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The basic trypsin inhibitor of bovine pancreas. III. A progress report on the tryptic peptides
Vol. 17, No. 6, 1964
BIOCHEMICAL
AND BIOPHYSICAL
THE BASIC TRYPSIN INHIBITOR
RESEARCH COMMUNICATIONS
OF BOVINE PANCREAS. III.
A
PROGRESSREPORT ON ME TRYPI'IC PEPTIDES* Beatrice
Kassell
and M. haskowski,
Sr.
Biochemical Laboratory for Cancer Research, Marquette School of Medicine, Hilwaukee, Wisconsin 53233
University
Received October 8, 1964 A derivative
of the basic trypsin
(1936) has been digested
all
present
in high yield
count for the 58 amino acids of the protein. a pentadecapeptide,
which contains
of Kunitz and Northrop
and the peptides
with trypsin,
Nine peptides,
analyzed.
inhibitor
all
isolated
in the digest,
The N-terminal
4 proline
residues
ac-
peptide
is
of the protein.
as ~811 as of some of the smaller
The sequence of this peptide,
and
ones,
has been determined. The preparation previously
(Kassell
of trypsin et al.,
inhibitor
to Crestfield tides
separated
analysis 1961)
--et al.
(RCM) derivative
It was digested
on Dowex 50-X2 (Margoliash
was carried
out as previously
using 24 or 72 hour hydrolysis.
terminal
analysis:
Conrat et sl., (1962),
(1963).
The S-S linkages
1963).
were reduced and the uarboxymethyl
w8re described of the inhibitor prepared
with trypsin
and smith,
described
according and the pep-
1962).
(Kassell
Amino acid
and Lsskowski,
Three methods Yere used for N-
a) the Sanger fluorodinitrobensene
1955), b) the subtractive
and c) following
hydrolyzed
and trypsin
method (Fraenkel-
Edm8n method of Konigsberg
(b) the phenylthiohydantoin
to free amino acids (Fraenkel-Conrat
(PTH) derivatives
et al.,
were
1955), which u8re
* This work was supported by grants from the National Institutes of Health (A-535) and the National Science Foundation (~-7581). The excellent work of Mrs. Milka Radicevic in the operation of the amino acid analyser 3s gratefully acknowl8dged. 792
c g.
Vol. 17, No. 6, 1964
6lOCiiEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
determined on the automatic amino acid analyzer. were identified
by hydrazinolysis
C-terminal amino acids
(Iiiu and Fraenkeldonrat,
1955).
A.rg-Pro-Asp-Phe-Cy8-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-QS-Iys (also someAla-Lys + Arg)
:12jAla-Lys-Arg Ileu-Ileu-Arg (3)
(4)
Ala-Arg fia,Av,Phe,Tyr2,Ws Ala,Asp,CyS,Glu,Meth,Ser,Arg Al.a,CyS2,Glu,Gly3,Leu,Phe,Thr,Tyr,Val,Arg b+,Phe,Lys (CySJW2,Th) fla
:56)) (7) (8) (9)
Figure 1.
The 9 main peptides of the tryptic
The three amino
acids at the N-terminus of the RCM-inhibitor
termined by method c above) are Arg-Pro-Asp. is alanine. of additional
digest.
Fig. 1 showsthe main tryptic peptides,
(de-
The C-terminal amino acid
peptides.
Small quantities
consisting of parts of these peptides, were
also obtained.
The sequences of peptides (3) end (4) are assumedfrom
the specificity
of trypsin.
and (2) will
The sequence determination of peptides (1)
be described in detail.
EFFLUENT,
ML
Figure 2. Chromatography of the products of partial~acid hydrolysis of peptide (1) on Douex l-X2. c01~mn95 x 1 a, 38O, load 78 mg, fractions 2 ml. The starting buffer was 250 ml of pyridine-N-ethylmorpholine-acetic acid, pB 9.3, in a constant volume gradient bottle; the gradient uas 03-2 M acetic acid (Schroeder et al., 1962). The peaks are ident3.fied in Fig. 3.
In peptide (1) srginine is the N-terminal. found by method (a). usine
is the C-terminal.
Partial
acid hydrolysis
793
(&hults
et al.,
Vol. 17, No. 6, 1964
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COi’AhWNlCATlONS
1962) was used to obtain smaller pieces. in 78 ml of 0.03 M HCl, previously evaczlated tubes, atlOs"
In addition
in Pig. 3. (Arg-Pro), peptide),
in 4 sealed,
The digest was lyophilised
in Fig. 2.
peak 1
acid) and peak 7 (the remsining 12-membered
breakdown of peptide 7 occurred, yielding
below #7 in Fig. 3.
and
The peptides isolated are given
to the 3 components expected, i.e.,
peak !5' (aspartio further
78 mg, was heated
deaerated with nitrogen,
for 10 hours.
chramatographed as indicated
Peptide (l),
These provide confirmation
the peptides
of the sequence, Peptide
Arg(Pro,Asp,Phe,CMCyS,Leu,Glu,Pro,Pro,Tyr,Thr,Gly,Pro,CMCyS)13rs 0.03
J
Arg-Prc (1 and la)
M HCl, lOso, 10 hrs
Phe-CM S-Leu-Glu-Pro(Pro,Tyr,Thr,Gly,Pro,CMCyS)Iys --Gc-++j Pro-Pro-Tyr-Thr-Gly-Pro-CMCyS-Iye ASP (5) +-3+-d++ ~o,Pro9Tyr,~,W (4)
(7)
(3)
(Pro,CMCyS)Vs (2) Phe,CMCyS,Leu,Glu (8)
Thr,Gb,Pro
CMCyS,Leu,Glu,Pro,F'ro,Tyr
(3A)
(C-l)
Horizontal. arrows Figure 3. Protocol for sequence determination. indicate stepwise degradation by Edman's method summarizedin Tables I (peak 3) and II (peak 7). 3A was derived by further
acid hydrolysis
of peptide 3 (heating in a boil-
ing water bath in 5.7 N HCl far &I minutes) and was the only peptide ison lated after g
a&,
this procedure in pure form by paper electrophoresis
1959).
Peptide Cl was isolated
from a chymotryptic
(Katz
digest to
be described elsewhere.
The small peaks not identified
on analysis to be either
free amino acids or mixtures present in quanti-
ties too amall to separate.
Peak 6 is impure starting
Peptides 3 and 7 were used for diaated by the arrows of Fig. 3. II respectively. off (last
in Fig. 2 proved
peptide.
the stepdse Edmandegradation,
in-
The results are shownin Tables I and
In each case, identification
column) agreed with the subtractive 794
of the P'IH compoundsplit method. As expected,
Vol. 17, No. 6, 1964
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PTfGthreonine and Pl!H-carboxymethylcysteine smino acids.
were not recovered as free
The composite results establish the sequence given in
Fig. 1 for peptide 1. TABJXI.
EdmanDegradation of Peak 3 Free Amino Acid from mi
Molar Ratio of Amino cids Remaining Step
cM-cp~
Gly
Ip
0.90
0.93
1.00
2.60
1.05
0.89
1.00
0.79
0.65
0.98
0.80
0.60
se-
11.93
0~58
0.66
PI-0
1.00
00.87
0.61
oe.54
Pi-0
0.97
1.00
1.12
0082
rl- 0
m
1.31
1.75
1.00
1.16
cltr
1.17
cl0.31
1.00
1.43
1.00
0.86
TABI Molar
S-W
II.
None GUY
!O tr
Pro
RdmanDegradation of Peak 7
Ratio of Amino Acids Remaining Thr he GF
IFree Amino eAcid from Pm
:kI-C@
Glu
l&l
0.91
1.00
.19
2.69
1.09
0.97
---
1.81
0.98
3.82
a
2.82
0.89
0.89
Phe
1.0 El
0.96
0.82
2.92 0.90
0.82
None
0.92
0.97
0.80
'2.80
0.87
0.89
r.eu
0.94
El.1
0058
'2.78
0.78
0.80
Glu
0.M
I2015 0.80
0.70
Pro
o.s1* *
1 Pro
Includes somecysteic
acid.
the sequence of peptide 2 of Pig. 1 uas established by the subtractive
Edmanmethod with the results
in Table III.
795
Vol. 17, No. 6, 1964
BlOCHEMlCAt
Subtractive
TABLE III.
AND BlOPHYSlCAt
Edman Degradation
Molar Ratio
SteP
1.00
1
piq
2
Discussion tryptic
minal
In contrast
digestion
(Kassell
at all
the linkages
digested
Arg-Pro linkage,
195h, Hirs t
G?s
&.,
ACTH (Bell,
participation
of proline
is not unique,
1963) and aatalase
inhibitor
1956), the RCM-derivative
1963).
It
to trypsin
1956), clupeine
hormone (Lee et al.,
residues
center"
was
(Bell, doubtful
the blocking
and RCM-inhibitors
(Hirs et al.,
tc
except the N-ter-
seems rather
account for
in a more complex "inhibiting
of the inhibitor,
collagens,
of native
1961).
of the
are inactive
sequences occur in many proteins,
stimulating
The concentration fourth
1.00
of the basic amino acids,
ribonuclease
1962) and melsnocyte
0.07 c-7
since oxidized
and Iys-Pro
1954),
1.11
and Laskowski,
Jones e al.,
center of trypsin,
and since &g-Pro
:::
to the resistance
that the sequence Arg-Pro can by itself active
hz
which is known to be resistant
1956,
2
1.01
0.09
ILI
of Peptids
of Amino Acids Remaining
Ala 0
RESEARCH COMMUNICATIONS
e.g. (Ando et -- al.,. However,
is not excluded.
found in the N-terminal
while unoommon in proteins
other than the
e*g. cytochrome C-551 (Gray and Hartley,
(Schroeder e al.,
1964)
contain
regions
even higher
in proline.
REFERENCES Ando, T., Iwai, K., I&ii, S.-I., Azegami, M. wd Nakahara, C., Biochem. Biophys. Acta g, 628 C1962). Crestfield, A. M., Moore, S. and Stein, W. H,, J. Biol. Ghem. 2& (1963)
l
Fraenkel-Conrat, H., Harris, J. I. and Levy, Methods of Biochemical An&&s, Vol. II, Inc., New York, 19.55, pb 359. Gray, W. R. and Hartley, B, Se, Bioohem. J,, Kassell, B. and Laskowski, M., Sr., J. Biol. Kassell, B. and Laskowski, M., Sr., J. Biol. 796
622
A. L., in Il. Glick (Editor), Interscience Publishers, 89 379 (1963); I!&rn. 219, 203 (1956). Chem. B, 1996 (1.961.).
Vol. 17, No. 6, 1964
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Kassell, B., Radicevic, M., Berlow, S., Peanasky, H. J. and Laskowski, M., Sr., J. Biol. Chem.238, 3274 (1963). Katz, A. M., Breyer, W. J. and Anfinsen, C. B., J. Biol. Chem. 234,
2897 (1959).
Konigsberg, W. and Hill, R. J., J. Biol. Chem. 237, 2547 (1962). Kunitz, M. and Northrop, J. H., J. Gen. Physiol. l9, 991 (1936). Lee, T. H., Lerner, A. B. and Buettner-Janusch, V., J. Biol. Chem. 236,
1390 (1961).
Margoliash, E.. and &nith, E.L., J. Biol. Chem. 237, 2151 (1962). Niu, C.-I. and Fraenkel-Conrat, H., J. Am. Chem.oc. 77, 5882 (1955). Schroeder, W. A., Jones, R. T., Cormick, J. and McCalla, K., Anal. Chem.
34, 1570 (1962).
Schroaer, W. A., Shelton, R. J., Shelton, J. B. and Olson, B. M., Biochim. Biophys. Acta 89, 47 (1964). Schultz, J., Allison, H. and Grice, M., Biochemistry I., 694 (1962).
797
BIOCHEMICAL
AND BIOPHYSICAL
THE BASIC TRYPSIN INHIBITOR
RESEARCH COMMUNICATIONS
OF BOVINE PANCREAS. III.
A
PROGRESSREPORT ON ME TRYPI'IC PEPTIDES* Beatrice
Kassell
and M. haskowski,
Sr.
Biochemical Laboratory for Cancer Research, Marquette School of Medicine, Hilwaukee, Wisconsin 53233
University
Received October 8, 1964 A derivative
of the basic trypsin
(1936) has been digested
all
present
in high yield
count for the 58 amino acids of the protein. a pentadecapeptide,
which contains
of Kunitz and Northrop
and the peptides
with trypsin,
Nine peptides,
analyzed.
inhibitor
all
isolated
in the digest,
The N-terminal
4 proline
residues
ac-
peptide
is
of the protein.
as ~811 as of some of the smaller
The sequence of this peptide,
and
ones,
has been determined. The preparation previously
(Kassell
of trypsin et al.,
inhibitor
to Crestfield tides
separated
analysis 1961)
--et al.
(RCM) derivative
It was digested
on Dowex 50-X2 (Margoliash
was carried
out as previously
using 24 or 72 hour hydrolysis.
terminal
analysis:
Conrat et sl., (1962),
(1963).
The S-S linkages
1963).
were reduced and the uarboxymethyl
w8re described of the inhibitor prepared
with trypsin
and smith,
described
according and the pep-
1962).
(Kassell
Amino acid
and Lsskowski,
Three methods Yere used for N-
a) the Sanger fluorodinitrobensene
1955), b) the subtractive
and c) following
hydrolyzed
and trypsin
method (Fraenkel-
Edm8n method of Konigsberg
(b) the phenylthiohydantoin
to free amino acids (Fraenkel-Conrat
(PTH) derivatives
et al.,
were
1955), which u8re
* This work was supported by grants from the National Institutes of Health (A-535) and the National Science Foundation (~-7581). The excellent work of Mrs. Milka Radicevic in the operation of the amino acid analyser 3s gratefully acknowl8dged. 792
c g.
Vol. 17, No. 6, 1964
6lOCiiEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
determined on the automatic amino acid analyzer. were identified
by hydrazinolysis
C-terminal amino acids
(Iiiu and Fraenkeldonrat,
1955).
A.rg-Pro-Asp-Phe-Cy8-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-QS-Iys (also someAla-Lys + Arg)
:12jAla-Lys-Arg Ileu-Ileu-Arg (3)
(4)
Ala-Arg fia,Av,Phe,Tyr2,Ws Ala,Asp,CyS,Glu,Meth,Ser,Arg Al.a,CyS2,Glu,Gly3,Leu,Phe,Thr,Tyr,Val,Arg b+,Phe,Lys (CySJW2,Th) fla
:56)) (7) (8) (9)
Figure 1.
The 9 main peptides of the tryptic
The three amino
acids at the N-terminus of the RCM-inhibitor
termined by method c above) are Arg-Pro-Asp. is alanine. of additional
digest.
Fig. 1 showsthe main tryptic peptides,
(de-
The C-terminal amino acid
peptides.
Small quantities
consisting of parts of these peptides, were
also obtained.
The sequences of peptides (3) end (4) are assumedfrom
the specificity
of trypsin.
and (2) will
The sequence determination of peptides (1)
be described in detail.
EFFLUENT,
ML
Figure 2. Chromatography of the products of partial~acid hydrolysis of peptide (1) on Douex l-X2. c01~mn95 x 1 a, 38O, load 78 mg, fractions 2 ml. The starting buffer was 250 ml of pyridine-N-ethylmorpholine-acetic acid, pB 9.3, in a constant volume gradient bottle; the gradient uas 03-2 M acetic acid (Schroeder et al., 1962). The peaks are ident3.fied in Fig. 3.
In peptide (1) srginine is the N-terminal. found by method (a). usine
is the C-terminal.
Partial
acid hydrolysis
793
(&hults
et al.,
Vol. 17, No. 6, 1964
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COi’AhWNlCATlONS
1962) was used to obtain smaller pieces. in 78 ml of 0.03 M HCl, previously evaczlated tubes, atlOs"
In addition
in Pig. 3. (Arg-Pro), peptide),
in 4 sealed,
The digest was lyophilised
in Fig. 2.
peak 1
acid) and peak 7 (the remsining 12-membered
breakdown of peptide 7 occurred, yielding
below #7 in Fig. 3.
and
The peptides isolated are given
to the 3 components expected, i.e.,
peak !5' (aspartio further
78 mg, was heated
deaerated with nitrogen,
for 10 hours.
chramatographed as indicated
Peptide (l),
These provide confirmation
the peptides
of the sequence, Peptide
Arg(Pro,Asp,Phe,CMCyS,Leu,Glu,Pro,Pro,Tyr,Thr,Gly,Pro,CMCyS)13rs 0.03
J
Arg-Prc (1 and la)
M HCl, lOso, 10 hrs
Phe-CM S-Leu-Glu-Pro(Pro,Tyr,Thr,Gly,Pro,CMCyS)Iys --Gc-++j Pro-Pro-Tyr-Thr-Gly-Pro-CMCyS-Iye ASP (5) +-3+-d++ ~o,Pro9Tyr,~,W (4)
(7)
(3)
(Pro,CMCyS)Vs (2) Phe,CMCyS,Leu,Glu (8)
Thr,Gb,Pro
CMCyS,Leu,Glu,Pro,F'ro,Tyr
(3A)
(C-l)
Horizontal. arrows Figure 3. Protocol for sequence determination. indicate stepwise degradation by Edman's method summarizedin Tables I (peak 3) and II (peak 7). 3A was derived by further
acid hydrolysis
of peptide 3 (heating in a boil-
ing water bath in 5.7 N HCl far &I minutes) and was the only peptide ison lated after g
a&,
this procedure in pure form by paper electrophoresis
1959).
Peptide Cl was isolated
from a chymotryptic
(Katz
digest to
be described elsewhere.
The small peaks not identified
on analysis to be either
free amino acids or mixtures present in quanti-
ties too amall to separate.
Peak 6 is impure starting
Peptides 3 and 7 were used for diaated by the arrows of Fig. 3. II respectively. off (last
in Fig. 2 proved
peptide.
the stepdse Edmandegradation,
in-
The results are shownin Tables I and
In each case, identification
column) agreed with the subtractive 794
of the P'IH compoundsplit method. As expected,
Vol. 17, No. 6, 1964
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PTfGthreonine and Pl!H-carboxymethylcysteine smino acids.
were not recovered as free
The composite results establish the sequence given in
Fig. 1 for peptide 1. TABJXI.
EdmanDegradation of Peak 3 Free Amino Acid from mi
Molar Ratio of Amino cids Remaining Step
cM-cp~
Gly
Ip
0.90
0.93
1.00
2.60
1.05
0.89
1.00
0.79
0.65
0.98
0.80
0.60
se-
11.93
0~58
0.66
PI-0
1.00
00.87
0.61
oe.54
Pi-0
0.97
1.00
1.12
0082
rl- 0
m
1.31
1.75
1.00
1.16
cltr
1.17
cl0.31
1.00
1.43
1.00
0.86
TABI Molar
S-W
II.
None GUY
!O tr
Pro
RdmanDegradation of Peak 7
Ratio of Amino Acids Remaining Thr he GF
IFree Amino eAcid from Pm
:kI-C@
Glu
l&l
0.91
1.00
.19
2.69
1.09
0.97
---
1.81
0.98
3.82
a
2.82
0.89
0.89
Phe
1.0 El
0.96
0.82
2.92 0.90
0.82
None
0.92
0.97
0.80
'2.80
0.87
0.89
r.eu
0.94
El.1
0058
'2.78
0.78
0.80
Glu
0.M
I2015 0.80
0.70
Pro
o.s1* *
1 Pro
Includes somecysteic
acid.
the sequence of peptide 2 of Pig. 1 uas established by the subtractive
Edmanmethod with the results
in Table III.
795
Vol. 17, No. 6, 1964
BlOCHEMlCAt
Subtractive
TABLE III.
AND BlOPHYSlCAt
Edman Degradation
Molar Ratio
SteP
1.00
1
piq
2
Discussion tryptic
minal
In contrast
digestion
(Kassell
at all
the linkages
digested
Arg-Pro linkage,
195h, Hirs t
G?s
&.,
ACTH (Bell,
participation
of proline
is not unique,
1963) and aatalase
inhibitor
1956), the RCM-derivative
1963).
It
to trypsin
1956), clupeine
hormone (Lee et al.,
residues
center"
was
(Bell, doubtful
the blocking
and RCM-inhibitors
(Hirs et al.,
tc
except the N-ter-
seems rather
account for
in a more complex "inhibiting
of the inhibitor,
collagens,
of native
1961).
of the
are inactive
sequences occur in many proteins,
stimulating
The concentration fourth
1.00
of the basic amino acids,
ribonuclease
1962) and melsnocyte
0.07 c-7
since oxidized
and Iys-Pro
1954),
1.11
and Laskowski,
Jones e al.,
center of trypsin,
and since &g-Pro
:::
to the resistance
that the sequence Arg-Pro can by itself active
hz
which is known to be resistant
1956,
2
1.01
0.09
ILI
of Peptids
of Amino Acids Remaining
Ala 0
RESEARCH COMMUNICATIONS
e.g. (Ando et -- al.,. However,
is not excluded.
found in the N-terminal
while unoommon in proteins
other than the
e*g. cytochrome C-551 (Gray and Hartley,
(Schroeder e al.,
1964)
contain
regions
even higher
in proline.
REFERENCES Ando, T., Iwai, K., I&ii, S.-I., Azegami, M. wd Nakahara, C., Biochem. Biophys. Acta g, 628 C1962). Crestfield, A. M., Moore, S. and Stein, W. H,, J. Biol. Ghem. 2& (1963)
l
Fraenkel-Conrat, H., Harris, J. I. and Levy, Methods of Biochemical An&&s, Vol. II, Inc., New York, 19.55, pb 359. Gray, W. R. and Hartley, B, Se, Bioohem. J,, Kassell, B. and Laskowski, M., Sr., J. Biol. Kassell, B. and Laskowski, M., Sr., J. Biol. 796
622
A. L., in Il. Glick (Editor), Interscience Publishers, 89 379 (1963); I!&rn. 219, 203 (1956). Chem. B, 1996 (1.961.).
Vol. 17, No. 6, 1964
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Kassell, B., Radicevic, M., Berlow, S., Peanasky, H. J. and Laskowski, M., Sr., J. Biol. Chem.238, 3274 (1963). Katz, A. M., Breyer, W. J. and Anfinsen, C. B., J. Biol. Chem. 234,
2897 (1959).
Konigsberg, W. and Hill, R. J., J. Biol. Chem. 237, 2547 (1962). Kunitz, M. and Northrop, J. H., J. Gen. Physiol. l9, 991 (1936). Lee, T. H., Lerner, A. B. and Buettner-Janusch, V., J. Biol. Chem. 236,
1390 (1961).
Margoliash, E.. and &nith, E.L., J. Biol. Chem. 237, 2151 (1962). Niu, C.-I. and Fraenkel-Conrat, H., J. Am. Chem.oc. 77, 5882 (1955). Schroeder, W. A., Jones, R. T., Cormick, J. and McCalla, K., Anal. Chem.
34, 1570 (1962).
Schroaer, W. A., Shelton, R. J., Shelton, J. B. and Olson, B. M., Biochim. Biophys. Acta 89, 47 (1964). Schultz, J., Allison, H. and Grice, M., Biochemistry I., 694 (1962).
797