 95 cm) of Sephadex G-100 in 0.04 M a m m o n i u m bicarbonate, p H 72. °

[22]

CHICKEN PEPSINOGEN AND CHICKEN PEPSIN

355

colu.mn, the absorbance dropped to 0.1. Chicken pepsinogen was then eluted with a logarithmic gradient formed by running a solution 0.05 M in boric acid and 0.5 M in NaC1 adjusted to p H 7.6 with 4 N N a O H into a closed mixing vessel containing 450 ml of the 0.05 M borate, 0.1 M NaC1 buffer, p H 7.6. Gradient elution was carried out at 120 ml/hour, the absorbance at 280 nm, NaC1 concentration in the effluent was determined, and the effluent was collected in 10-ml fractions. Chicken pepsinogen emerged as a large peak with its center at 0.28 M NaC1 (Fig. 2). A small protein peak (or shoulder) containing potential proteolytic activity preceded the main peak and emerged from the column at about 0 . 1 5 M NaC1. This material was either added to the next batch to be chromatographed or discarded. The fractions containing the main peak were pooled and desalted in 150-200 ml portions on a 4 X 50 cm column of an ion-retardation resin (Biorad A G l l A S ) . The column was eluted with water at a rate of 200 m l / h o u r and fractions of 25 ml were collected. The protein peak in the effluent was located by monitoring the absorbance at 280 nm, and the appropriate fractions pooled and lyophilized. Step 5. Gel Filtration on Sephadex G-IO0. One half of the lyophilized powder was dissolved in 15 ml of 0.04 M ammonium bicarbonate buffer, pH 7.3, and applied to a 1.8 X 95 cm column of Sephadex G-100 equilibrated with the same buffer. The column was eluted at a rate of 20 ml/hour and the effluent collected in 10-ml fractions. A minor protein peak appeared in the breakthrough volume and the chicken pepsinogen was eluted as a single peak with constant specific activity (Fig. 3). The fractions containing the zymogen were pooled and lyophilized. See the table for a summary of the purification of chicken pepsinogen. RECOVERY OF POTENTIAL PEPTIC ACTIVITY IN THE PURIFICATION OF CHICKEN PEPSINOGEN

Potential peptic activity Step in isolation procedure 1 2 3 4 5

Purification step Chicken forestomachs 100 g Crude extract Precipitate obtained by addition of acetone. After dissolution and dialysis. After chromatography on DEAE-eeUulose. After gel filtration on Sephadex G-100.

Total Volume (PU Yield (ml) X 1@) (%)

Specific [PU]/mg protein

-550 190

29.2 30.0 24.7

100 103 85

1.1 × 103

210

22.5

77

3.1 X 103

40

18.0

61

3.5 X 10~

356

~,

AcmIc PRO~AS~,S

[22]

Preparation of Chicken. Pepsin Preparation o] Chicken Pepsin ]rom Chicken Pepsinogen. One hundred milligrams of chicken pepsinogen was dissolved in 7 ml 0.005 M phosphate buffer, pH 7.5. The zymogen was activated by acidifying the solution to pH 1.8 with 1 N HC1, and keeping the acid solution for 15 minutes at room temperature. Chicken pepsin was separated from the peptide fragment split off during activation by filtration through a 2 X 83 cm column.of Sephadex G-100 (Fig. 4). Elution was carried out with 0.001 N HC1, and the pepsin in the effluent was located by determination of the absorbance at 280 nm and determination of activity on aliquots of the effluent. The pepsin-containing fractions were pooled and lyophilized. Preparation o] Chicken Pepsin from Chicken Forestomachs. One hundred grams of chicken forestomachs was ground in a meat grinder and stirred for 3 hours with 300 ml of a solution containing 30 g NaC1 and 7 g NaHCO~ per liter. The slurry was filtered through two layers of gauze and the filtrate was acidified to pH 2 with 3 N HC1. The suspension was centrifuged for 30 minutes at top speed (ll,000 g) in a Sorvall RC-2 centrifuge employing the large head. The clear supernatant was decanted "-'-tl

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Effluent, ml

Fla. 4. Separation of chicken pepsin from the peptides formed during the activation of chicken pepsinogen. A Sephadex G-100 column (2 X 83 cm) was used and elution was carried o u t with HC1 0.001 N ?

[22]

CHICKEN PEPSINOGEN AND CHICKEN P E P S I N

357

and 28 g of solid NaC1 were added for each 100 ml supernatant. The mixture was stirred for 30 minute at room temperature, and the precipitated crude pepsin was collected by centrifugation at 11,000 g for 15 minutes, and lyophilized. The dry powder thus obtained contained about 10-15% by weight of chicken pepsin and about 50% by weight NaC1. The recovery of activity was 45 ± 5%. Two grams of this crude pepsin preparation was mixed with 3.4 ml HC1 0.02 N in a Sorvall centrifuge tube and the mixture stirred with a glass rod until all lumps disintegrated and a smooth suspension was obtained. The suspension was centrifuged for 15 minutes at 27,000 g and the clear supernatant discarded. The precipitate was mixed with 10 ml 0.02N HC1 and stirred for 5 minutes. The suspension was centrifuged for 30 minutes at 27,000 g and the supernatant filtered through a Sephadex G-100 column as described in the previous section. Properties 8-~

Stability. Chicken pepsinogen is stable at 25 ° between pH 7 and pH 10.5 and is irreversibly inactivated in more alkaline solutions. It is stable in solution in 0.1 M phosphate buffer, pH 7.6, up to 55 ° and is inactivated at higher temperatures, the inactivation being partly reversible on cooling to 25% Chicken pepsin is stable at 25 ° between pH 1 and pH 8. In more alkaline solutions it is inactivated, with concomitant autolysis, at a rate which increases with pH and temperature. Purity and Physical Properties. Chicken pepsinogen and chicken pepsin are homogeneous on disc electrophoresis at pH 7.6. The zymogen sediments in 0.05 M Tris-HC1 buffer, pH 7.5, 0.1 M in KC1 as a monodisperse material whereas the enzyme yields an asymmetric sedimentation boundary indicating some aggregation. The molecular weight of chicken pepsinogen calculated from its sedimentation and diffusion coefficients (S2o,~ ----3.58; D2o = 8.28 10-5 cm 2 sec; I~ assumed 0.75), from gel filtration, and from amino acid composition are all nearly 43,000. The molecular weight of chicken pepsin calculated from gel filtration and from amino acid composition is nearly 35,000. Composition. CHICKEN PEPSINOGEN. Lysls, Hiss, Argo, Asp43, Thr28, Ser39, Glu~o, Pro19, Gly32, Alals, 1~ Cyst, Va128, Metg, Ile23, Leu3o, Tyr24, Phe2~, Trps. Amide NH3-32. Glucosamine-2, Hexoses 6-7. CHICKEN PEPSIN. Lyss, Hiss, Arg4, Asp35, Thr2~, Ser~5, Glu23, Pro~4, Gly27, Ala~5, 1//2 CysT, Va121, Metg, Ile2o, Leu~o, Tyr2o, Phe~s, Trps, Amide NH~-20. Glucosamine-2, hexoses 6-7. Inactivators. Chicken pepsin contains one free sulfhydryl group and is inactivated with mercuric chloride and on oxidation with ferricyanide.

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).