On the two anionic chymotrypsinogens of porcine pancreas

82

BIO('HIMI(A E'f BI()PH'(SICA ACTA

BBA 35303 ON THE TWO ANIONIC CHYMOTRYPSINOGENS OF PORCINE PANCREAS"

D. GRATECOS, O. GUY, M. R O V E R Y ANn P. 1 ) E S N U E L L E

Centre de Biochirnie et de Biologie Moldculaire ( C N R S ) , Marseille "France) (Received J u n e i8th, 1968)

SUMMARY

A second anionic chymotrypsinogen (chymotrypsmogen B) was identified in porcine pancreas and purified from pancreatic juice by DEAE-cellulose chromatography and two Sephadex filtrations in buffers of different ionic strength. Its molecular weight (about 26 ooo) and amino acid composition were found similar to those of already known bovine chymotrypsinogen A, bovine chymotrypsinogen B and porcine chymotrypsinogen A. Like these three zymogens, its molecule is composed of a single peptide chain with an N-terminal half-cystine and a C-terminal asparagine. Activation by trypsin is associated with the cleavage of the I5th bond in the sequence Argl~Ile16-Va117. The short chain of 15 residues arising from the cleavage has the same amino acid composition and probably the same sequence as porcine chymotrypsinogen A. The other anionic chymotrypsinogen of porcine origin (porcine chymotrypsinogen C) can also be purified from pancreatic juice by passage through DEAEcellulose, Sephadex G-ioo and CM-Sephadex. Sephadex filtration indicated for this protein a molecular weight of about 23 ooo. However, results obtained by ultracentrifugation and the amino acid composition were consistent with a markedly higher value (about 29 ooo). I t was confirmed that porcine chymotrypsinogen C and Fraction I I of bovine procarboxypeptidase A had a similar amino acid composition and that both were activated by the cleavage of an arginyl-valine bond. As for other chymotrypsinogens, this bond was the first basic bond in the N-terminal sequence of the molecule. But a preliminary investigation suggested that it was probably the I3th instead of the I5th, and that the amino acid composition of the short chain arising during activation was different. It appears, therefore, that two sub-groups can be discerned in the chymotrypsinogen group, one composed of bovine and porcine chymotrypsinogens A and B, and the other of bovine Fraction I I and porcine chymotrypsinogen C.

* This work is p a r t of the Science degree Thesis which will be s u b m i t t e d this year to tile Science F a c u l t y of the Aix-Marseille University by D. GRATECOS u n d e r No. A.O. 25o2. Abbreviation: A T E E , acetyl-L-tyrosine ethylester.

Biochim. Biophys. Acta, 175 (t969) 82 96

TWO ANIONIC PORCINE PANCREATIC CHYMOTRYPSINOGENS

83

iNTRODUCTION

Acetyl-L-tyrosine ethylester (ATEE) is one of the synthetic substrates commonly used for the determination of chymotrypsin activity. Bovine pancreas has been shown for m a n y years to contain three zymogens with potential* ATEE-splitting activity: a cationic chymotrypsinogen A, an anionic chymotrypsinogen B and one of the subunits 1 (Fraction II) of anionic procarboxypeptidase A. Porcine pancreas is also known to contain a cationic chymotrypsinogen A which has recently been purified from acidic extracts 2. This zymogen accounts for about 70% of the total activity of the tissue against ATEE. The remainder, namely 30%, is found in the anionic fraction obtained by chromatography on D E A E or CM-cellulose of the proteins extracted at neutral p H (ref. 3). Therefore, one or several anionic zymogens with chymotrypsinogen-like properties can also be expected to exist in porcine pancreas. A first anionic chymotrypsinogen of porcine origin was purified some years ago by FOLK AND SCHIRMER4 from an acetone powder of pancreas. It was designated chymotrypsinogen C rather than chymotrypsinogen B because of the abnormally high activity of the corresponding enzyme against leucyl bonds in synthetic substrates and peptides. Its molecular weight (3I 80o) was also reported to be markedly higher than for other chymotrypsinogens and its amino acid composition was different. It was suggested by the authors that chymotrypsinogen C and Fraction I I were homologous proteins. One would be free in porcine pancreas, whereas the second would be associated in bovine pancreas with two other compounds to form trimeric procarboxypeptidase A. This hypothesis was founded upon striking similarities existing between the amino acid composition of the two zymogens and the relative activities of the corresponding enzymes towards aromatic and leucyl bonds. In spite of the fact that no indication was obtained by FOLK A~
i. Activation of zymogens For impure fractions containing trypsin inhibitors, 2oo-4oo #g protein in 1.5 ml o. I M Tris-HC1 buffer (pH 8.o) were incubated at 25 ° for 30 Inin with 400 #g crystalline trypsin (Worthington). For more purified fractions, the amount of protein and trypsin could be reduced to IOO and 4 ° #g, respectively. Full activation occurred in all cases. 2. Evaluation of enzymatic activities Activity against the substrate A T E E was measured potentiometrically according * Activity appearing after activation of the zymogen by trypsin.

]3iochim. Biophys. Acta, 175 (1969) 82 96

~4

1). (;!{ VII,S('I)N

CI (I/.

to a technique already described a. One A T E E unit was defined as the amount o1 enzyme(s) splitting I /,mole A T E E per rain under the conditions (d the assays. An absorbance coefficient E~ei6,~~ 23.8 (ref. 4) was used for the calculation of the spe( ilic activities of the purified zymogens. Carboxypeptidase A activity was measured spectrophotometrically a against the substrate carbobenzoxyglycyl-L-phenylalanine in a (;ilford Multiple Sample Absorbance Recorder Model 2oo0. One ml of a IO mM carbobenzoxyglycyl-L-phenylalanine solution in a 25 mM Tris buffer (pH 7.6) o.I M in NaC1 was introduced into the cuvette. After equilibration at 35 °, Ioo 2oo ~1 of the activated sample were added and the linear decrease in A 2as m# was recorded for 3 lnin. One carboxypeptidase A unit was defined as the a m o u n t of enzyme hydrolyzing I ~mole carbobenzoxyglycyl-L-phenylalanine per rain. The hydrolysis of I #mole carbobenzoxyglycyl-L-phenylalanine induced, under the conditions of the assays, a decrease of o.o 9 absorbance unit. Carboxypeptidase B activity was also measured spectrophotometrically: at 2 5 using a I mM solution of the substrate hippuryl-L-arginine in tim same buffer as above. IO 2oo/,1 of the activated sample were added and tim increase in A.,a4 ma was recorded for 3 rain. The carboxypeptidase B unit was expressed in #moles hippuric acid liberated per rain (E7..... ~ o f a I mM hippuric acid solution at 254 nv~, o.36). RESULTS

Existence of two anionic chymotrypsinogens in porcine pancreas The first step of the purification of bovine chymotrypsinogens A and B and porcine c h y m o t r y p s i n o g e n A is to prepare an acidic extract of fresh pancreas. In contrast, bovine Fraction I I and porcine chylnotrypsinogens B and C are irreversibly denatured b y acid and t h e y must be purified from an acetone powder or from pancreatic juice. The existence of two anionic chymotrypsinogens in an acetone powder of porcine pancreas is shown b y the following experiment : The powder (zo g), prepared as described b y FOLK A N D SCHIRMER4, was extracted for 9 ° min with zoo ml water in the cold*. One mg trypsin inhibitor from Soya beans was added per ml clear extract. 2o-ml Samples were charged into a 1.8 cm >< 15 cm DEAE-cellulose column equilibrated with a Tris acetate buffer (pH 6.o), Io mM in Tris. The unretarded cationic peak was discarded and the whole anionic protein fraction was eluted b y raising to o. 4 M the concentration of NaC1 in the buffer. This fraction contained about 3 o % of the total A T E E - s p l i t t i n g activity of the original extract. Stability was somewhat improved b y the elimination of the cationic trypsinogen. The fraction was concentrated and freed of nucleic acids b y precipitation in 0.8 satd. (NH4)2SO 4. After centrifugation, the sediment was taken up in a small volume of water. The solution was dialyzed against a Tris acetate buffer (pH 6.o) IO mM in Tris and charged into a DEAE-cellulose column equilibrated with the same buffer. Elution was performed at p H 6.o b y a linear concentration gradient of NaC1 from o to o. 4 M. All the fractions were tested for potential activity against A T E E , carbobenzoxyglycyl-L-phenylatanine and hippuryl-L-arginine with the results presented in Fig. I. Fig. I shows t h a t two incompletely separated peaks with potential A T E E * All assays

were carried

o u t a t o 2 °.

]3iochim. Biophys. 4c[a, r 7 5 ( i 0 6 9 ) 82 9 6

"1t~,O ANIONIC PORCINE PANCRFATIC CHYMOTRYFSINOGENS

3°l

~5

ChTgB

/"

o

o ProC 2O

E u~ n L)

;hi

.,,

n

I

~o

0.5

u

i 75 Fraction

100 No.

Fig. [. C h r o m a t o g r a p h y on DEAE-cellulose at p H 0.o of the anionic proteins of porcine pancreas. 20o mg of anionic proteins were charged into a DEAE-cellulose column (i.o cm × 4 ° cm; W h a t m a n D E II ; I.o mequiv/g) equilibrated with a T r i s - a c e t a t e buffer (pH 6.o) Io mM in Tris. The NaC1 concentration gradient used for elution is indicated by a dashed line. Fraction volume 2. 4 ml. l~otential activities of the fractions against A T E E (chymotrypsinogens) (O), CGP (proc a r b o x y p e p t i d a s e A) ( d ) and HA (procarboxypeptidase B) ((;). ChTg, c h y m o t r y p s i n o g e n ; ProCp, i)rocarboxypeptidase; HA, hippuryl-L-arginine; C(;I ), carbobenzoxy glycyl-L-phenylalanine.

splitting activity emerged from the column for a NaC1 molarity of o.23 and 0.3o, respectively. Fractions 55 75 under the first peak and Fractions 76-95 under the second were pooled. The two solutions were concentrated by precipitation in o.8 satd. (NH4)2SO4, dialyzed and submitted to another chromatography under the same conditions. The new peaks were still impure and consequently unsymmetrical. But, elution of the top fraction occurred again for a o.23 and o.3o NaCI molarity. Hence, porcine pancreas could be assumed to contain two distinct anionic chymotryFsinogens designated from now on as chymotrypsinogens B and C. Similar results have been obtained so far with five lots of acetone Fowder, each prepared from a single pancreas and with seven lots of pancreatic juice (see later), each collected from a single animal. This observation seems to rule out the possibility of genetic variations inducing the biosynthesis of either chymotrypsinogen B or chymotrypsinogen C. Both zymogens appear to be produced simultaneously by porcine pancreas.

Puri3cation of ehymotrypsinogens B and C from pancreatic juice Early in this study, it became apparent that porcine pancreatic juice would be a better source than acetone powder for the complete purification of chymotrypsinogens B and C. The preparation of acetone powders requires large volumes of solvents and it is often associated with partial activation of the zymogens. Pancreatic juice is free of cellular proteins and nucleic acids, and it is quite stable after lyophilization when kept at -- 15 ° for several weeks. Large samples of juice were collected by cannulation of the Wirsung duct in young pigs weighing about 7 ° kg. Most of them were gifts from Drs. A. RI~RAT and A. AUMMIRE, Station de Recherches sur l'I~21evage des Porcs, Jouy-en-Josas (France), Biochim. Bioph3's..dcta, t75 ([969) 82 96

S()

I). GI~:VIECO,~ el a[.

to whom we are deeply indebted. Others were obtained through the courtesy of Dr. C. M. W. HIRS, Brookhaven Laboratories (U.S.A.). Their average protein content was about 25 °//o as iudged bv~ A,as0 m~. Their direct ATEE-splitting activity, was not higher than o.5-1c~ of the maximal value observed after full activation by trypsin. Complete purification of the two chymotrypsinogens B and C was achieved in only three steps. Chromatography on DEAE-cellulose at p H 6.0 Lots (15 g) of lyophilized juice containing about 4 g proteins (spectrophotometric evaluation at 280 m/z) were dissolved in 3o ml cold water. After addition of 15o mg Soybean trypsin inhibitor, the solutions were dialyzed overnight against 2o 1 of the Tris-acetate buffer (pH 6.o) IO mM in Tris already used before. They were submitted to chromatography on DEAE-cellulose under the same conditions as the pancreas extracts, except for the use of a larger column and a slower NaC1 gradient. The diagram reproduced in Fig. 2 shows that two peaks with potential ATEE-splitting

3 t

100~ ~.O-

,

I

ChTgi~~ Lipase -~ oxyribo~I ,,,-.De ~...,nuclea ~ i "~ I I

-g

g 0.5-

L

I

C)

5"

"'-.---...'

Cationic ', e n z y m e s ,

50

loo

-

Fracllon No.

15o

Fig. 2. C h r o m a t o g r a p h y of porcine pancreatic juice on DEAE-cellulose at p H 0.o, The conditions of the c h r o m a t o g r a p h y are the same as in Fig. i, except for the use of a larger column (3-5 cm x 4o cm) and a slower NaC1 gradient. F r a c t i o n volume z 3 ml. Potential activities in the fractions against A T E E (Q), CGP ( + ) and H A (@) as in Fig. i. The dashed line indicates the total protein c o n t e n t of the fractions (A2,o rna). Abbreviations: see legend of Fig. ~.

activity were again obtained for NaC1 molarities not substantially different from those already observed with pancreas extracts (o.2o and o.27 instead of o.23 and o.3o). The two chymotrypsinogens were freed of cationic enzymes, lipase and deoxyribonuclease and were well separated from each other. But, they were eluted in the same region as the procarboxypeptidases A and B. Chromatography on Sephadex at low ionic strength FOLK AND SCHIRMER4 attempted to separate chymotrypsinogen C from the procarboxypeptidases by several filtrations through Sephadex G-Ioo. A better separation and higher yields were obtained here b y the successive use of Sephadex and CM-Sephadex. Moreover, the purification of chymotrypsinogen B on Sephadex G-Ioo was greatly facilitated b y the observation that the zymogen underwent an ionic t3iochim. Biophys..4cta, 175 (1909) 82 9G

TWO ANIONIC PORCINE PANCREATIC CHYMOTRYPSINOGENS

87

strength-dependent association-dissociation equilibrium at pH 6.o. By varying the ionic strength of the buffer, complete purification could be achieved by two filtrations only. Collected Fractions 11o-147 under the chymotrypsinogen B peak and 148-17o under the chymotrypsinogen C peak in Fig. 2 (potential specific activity against ATEE, IOO and 35, respectively) were concentrated by precipitation in 0.8 satd. (NH4)2504, dialyzed against water and filtrated through Sephadex G-Ioo in a sodium citrate buffer (pH 6.0) 30 mM in citrate (I 0.3). The separation obtained under these conditions for the fraction containing chymotrypsinogen B is given by Diagram a in Fig. 3. It is given in Fig. 4 a for the second fraction corresponding to chymotrypsinogen C.

I

i

r :c.o, \ ,,/;/: :I, "./... ,' ,

./<

~

250-

J

b

; ,

~ ~

~50"

,25 25-

1

• / ¶42o

,,.,,,,

I0

;/

~

I r~

f'~ ChTgB

'' ProcpA: ',,

2.0

o.o

~,X ~+

o~'~o (,,,

"~

i~

o

['~ ChTg B ,' ',ProCpA

| ~

/

~

i/

i/ .. . . . . . . . . ;

~ ,, 36o•

~',

/\ /,'",

", •,:

° ;F~
2 Re~el~t]on volumes

\

•400

",. \

"...\ "-.

Fig. 3. Purification of porcine c h y m o t r y p s i n o g e n B. a. The column (3.o cm x t9o cm; retention volume (Vo), 500 ml) of Sephadex G - i o o was equilibrated and eluted by a sodium citrate buffer (pH 6.0), 30 mM in citrate (I 0.3). I t was charged with 700 mg proteins from Fractions ItO 147 in Fig. 2. Flow rate ,51 ml/h. Same s y m b o l s as in Figs. i and z. b. R e c h r o m a t o g r a p h y on Sephadex G-ioo of the c h y m o t r y p s i n o g e n B peak (400 500 m g proteins) in a. The column (3.0 cm x 19o cm; same Vo as above) was equilibrated with and eluted by a Tris acetate buffer (pH 6.0) o.i M in Tris and 0. 4 M in NaCI (I 0.5). Same flow rate and same s y m b o l s as in a. The n u m b e r s along the c h y m o t r y p s i n o g e n 13 peak indicate the potential specific activity of the fractions against A T E E . Abbreviations: see legend to Fig. i.

Fig. 3a shows that when a solution of chymotrypsinogen B was filtrated through Sephadex G-IOO at low ionic strength, the zymogen emerged from the column at the end of the second retention volume. Its apparent molecular weight, therefore, was about 50 000-60 ooo under these conditions. Procarboxypeptidase B emerged at the beginning of the third volume (molecular weight range 25 ooo 3 ° ooo). Procarboxypeptidase A gave two peaks, as if two forms of the zymogen with different molecular Biochim. Biophys. Acta, 175 (1969) 82-96

cS~

l). ( ; I L \ r l , ; t ( ) s

('t # / .

weights were present. ('hymotrypsinogen B was treed of procarboxypeptidase B and of the "lighter" form of procarboxypeptidase A, but it was still contalninated with the "heavier" form. During a similar filtration at low ionic strength (Fig. 4 a ) , chymotrypsinogen d was eluted in the third retention volume on the right of a broad protein peak. A good separation from a number of contaminants (chymotrypsinogen B, procarboxypeptidases A and B) was obtained.

Final pz~rificatio~ of c@,motrypsinoge~ B The above experiments might have suggested that porcine chymotrypsinogens B and C had widely different molecular weights. But, a more plausible hypothesis was to assume that chymotrypsinogen B molecules associate at pH 6.o in solutions of low ionic strength. This hypothesis was confirmed by Fig. 3b. When the mixture of chymotrypsinogen B and procarboxypeptidase A obtained on Sephadex at low ionic strength was filtrated at higher ionic strength (Tris acetate buffer (pH 6.o) o.I M in

Lu o_ oo '~

5

5o Io

//

zo. SO

:

,'

~J =

//

c h ~ g Bf , . o - ' , . . I' P~ocpA / M " "

'11

2S 5; ',

/ l

O.C

,--

/ /~

'" "'"

•

"- . . . . %+ /~

O /

I

. ~il (,~1:I

Retention II~. vol . . . . ~%-

2

b

t",

I

~O

,

I

,

i

/.~-

/ ~ChTgC

:t,,, oo

~

,'/ e, 165 ,o \ ,,

' ,oo/;1~

,,o

i/'t,o',,~o

;/ ,~o,~

/ ~ o.

,

~.

ProCpB

1

20

/

,,.'~;_U'".... 20

L'--" 40

/'

....

r °3

......, ';t...,:....t: ,oo

60 Fract]on No.

i"ig. 4. P u r i f i c a t i o n of porcine c h y m o t r y p s i n o g e n C. a. F i l t r a t i o n t h r o u g h S e p h a d e x O - i o o a t low ionic s t r e n g t h of F r a c t i o n s [48 i 7 o c o r r e s p o n d i n g to c h y m o t r y p s i n o g e n C in Fig. i . The c o n d i t i o n s are t h e s a m e as for t h e first S e p h a d e x c h r o m a t o g r a p h y of c h y m o t r y p s i n o g e n B (in Fig. 3a). After c o n c e n t r a t i o n of the f r a c t i o n s b y p r e c i p i t a t i o n in o.8 sald. (NH4)2SO 4 a n d dialysis, a v o l u m e c o n t a i n i n g 90o mg p r o t e i n s w a s f i l t r a t e d t h r o u g h a 3 c m × I9o cm S e p h a d e x G - i o o c o l u m n e q u i l i b r a t e d w i t h a n d e l u t e d b y a s o d i u m c i t r a t e buffer (pH 6.o) 3 ° mM in c i t r a t e (I o.3). l'(,, 500 ml. Flow rate, 5~ ml/h. b. C h r o m a t o g r a p h y on C M - S e p h a d e x a t p H 6.0. The f r a c t i o n s c o n t a i n i n g c h y m o t r y p s i n o g e n C in a were c h r o m a t o g r a p h e d in a 2.o cm × 5 ° c m c o l u m n of CMS e p h a d e x C 5o ( P h a r m a c i a , LTppsala) e q u i l i b r a t e d w i t h a n d w a s h e d b y a T r i s - a c e t a t e buffer (pH 6.o) o.o5 M in Tris. FAution of c h y m o t r y p s i n o g e n C w a s o b t a i n e d by a l i n e a r increase of t h e Tris m o l a r i t y fro m o.o 5 to 0. 3 M. F r a c t i o n v o l u m e 9.5 ml. The n u m b e r s a l o n g t h e c h y m o t r y p s i nogen C p e a k i n d i c a t e t h e p o t e n t i a l specific a c t i v i t y of t h e f r a c t i o n s a g a i n s t A T E E . A b b r e v i a t i o n s : see legend to Fig. i. Fig. 5- Disc e l e c t r o p h o r e s i s a t p H 8.0 of c h y m o t r y p s i n o g e n s B (left) a n d C (right).

Biochim. Biophvs. Acta, ]75 (1969) 82 9(7

TWO ANIONIC PORCINE PANCREATIC CHYMOTRYPSINOGENS

89

Tris and o.4 M in N a G ; I o.5) , chymotrypsinogen B emerged at the beginning of the third retention volume and it was now completely free of the heavier form of procarboxypeptidase A which remained in the second volume. The fractions of the chymotrypsinogen B peak marked by a horizontal bar in Fig. 3 were pooled. After concentration by vacuum dialysis, the solution (potential specific activity against ATEE, 38o) was found homogeneous by disc electrophoresis at pH 8.6 (left tube in Fig. 5). Homogeneity was also demonstrated by electrophoresis on starch gel at pH 8.2. The overall yield from the first separation on DEAE-cellulose (Fig. 2) was 25 o(). From 15 g lyophilized juice (4 g protein), 6o-7o mg pure chymotrypsinogen B were usually obtained. The preparations were kept frozen at --15 °.

Final purification of chymotrypsinogen C Chymotrypsinogen C, already free of chymotrypsinogen B, procarboxypeptidase A and procarboxypeptidase B by filtration through Sephadex at low ionic strength (in Fig. 4a), was further purified by a chromatography on CM-Sephadex in a Trisacetate buffer (pH 6.o) o.o5 M in Tris (Fig. 4b). Under these conditions, a large protein peak composed of still unidentified proteins emerged just after the break-through volume of the colunm. Elution of chymotrypsinogen C was induced by an increase in the buffer concentration at the same pH. The fractions indicated by a horizontal bar were pooled. After concentration by vacuum dialysis, the preparation (potential specific activity against ATEE, 15o ) was found homogeneous by disc electwphoresis at pH 8.6 (right tube in Fig. 5) and starch-gel electrophoresis at pH 8.2. The overall yield from the first separation on DEAE-cellulose (Fig. 2) was 40%. From 15 g lyophilized juice (4 g total proteins), 60-7o mg pure chymotrypsinogen C were usually obtained.

Molecular properties of chymotrypsinogen B Tile molecular weight of chymotrypsinogen B was compared to those of already known zymogens of the same group by filtration through a I cm × 147 cm Sephadex G-ioo column at high ionic strength (Tris acetate buffer (pH 6.0) o.I M in Tris and 0.4 M in NaC1). Table I gives the elution volumes observed and the corresponding molecular weights. According to Table I, the apparent molecular weight of porcine chymotrypsinogen B did not appear to be substantially different from the others (about 26 ooo). TABLE

I

MOLECULAR VVEIGHT OF CHYMOTRYPSINOGEN B BY THE SEPHADEX TECHNIQUE

Protei~

Bovine A Bovine B Porcine A Porcine B

Conc*z. (mg/ml)

Elutio~ vol.

0 io TO io 0 lo

79.0 81.2 78.3 8o.3 81.o 76.2

Mol. wt.

(ml)

25 674*

about about

25 754* 25 ooo** 26 ooo

* C a l c u l a t e d f r o m t h e s e q u e n c e S , 9. ** E s t i m a t e d b y S e p h a d e x a n d u l t r a c e n t r i f u g a t i o n a .

Biochim. Biophvs. dcta, i 7 5 (1969) 82 9 6

00

I). (;RATE(OS ~'[ a[.

Moreover, since the experiments were perfl)rmed at high ionic strength and since similar elution volumes were observed at two concentrations (6 and IO ing/ml), the value of 26 ooo indicated in Table I for the molecular weight of the zymogen could be considered as corresponding to the monomer. In contrast, when a buffer of low ionic strength was used (o.o3 M sodium citrate (pH 6.o)) the elution volume and consequently the apparent molecular weight of the protein strongly varied with the concentration of the solution charged into the colunm (26 ooo, 33 ooo, 43 5 oo, 56 ooo TABLE 11 AMINO ACID COMPOSITION OF CHYMOTRYPSINOGENS B AND C Amino acid

Number of residues in Chymotrypsinogen B (26 000 g) Experimental Nearest integral number

Ala Arg Asp Cys Glu Gly His lle Leu Lys Met Phe Pro Ser Thr Trp Tvr Val Total

21.88 7.65 19.97 9-57 14.94 22.02 2.79 t 1.24 16.56 5.7 ° i .97 7.9 2

13.66 28.26 17.39 12.6 5 4.15 24.56

22 8 20 lo 15 22 3 i1 16 17 (i 2 8 14 28 ~7 13 4 24 -25

Chymotrypsincgen C (29 ooo g) Experimental Nearest integral number

R~f. 4

14.94 8.89 25.22 1o.34 26.35 20.55 6.09 13.76 21.68 7.15 1.02 4.15 13. 5 22.48 16.7I 12.o 9 ~.28 23.03

10 8 29 lO 26 27 5 14 21 22 8 1 5 14 24 15 13 7 2i

243-245

15 9 25 io 26 20 27 0 14 22 7 ~ 4 13 14 22 23 17 12 6 23 258-26~

a n d 69 000 f o r c o n c e n t r a t i o n s o f I, 2, 4, I 0 a n d 20 m g / m l , r e s p e c t i v e l y ) . I t will b e s e e n l a t e r (Fig. 6) t h a t a v a l u e o f a b o u t 26 000 is also c o n s i s t e n t w i t h t h e a m i n o a c i d c o m p o sition of the protein. The a m i n o acid c o m p o s i t i o n i n d i c a t e d b y Table II for c h y m o t r y p s i n o g e n B was d e r i v e d f r o m a s e r i e s o f a n a l y s e s b y t h e a u t o m a t i c t e c h n i q u e o f SPACKMAN, STEIN AND MOORE 1° a f t e r 24, 48 a n d 7 2 h h y d r o l y s e s o f t h e p r o t e i n in t r i d i s t i l l e d HC1. A v e r a g e v a l u e s w e r e t a k e n i n t o a c c o u n t e x c e p t f o r s e r i n e a n d t h r e o n i n e for w h i c h a l i n e a r e x t r a p o l a t i o n to zero t i m e was used, and for valine a n d isoleucine for which only m a x i m a l v a l u e s a f t e r 72 h h y d r o l y s i s w e r e r e t a i n e d . H a l f - c y s t i n e w a s d e t e r m i n e d as cysteie acid after performic oxidation. Tryptophan was estimated by spectrophotometry n. M o r e o v e r , a n N - t e r m i n a l h a l f - c y s t i n e r e s i d u e c o u l d b e i d e n t i f i e d in c h y m o t r y p s i n o g e n B b y t h e d a n s y l t e c h n i q u e 12 a f t e r p e r f o r m i c a c i d o x i d a t i o n o f t h e p r o t e i n . Biochim, Biophys. Acta, 175 (I969) 82-96

TWO ANIONIC PORCINE PANCREATIC CHYMOTRYPSINOGENS

91

Hydrazinolysis gave negative results. However, during incubation of the oxidized protein with carboxypeptidase A in presence of I mM diisopropylfluorophosphate, I mole per mole asparagine was freed with lower amounts of alanine, valine, glutamine, isoleucine and histidine. It could therefore be concluded that porcine chymotrypsinogen B was composed of about 245 residues arranged along a single chain with an N-terminal half-cystine and a C-terminal asparagine.

Activation of chymotrypsinogen B In order to see which bond was cleaved during activation, a solution (5 mg/ml) of the zymogen was incubated at o ° (pH 7.9) for 15 rain with trypsin (lO% by weight). /5-Phenyl propionate was added to a o. i M concentration with the purpose of avoiding a possible autolysis of the primary chymotrypsin 1~. Activation was maximal under these conditions. After a I5o-min incubation with 2o mM diisopropylfluorophosphate at o °, the mixture was desalted by passage through Sephadex G-25 (coarse) equilibrated with water, and lyophilized. The short chain presumably formed during activation (Chain A) was liberated by performic acid oxidation, extracted with water and purified on a 1. 4 cm × 4 ° cm Sephadex G-5o (medium) column equilibrated with 5 mM HC1 (ref. 5). As for other chymotrypsinogens, monitoring of the eluate at 23o Ink* revealed the presence of a relatively short peptide in the third retention volume of the column. This peptide did not absorb at 28o m#. After further purification b y electrophoresis on paper (35 V/cm; pyridine-acetate buffer (pH 3.6), o.o4 M in pyridine) and chromatography (butanol-pyridine-acetic acid-water (6o :4o:12:48, by vol.), the peptide had the following amino acid composition: Alal, CySO3Hx, Gly2, Ilel, Leu2, Pr%, Ser2, Val 2. Moreover, one equivalent of free arginine was identified in the last fractions emerging from the Sephadex G-25 column (see above). This arginine should be Cterminal in a chain arising from trypsin attack and it was probably split o f f b y a trace of carboxypeptidase B remaining in the preparation. Therefore, the intact Chain A could be assumed to contain 15 residues as for bovine chymotrypsinogens A and B and porcine chymotrypsinogen A. It had the same amino acid composition as in this latter zymogen and probably also the same sequence. On the other hand, an N-terminal isoleucine residue was detected by the fluorodinitrobenzene technique in the insoluble oxidized material after extraction of Chain A b y water. The peptide D N P - I l e - V a l was also identified. Therefore, the I5th bond cleaved by trypsin during activation of porcine chymotrypsinogen B was a part of the same sequence Argl.~-Ii%6-Vall~ as in the three chymotrypsinogens listed above.

Molecular properties of chymotrypsinogen C The molecular weight and amino acid composition of chymotrypsinogen C, already evaluated by FOLK AND SCHIRMER4, were re-investigated because of the possible homology of the zymogen with bovine Fraction II. Two techniques were used for the determination of the molecular weight: Sephadex filtration and ultracentrifugation (sedimentation equilibrium and approach to the equilibrium (Archibald)). An additional value was derived from the amino acid composition of the protein indicated in Table II. Five or io mg/ml solutions of pure chymotrypsinogen C were filtrated through Sephadex G-ioo under the conditions used for the experiments reported in Table I. Biochim. Biophys. dcla, 175 (1969) 82-96

I). (;I
02

The elution wflume (83.2 ml) observed for this protein was definitely higher than for the other chymotrypsinogens, suggesting that its molecular weight was smaller than 26 ooo. More precisely, when the colunm was standardized with proteins of known molecular weight (ribonuclease, bovine chymotrypsinogen A and pepsin), a value not exceeding 23 ooo was obtained, instead of 3I 8oo as indicated by FOLK aND ScH n~.~mrd. However, the results of ultracentrifugation assays kindly performed bv Prof. J. REYNAUD, Facultfi de Mddecine, Marseille, were consistent with a higher value. Using the partial specific volume (o.724) calculated from the amino acid composition, sedimentation equilibrium experiments (Io 589 rev./min for IOO h) carried out with o.~ 5 o.4o mg/ml solutions in a 0.3 5I Tris buffer (pH 6.o) indicated a value of z8 8oo 5 Iooo after extrapolation to zero concentration. Assays by the technique of Archibald at IO 589, I I 573 and I2 59 ° rev./min for I6 96 min of 5 mg/ml solutions in the same buffer, gave an average value of 3o See. There was no indication of molecular association during these latter assays.

Bovine

B

c 5 g

~xx

'orclne C

VVO2-o~

I 1

25' 1264 282 :~b

35

25.5

MOI wt. xlO 3

Fig. (). B e s t lit b e t w e e n a m i n o acid c o m p o s i t i o n a n d molecular weight.

Finally, tile best fit between amino acid composition and molecular weight was evaluated by a method developed by Dr. M. DELAAGEin this laboratory 14. If ni is the number of residue i in I mole protein and Ni is the nearest integer, the entire composition of the protein can be compared for any molecular weight value to the nearest integral composition by means of the expression : ~'i log~ Ni

When this expression is plotted against molecular weight, any minimum of the plot will indicate a possible fit, the lowest minimum corresponding to the best fit. The basic assumption is that all residues are estimated with the same degree of confidence. A recently published amino acid analysis of bovine chymotrypsinogen B (ref. 5) was used to check the validity of the method. The plot reproduced in Fig. 6 for this zymogen indicated a sharp minimum for 25 5oo, a value very close to the molecular weight (25 754) calculated from the sequence 9. Similarly, an excellent agreement was found for porcine chymotrypsinogen B between the value derived above from Sephadex filtration assays (about 26 ooo) and the position of the minimum in the corresponding plot in Fig. 6 (26 4oo). The value observed under the same conditions and with the Biochim. t~iophys. Acta, i75 (1969) 82 96

TWO ANIONIC PORCINE PANCREATIC CHYMOTRYPSINOGENS

93

same assumptions for porcine chymotrypsinogen C was 28 2oo. It could therefore be concluded that the result given in this particular case b y the Sephadex filtration technique was for some unknown reason unreliable and that the average of the other values, namely 29 ooo, should be retained. Table II indicates the amino acid composition of chymotrypsinogen C. The results previously published by FOLK AND SCHIRMER for the same protein are also included after re-calculation for a molecular weight of 29 ooo. Both analyses are in substantial agreement.

Activation of chyrnotrvpsinoge~, C Preliminary experiments have confirmed 4 that the activation of this zymogen induces the appearance of an N-terminal valine residue. Moreover, the short chain formed during activation was studied b y the technique described above for chymotrypsinogen B. The purified peptide isolated after performic acid oxidation appeared to contain 13 residues only (Alal, Argt, ASpl, CySOaH1, Gly 1, Leu 1, Phe 1, Pro 3, Ser2, Vall) , instead of the expected 15. A special investigation on the possible presence of t r y p t o p h a n in the chain has not yet been carried out. Sequence determinations are presently in progress. DISCUSSION

Like bovine pancreas, porcine pancreas contains three zymogens with potential ATEE-splitting activity : a cationic chymotrypsinogen A purified from acidic extracts of the tissue, and two anionic chymotrypsinogens (B and C) purified from acetone powder and pancreatic juice. Lyophilized pancreatic juice is a good source for the purification of porcine chymotrypsinogens B and C. From 15 g samples containing about 4 g total proteins, 6o-7o mg of each zymogen can be isolated in a homogeneous form. The first step of the procedure is a chromatography on DEAE-cellulose at p H 6.o which separates the three zymogens, A, B and C, from each other (Fig. 2). Taking into account the 85 % overall loss in ATEE-splitting activity during this chromatography and supposing that the loss is the same for the three zymogens, their proportions in porcine juice can be roughly calculated from the specific A T E E activities of the pure products. These proportions are, respectively, I o - I 5 , 4-6 and 3 - 4 % of the total proteins in the juice. The corresponding proportions in bovine juice have been reported to be I6-22°/0 for chymotrypsinogen A (refs. 15 and 3) and one fourth of this value, namely 4 - 6 % , for chymotrypsinogen B (ref. 3). On the other hand, since trimeric procarboxypeptidase A represents 3o% of the total proteins in bovine juice15,16, about one-third (IO%) m a y be attributed to Fraction II. These figures probably do not represent more than a rough approximation since enzyme levels are known to vary within large limits in rat pancreas and pancreatic juice as a function of the diet ingested by the animals 17,1s. It is of interest that the efficiency of the Sephadex filtration technique for the purification of a protein is very much increased when two sets of experimental conditions can be found under which the rate of migration of the protein is different. When prepared from fresh pancreas, porcine pancreatic lipase was already observed to migrate unretarded through Sephadex G-2oo (ref. 19). This abnormal behavior was caused by the binding of the enzyme to large lipid micelles. After cleavage of the cornBiochim. Biophys. Acta, 175 (I969) 82~:)6

94

D. ( ; R A T E C ( ) 8

¢[ tl[.

plex, the migration rate became consistent with the real molecular weight of the enzyme (about 45 ooo). In the present study, the ionic strength dependence of the association-dissociation equilibrium of porcine chymotrypsinogen B at pH 6.o was successfully used for the purification of the zymogen on Sephadex. When dissolved in a buffer of low ionic strength, the zymogen emerged from Sephadex G-ioo with the second retention volume. At higher ionic strength, it emerged with the third volume ; whereas, the contaminants originating from the first filtration remained in the second volume. The association-dissociation equilibrium of porcine chymotrypsinogen B was also found to be concentration-dependent at low ionic strength. Hence, a correct evaluation of the molecular weight of the monomer by the Sephadex technique would have required under these conditions, either the use of very dilute solutions, or an extrapolation to zero concentration. No concentration dependence was observed at higher ionic strength. Another interesting but still unexplained observation was that porcine procarboxypeptidase A gave two distinct peaks on DEAE-cellulose (Fig. 2) and Sephadex (Figs. 3 and 4). These peaks emerged from the Sephadex G-ioo colunm, respectively, at the end of the second retention volume and at the beginning of the third, as if the molecular weights of the corresponding forms were in the 50 000-60 ooo and 25 ooo3o ooo range. Experiments are in progress to clarify this point. The technique of filtration through Sephadex is often used for evaluating the molecular weight of proteins. Obviously, the rate of nfigration of any molecule through Sephadex is not a function of its weight but of its Stoke's radius. This is probably the reason why it is recommended to standardize the column with known compounds, that are as chemically related as possible to the compound under investigation. The exanlple of porcine chymotrypsinogen C is a serious warning against the view that the TABLE

I[I

AMINO ACID COMPOSITION

OF SOME CHYMOTRYPSINOGENS

Mol. wt. normalized

to 26 ooo.

Amino acid

Bovine A (re/. 8)

Bovine B (re). 9)

Porcine 4 (roy. 2)

Porcine B ( T h i s work)

OF BOVINE

Bovine Fractio~z II (ref. 21)

AND PORCINE

Porcine C ( T h i s work)

Ala

22

23

22

22

15

[()

13

Arg Asp

4 23

5 20

5 6 2i

8 20

8 9 2 4 25

8 23

Cys

1o

IO

IO

IO

8

Glu Gly His lie I.eu

15 23 2 1o t9

18 23 2 9 19

17

15

22

22

22

22

1o i t 19

3 it 16-17

5 13 2 0 21

i ,ys

14

II

I I

6

7

4 7

2 6

~ 8

I 7

Met Phe

'2 6

2

[O

24 24 5 0 12 19 20 6

7

t 4

Pro

9

i3

~6

14

I l

12

Ser Thr Trp Tyr Val

28 23 8 4 23

22 23 8 3 25

24 20 8 5 25

28 J7 13 4 24 25

14 16 13 7 19

20 t7 11 6 2~

Biochim. Biopkys. Acta, I 7 5 (~969) 8 2 9 6

ORIGIN

TWO ANIONICPORCINE PANCREATIC CHYMOTRYPSINOGENS

95

Sephadex technique always gives reliable results provided that this condition is fulfilled. When filtrated through Sephadex G-Ioo in a buffer of high ionic strength, porcine chymotrypsinogen C emerged later than bovine and porcine chymotrypsinogens A and B, in spite of the fact that ultracentrifugation assays and the amino acid composition found by analysis indicated for this protein a significantly larger molecular weight. The origin of this discrepancy is unknown. Tile anfino acid compositions of the six chymotrypsinogens of bovine and porcine origin are given in Table I I I . For purpose of comparison, the molecular weights of the zymogens in this table are normalized to 26 ooo, a value very close to those calculated from the sequence for bovine chymotrypsinogens A (ref. 8) and B (ref. 9)- The values in Table I I I show that all zymogens belong to the same group as far as their overall amino acid composition is concerned. Their molecular weights are also of the same order. All of them are activated by trypsin through the cleavage of the first basic bond in the N-ternfinal sequence 2°. Half-cystine is invariably the N-ternfinal residue through which the short chain formed during activation remains attached to the rest of the molecule. Moreover, tile resulting enzymes are specific for apolar bonds (aromatic and sometimes aliphatic) and the catalytic sites of most of them are already known to be similar. However, as suggested by FOLK AND SCmRMER4, two sub-groups m a y be discerned, one composed of bovine chymotrypsinogens A and B and porcine chymotrypsinogen A, the other of bovine Fraction I I and porcine chymotrypsinogen C. I f the distance separating the composition of two proteins is expressed by the sum(s) of the individual differences at the level of each residue*, the results listed in Table I I I show that porcine chymotrypsinogen C resembles bovine Fraction I I (s value 27 32) more than chymotrypsinogen A of the same species (s value 54-6o). Moreover, the zymogens of the second sub-group appear to have a somewhat higher molecular weight and the enzymes to have a much higher activity toward leucine substrates. In fact, some variations of lesser magnitude have already been observed in the relative activities of the enzymes in the first sub-group towards aromatic and leucyl bonds. Some leueyl bonds that are poorly attacked by bovine chymotrypsin A are readily cleaved by bovine chynmtrypsin B and porcine chymotrypsin A (ref. 2o). Considering now the new anionic chymotrypsinogen B isolated from porcine pancreas, it is probably significant that, in spite of striking differences at the level of arginine, histidine, lysine and tryptophan, this zymogen resembles the first group (is value 3o 35 for the pair porcine B-porcine A) more than the second (s value 53 56 for the pair porcine B porcine C). Its molecular weight is also in the san-~e range as those for the zymogens of the first sub-group. Its specific ATEE-splitting activity is found approximately at the same level as for bovine chymotrypsinogen A (about 5oo), bovine ehymotrypsinogen B (about 4oo) and porcine chymotrypsinogen A (about 4oo). In contrast, the specific activity of porcine chymotrypsinogen C is definitely lower (about i8o) under the same conditions. In this respect, the new zymogen appears to originate from the same line as bovine chymotrypsinogens A and B and porcine chymotrypsinogen A. However, * This m o d e of e x p r e s s i o n is v e r y t e n t a t i v e since it does n o t t a k e i n t o a c c o u n t possible subs t i t u t i o n s of a s p a r t i c a n d g l u t a m i c acid residues by t he amides. Moreover, it gives t h e s a m e w e i g h t to a n y residue while some are e s p e c i a l l y i m p o r t a n t for t h e c o n f o r m a t i o n of t he mol e c ul e s and their catalytic activity.

Biochim. Biophvs. ,4cfa, I75 (~069) 82 9(7

96

I). GRATECOS el a[.

bovine chymotrypsinogens A and B are so chemically similar (one substitution only for 5 residuesS) that it is difficult to decide whether this zymogen is of the A or B type (s value 4o-42 for the pair porcine B-bovine A; 39-4o for the pair porcine B bovine B). The filiation of the previously purified porcine chymotrypsinogen A is also uncertain. It must be emphasized that the designation of both zymogens rests solely upon considerations of electric charges. Perhaps more precise information will be obtained when the sequences of all proteins are known. ACKNOWLEDGEMENTS

We are indebted to Mrs. F. C. STEVENS who participated in the initial stages of this work, to Mrs. A. GUIDONI who performed the amino acid analysis and to Miss G, BI~RENGIER for her skilful technical assistance. Financial help from Ddl6gation G6n6tale ~ la Recherche Scientifique et Technique (Convention No. 66.00.056) and National Institutes of Health (Grant AM 04642) is gratefully acknowledged. REFERENCES I J. BROWN, R. GREENSHIELDS, M. YAMASAKI AND H. NEURATH, Biochemistry, 2 (I963) 867 2 M. CHARLES, D. GRATECOS, M. ROVERY AND P. DESNUELLE, Biochim. Biophys. Acta, 14o (1967) 395. 3 M. CHARLES, Biochim. Biophys. Acla, 92 (1964) 319. 4 J- E. FOLK AND W. E. SCHIRMER, J. Biol. Chem., 240 (1965) 181. 5 0 . GuY, D. GRATECOS, M. ROVERY AND P. DESNUELLE, Biochim. Biophys. Acta, 115 (1966) 404 . 6 H. NEURATH AND C. W. SCHWERT, Chem. Rev., 46 (196o) 69. 7 J" E. FOLK AND J. A. GLADNER, J. Biol. Chem., 231 (1958) 379. 8 B. S. HARTLEY, Nature, 2Ol (1964) I284. 9 L. B. SMILLIE, A. FURKA, N. NAGABHUSHAN, ]4. J. STEVENSON AND L. O. })ARKES, Nature, 218 (1968) 344IO l). ]-t. SPACKMAN, VV. H. STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 119o. I I J. R. SPIES AND D. C. CHAMBERS, Anal. Chem., 21 (1949) 1249. i2 B. S. t-IARTLEY AND W. R. GRAY, Biochem. J., 89 (1963) 379. 13 F. R. BETTELHEIM AND H. NEURATH, J. Biol. Chem., 212 (1955) 241. 14 M. DELAAGE, Biochim. Biophys. Aeta, in the press. 15 J. J. GREENE, C. H. W. HIRS AND G. E. PALADE, J. Biol. Chem., 238 (1963) 2o54. 16 P. J. KELLER, E. COHEN AND H. NEURATH, J. Biol. Chem., 233 (1958) 344. I 7 A. BEN ABDELJLIL AND P. DESNUELLE, Biochim. Biophys. Acta, 81 (1964) 136. 18 J. P. REBOUD, G. MARCHIS-MOUREN, L. PASI~RO, A. COZZONE AND P. DESNUELLE, Biochim. Biophys. Acta, 117 (1966) 351. 19 L. SARDA, M. F. MAYLI~, J. ROGER AND t ~. DESNUELLE, Biochim. Biophys. Acta, 89 (1964) r83. 20 R. I'EANASKY, D. GRATECOS, J. BARATTI AND M. ROVERY, Biochim. Biophys. Acta, s u b m i t t e d for publication. 21 D. GRATECOS AND M. I~OVERY, Biochim. Biophys. ~4cta, 14o (1967) 41o.

Biochim. Biophys. Acta, 175 (1969) 82-96