Hydrolytic cleavage of pyroglutamyl-peptide Bond. V. Selective removal of pyroglutamic acid from biologically active pyroglutamylpeptides in high concentrations of aqueous methanesulfonic acid

Article abstract of DOI:10.1248/cpb.54.827

Application of aqueous methanesulfonic acid (MSA) for selective chemical removal of pyroglutamic acid (pGlu) residue from five biologically active pyroglutamyl-peptides (pGlu-X-peptides, X=amino acid residue at position 2) was examined. Gonadotropin releasing hormone (Gn-RH), dog neuromedin U-8 (d-NMU-8), physalaemin (PH), a bradykinin potentiating peptide (BPP-5a) and neurotensin (NT) as pGlu-X-peptides were incubated in either 70% or 90% aqueous MSA at 25°C. HPLC analysis of the incubation solutions showed that the main decomposition product was H-X-peptide derived from each pGlu-X-peptide by the removal of pGlu. The results revealed that the pGlu-X peptide bond had higher susceptibility than various internal amide bonds in the five peptides examined, including the Trp-Ser bond in Gn-RH, the C-terminal Asn-NH₂ in d-NMU-8, and the Asp-Pro bond in PH, whose acid susceptibility is well known. Thus, mild hydrolysis with high concentrations of aqueous MSA may be applicable to chemically selective removal of pGlu from pGlu-X-peptides for structural examinations.

Full text of DOI:10.1248/cpb.54.827

June 2006
Chem. Pharm. Bull. 54(6) 827—831 (2006)
827
Hydrolytic Cleavage of Pyroglutamyl-peptide Bond. V. Selective Removal
of Pyroglutamic Acid from Biologically Active Pyroglutamylpeptides in
High Concentrations of Aqueous Methanesulfonic Acid
Junko KOBAYASHI, Kazuhiro OHKI, Keiko OKIMURA, Tadashi HASHIMOTO, and Naoki SAKURA*
Faculty of Pharmaceutical Sciences, Hokuriku University; Kanagawa-machi, Kanazawa 920–1181, Japan.
Received December 26, 2005; accepted March 10, 2006
Application of aqueous methanesulfonic acid (MSA) for selective chemical removal of pyroglutamic acid
(pGlu) residue from five biologically active pyroglutamyl-peptides (pGlu-X-peptides, Xꢀamino acid residue at
position 2) was examined. Gonadotropin releasing hormone (Gn-RH), dog neuromedin U-8 (d-NMU-8),
physalaemin (PH), a bradykinin potentiating peptide (BPP-5a) and neurotensin (NT) as pGlu-X-peptides were
incubated in either 70% or 90% aqueous MSA at 25 °C. HPLC analysis of the incubation solutions showed that
the main decomposition product was H-X-peptide derived from each pGlu-X-peptide by the removal of pGlu.
The results revealed that the pGlu-X peptide bond had higher susceptibility than various internal amide bonds
in the five peptides examined, including the Trp-Ser bond in Gn-RH, the C-terminal Asn-NH in d-NMU-8, and
2
the Asp-Pro bond in PH, whose acid susceptibility is well known. Thus, mild hydrolysis with high concentrations
of aqueous MSA may be applicable to chemically selective removal of pGlu from pGlu-X-peptides for structural
examinations.
Key words pyroglutamylpeptide; chemical cleavage; methanesulfonic acid; selectivity; hydrolysis
Biologically active peptides bearing pyroglutamic acid Amino acid analysis was conducted on a 7300 Model amino acid analyzer
system (Beckman Instruments Ltd., Fullerton, CA, U.S.A.). The hydrolysis
of a synthetic peptide was performed by 6 M HCl vapor at 130 °C for 3 h.
Fast-atom bombardment mass spectra (FAB-MS) were obtained on a JMS-
DX300 mass spectrometer (JEOL Ltd., Tokyo, Japan). Optical rotations of
residue (pGlu) at the N-terminal are widely known. The re-
moval of pGlu residue from a pGlu-X-peptide (X, amino acid
residue at position 2) is required for the primary structure de-
termination by Edman degradation. Our previous studies in- the peptides were measured with a DIP-370 digital polarimeter (Nippon
dicated that pGlu-X peptide bond is highly sensitive to mild Bunko Co., Ltd., Tokyo, Japan). HP-TLC analysis was carried out on pre-
coated silica gel plates (Kieselgel 60; Merck, Darmstadt, Germany).
Peptides Syntheses of Gn-RH (1), PH (3), BPP-5a (4) and NT (5) (Fig.
acidic conditions, generating not only the ring opened prod-
uct (H-Glu-X-peptide) at the pyrrolidone moiety of pGlu, but
1) and various fragment peptides related to these biologically active peptides
also the cleavage product (H-X-peptide) at pGlu-X link-
(
Figs. 2, 5, 6, 8, 9) were carried out according to a method previously re-
1,2)
11)
age. High selectivity to remove pGlu-OH from synthetic ported for d-NMU-8 (2). Protected peptide resins were constructed by Boc
strategy either on a benzhydrylamine resin or a chloromethylated poly-
model pGlu-X-peptides was attained in concentrated hy-
3)
styrene resin (Peptide Institute Inc., Osaka, Japan) using a peptide synthe-
sizer (ABI 433A; Applied Biosystems, Foster City, CA, U.S.A.). Side chain
protected Boc-amino acid derivatives (Watanabe Chemical Industries Ltd.,
Hiroshima, Japan) were Tyr(BrZ), Arg(Tos), Asp(OcHex), Lys(Cl-Z),
drochloric acid and in 70% aqueous methanesulfonic acid
4,5)
(
MSA). The aim of this study was to compare the selectiv-
ity of the cleavage reaction at pGlu-X bond in aqueous MSA
with various internal peptide bonds in five biologically active Ser(Bzl), His(Bom) and Glu(OBzl). The protected peptide resins were
treated with anhydrous liquid hydrogen fluoride (HF) containing 10%
pGlu-X-peptides, containing well-known acid sensitive
anisole under ice cooling for 45 min in a Teflon HF apparatus (Peptide Insti-
amide bonds. The sequence of these peptides, namely, go-
tute Inc., Osaka, Japan). After evaporation of HF in vacuo, the residual pep-
6)
nadotropin-releasing hormone (Gn-RH; 1), dog neuromedin
tides were purified by HPLC, using a column of YMC-pack ODS-AM S-5
7)
8)
U-8 (d-NMU-8; 2), physalaemin (PH; 3), a bradykinin po- 120 Å (20ꢀ150 mm) with 0.1% TFA–acetonitrile (MeCN) solvent system.
9)
10)
tentiating peptide (BPP-5a; 4), and neurotensin (NT; 5),
Finally, the peptides were gel-filtered on a column of Toyopearl HW-40
(
5
super fine) (1.5ꢀ47 cm, Tosoh Co., Tokyo, Japan) with 25% MeCN in
ꢀ10 M hydrochloric acid as an eluent; and lyophilized. The amino acid
are shown in Fig. 1.
ꢁ3
analysis data of various synthetic peptides are shown in Table 1, and their
FAB-MS analysis data and characteristics are in Table 2.
Experimental
General and Apparatus HPLC analysis was performed on a module
consisted of a 7125 injector (Rheodyne Inc., U.S.A.), a 616 pump, a 600 s
controller, a 486 tunable absorbance detector, a 717 plus autosampler, and an
SDM solvent degas module (all from Waters Corp., Milford, MA, U.S.A.).
Cleavage Reaction of Peptides in Aqueous MSA Solutions of the bio-
ꢁ3
logically active peptides were prepared at a concentration of 10 M in either
0% or 90% methanesulfonic acid (MSA) under ice cooling. To examine the
7
Fig. 1. Biologically Active Pyroglutamyl-peptides (pGlu-X-Peptides)
∗
To whom correspondence should be addressed. e-mail: n-sakura@hokuriku-u.ac.jp
© 2006 Pharmaceutical Society of Japan

8
28
Vol. 54, No. 6
cleavage reactions, these peptide solutions were divided into ten aliquots
(100 ml each) in polypropylene tubes (2 ml), which were tightly capped and
kept at 25 °C in a thermostatically regulated apparatus. Samples were re-
moved from the apparatus at an appropriate time (shown in Figs. 2—9), neu-
tralized under cooling and kept in a freezer at ꢁ40 °C until analysis.
Identification of Peptide Fragments Produced Incubation solutions of
biologically active peptides (3—5 mg) kept at 25 °C in aqueous MSA solu-
tions (3—5 ml) were subjected to RP-HPLC employing a YMC-pack ODS-
AM (1ꢀ25 cm) column. Materials corresponding to each peak were col-
lected and lyophilized. Structures of the isolated peptide fragments were de-
duced by amino acid analysis and FAB-MS, and then confirmed by co-elu-
TM
tion with authentic synthetic peptide on analytical HPLC using a Puresil
C18 (4.6ꢀ250 mm) column.
HPLC Analysis of Peptides To determine the amounts of the remain-
ing starting materials and the cleavage products, aliquots (15 ml) of each of
the incubation solutions in aqueous MSA were subjected to RP-HPLC
analysis. The peak areas of the starting materials and their hydrolysates were
compared with those of synthetic peptide samples. HPLC analysis was per-
TM
formed using a Puresil C18 (4.6ꢀ250 mm) column, a gradient elution with
varying concentrations of MeCN [16 to 24% for Gn-RH (1), BPP-5a (4),
and NT (5), 16 to 28% for d-NMU-8 (2), 20 to 25.6% for PH (3)] in 0.1%
TFA for 40 min at a flow rate of 1 ml/min with detection at a wavelength of
210 nm. The HPLC analysis of the acid hydrolysates was repeated 4—6
times. The average values varied in the range of ꢂ2.5 to ꢂ5.0%.
Results and Discussion
Cleavage Reaction of Gn-RH (1) Gn-RH (1) degraded
rapidly in 70% aqueous MSA at 25 °C giving various decom-
posed products. Thus, the decomposition reactions of Gn-RH
(1) and its hydrolysates were examined in various concentra-
tion of MSA (10—90%) at 25 °C for 4 h. As shown in Fig. 2,
the increase of MSA concentration promoted the degradation
of Gn-RH (1). In 30% MSA, deamidation of the C-terminal
Gly-NH occurred predominantly, to produce Gn-RH-OH
2
(
1c), the amount of which decreased greatly in higher con-
3
4
centrations of MSA. In 70% MSA, Trp -Ser peptide bond
cleaved mainly, to produce Gn-RH (4—10)-NH (1e) and its
2
counterpart Gn-RH (1—3)-OH (1d). Decomposition reaction
of Gn-RH (1) was promoted in higher concentration of MSA,
4
but the cleavage at the N-terminal of Ser was suppressed in
9
0% MSA. The production of Gn-RH (2—10)-NH (1a)
2
markedly increased in 90% MSA. HPLC profiles of the incu-
bation mixtures of Gn-RH (1) in 70% and 90% MSA are
shown in Figs. 3A and B, respectively. As seen from the time
courses of Gn-RH (1) and its hydrolysis products during in-
cubation in 90% MSA at 25 °C (Fig. 4), pGlu-His peptide
bond predominantly cleaved to yield Gn-RH (2—10)-NH2
(1a) as a major product in 3 h, while Gn-RH (1—3)-OH (1d)
and Gn-RH (4—10)-NH (1e) were the minor products.
2
Reaction of d-NMU-8 (2) Decomposition of d-NMU-8
(
2) in 90% MSA at 25 °C yielded H-Phe-Leu-Phe-Arg-Pro-
12)
Arg-Asn-NH2 [NMU-8 (2—8)-NH (2a)]
product, and [Glu ]-NMU-8 (2b), d-NMU-8-OH (2c),
as the main
2
1
11)
13)
8
13)
[Asp ]-d-NMU-8-OH (2d), and d-NMU-8 (2—8)-OH (2e)
as minor products (Fig. 5B). However, in 70% MSA, the
deamidation products (2c—e) at the C-terminal Asn-NH of
2
d-NMU-8 (2) increased greatly, and the main decomposition
product was d-NMU-8-OH (2c) (Fig. 5A). The decomposi-
tion reaction of d-NMU-8 (2) proceeded much faster in 70%
MSA compared to 90% MSA.
Reaction of PH (3) Incubation of PH (3) in 70% MSA
at 25 °C for 12 h gave numerous peaks on HPLC chro-
matogram (Fig. 6), however, the main product was PH (2—
3
4
1
1)-NH (3a). Cleavage at Asp -Pro peptide bond to yield
2
PH (4—11)-NH (3f) occurred to a less extent than at pGlu-
2

June 2006
829
Table 2. FAB-MS Analysis and Characteristics of Synthetic Peptides
2
4
b)
FAB-MS
Found
[a]
HP-TLC
D
a)
Peptides
Formula
(cꢃ0.5)
(50% AcOH)
ꢄ
1
2
[MꢄH]
Rf
Rf
Gn-RH (2—10)-NH (1a)
C H N O
1071
1200
1183
453
748
949
1154
1283
1266
1284
1155
968
501
630
1561.9
1690.9
1673.9
1562.9
ꢁ25.2°
ꢁ28.4°
ꢁ40.8°
0.40
0.32
0.34
0.38
0.43
0.33
0.36
0.31
0.36
0.26
0.32
0.39
0.26
0.20
0.37
0.30
0.36
0.33
0.43
0.29
0.44
0.51
0.44
0.46
0.49
0.47
0.48
0.44
0.46
0.50
0.31
0.33
0.16
0.17
0.44
0.22
2
50 70 16 11
1
[Glu ]-Gn-RH (1b)
C H N O
5
5
77 17 14
Gn-RH-OH (1c)
Gn-RH (1—3)-OH (1d)
C H N O
55 74 16 14
c)
C H N O
ꢁ4.8°
2
2
24
6
5
Gn-RH (4—10)-NH (1e)
C H N O
ꢁ40.8°
ꢁ40.8°
2
33 53 11
9
d-NMU-8 (2—8)-OH (2e)
C H N O
4
5
68 14
9
c)
PH (2—11)-NH (3a)
C H N O S
ꢁ24.0°
2
53 79 13 14
1
c)
[
Glu ]-PH (3b)
C H N O S
ꢁ23.2°
5
8
86 14 17
PH-OH (3c)
C H N O S
ꢁ69.2°
ꢁ61.2°
ꢁ59.6°
5
8
83 13 17
1
[
Glu ]-PH-OH (3d)
C H N O S
58 85 13 18
PH (2—11)-OH (3e)
C H N O S
53 78 12 15
c)
PH (4—11)-NH (3f)
C H N O S
ꢁ14.8°
2
46 69 11 10
BPP-5a (2—5) (4a)
C H N O
ꢁ27.6°
ꢁ44.8°
ꢁ55.0°
2
5
36
6
5
1
[
Glu ]-BPP-5a (4b)
C H N O
3
0
43
7
8
c)
NT (2—13) (5a)
C H N O
7
3
116 20 18
1
[
[
[
Glu ]-NT (5b)
C H N O
123 21 21
ꢁ69.5°
ꢁ79.3°
ꢁ69.9°
7
8
5
Asp ]-NT (5c)
C H N O
78 120 20 21
5
Asp ]-NT (2—13) (5d)
C H N O
73 115 19 19
1
2
a) The structures of peptides are shown in the figure captions of Figs. 2, 5, 6, 8 and 9. b) Rf , n-BuOH : pyridine : AcOH : H O (30 : 20 : 6 : 24); Rf , n-BuOH : AcOEt :
2
AcOH : H O (1 : 1 : 1 : 1). c) cꢃ0.2.
2
Fig. 4. Time Courses for Gn-RH and Its Hydrolysates during Incubation
in 90% MSA at 25 °C
Fig. 2. Relationships between the Concentration of MSA and Gn-RH and
Its Hydrolysates after Incubation at 25 °C for 4 h
Fig. 3. HPLC Profiles for Gn-RH and Its Hydrolysates after Incubation in Fig. 5. Time Courses for d-NMU-8 and Its Hydrolysates during Incuba-
7
0% (A) or 90% (B) MSA at 25 °C for 4 or 3 h
tion in 70% (A) and 90% (B) MSA at 25 °C
TM
HPLC conditions: column, Puresil C18 (4.6ꢀ250 mm); elution, linear 40 min gra-
dient elution from 16 to 24% MeCN in 0.1% TFA; flow, 0.8 ml/min; detection, 210 nm.
Retention times (t , min) were shown in parenthesis.
R

8
30
Vol. 54, No. 6
Ala bond, however, the production of PH (4—11)-NH (3f) the hydrolysis of the other internal peptide bonds in Gn-RH
2
in 70% MSA was slightly higher than in 90% MSA (Figs. (1) were suppressed. However, in 70% MSA, the cleavage of
5
3
4
7
A, B). The deamidation at the side chain of Asn and at the an internal peptide bond of Trp -Ser , was the predominant
C-terminal Met-NH was insignificant. reaction. The cleavage of the peptide bond at the N-terminal
2
Reaction of BPP-5a(4) HPLC profile of the incubation of Ser was possibly via N-O rearrangement reaction followed
mixture of BPP-5a (4) in 70% MSA at 25 °C and the time by the hydrolysis of the ester bond as seen in concentrated
14)
courses of BPP-5a (4) and its hydrolysates are shown in Figs. hydrochloric acid. In 90% MSA, d-NMU-8 (2) yielded the
A and B, respectively. After 2 d, 24% BPP-5a (4) remained X-peptide [d-NMU-8 (2—8)-NH (2a)] as the main product.
8
2
in the incubate, and the yield of BPP-5a (2—5) (4a) was While, in 70% MSA, the greater decomposition of the C-ter-
1
8
5
6%, the ring-opened product [Glu ]-BPP-5a (4b) was less minal Asn -NH took place compared to the cleavage of
2
1
2
than 1%, and the other minor products were not determined.
pGlu -Phe bond. Concerning the decomposition of peptides
Reaction of NT (5) Incubation of NT (5) in 70% MSA bearing C-terminal Asn-NH , we recently reported the stabil-
2
at 25 °C for 24 h produced complex degradation mixture as ity of porcine neuromedin U-8 in detail, where it was shown
seen from the HPLC chromatogram (Fig. 9A). Main peak in that the Asn-NH portion was hydrolyzed to produce pep-
2
the chromatogram corresponded to NT (2—13) (5a). Four tides bearing Asn-OH, Asp-NH and Asp-OH in dilute acidic
2
15)
other peaks were also identified and the remaining small solution. The hydrolysis experiments of Gn-RH (1) in 10—
peaks were not determined. The time courses of the hydroly- 90% aqueous MSA (Fig. 3) showed that the deamidation re-
sis products are shown in Fig. 9B.
action at C-terminal amide portion was enhanced in lower
Selective cleavage of pGlu-X peptide bond was performed concentration of aqueous MSA. The reaction of PH (3)
in either 70% or 90% MSA at 25 °C for all pGlu-X-peptides yielded the X-peptide [PH (2—11)-NH (3a)] as a main
2
1
2
examined in this study. The reaction of Gn-RH (1) yielded product, demonstrating the cleavage of pGlu -Ala peptide
more than 60% of the X-peptide [Gn-RH (2—10)-NH (1a)] bond occurred to a greater extent than the internal peptide
2
3
4
after 3 h in 90% MSA at 25 °C. The cleavage reaction at bond of Asp -Pro , which has previously been reported to be
pGlu -His peptide bond proceeded so rapidly that the other one of the most susceptible bonds to acidic hydrolysis.
reactions such as the ring open reaction at pyrrolidone moi- Treatment of BPP-5a (4) with 70% MSA yielded H-Lys -
ety of pGlu , the deamidation of C-terminal Gly -NH and Trp-Ala-Pro -OH (4a), indicating that the cleavage occurred
1
2
16—18)
2
1
10
5
2
1
2
almost exclusive at pGlu -Lys peptide bond in 70% MSA.
NT (5) was decomposed by either a non-specific hydrolysis
Fig. 6. HPLC Profiles for PH and Its Hydrolysates after Incubation in
0% MSA at 25 °C for 0.5 d
7
Fig. 8. HPLC Profiles (A) and Time Courses (B) for BPP-5a and Its Hy-
drolysates during Incubation in 70% MSA at 25 °C
TM
HPLC conditions: column, Puresil C18 (4.6ꢀ250 mm); elution, linear 40 min gra-
dient elution from 20 to 25.6% MeCN in 0.1% TFA; flow, 1.0 ml/min; detection,
10 nm.
TM
HPLC conditions: column, Puresil C18 (4.6ꢀ250 mm); elution, linear 40 min gra-
dient elution from 16 to 24% MeCN in 0.1% TFA; flow, 1.0 ml/min; detection, 210 nm.
2
Fig. 7. Time Courses for PH and Its Hydrolysates during Incubation in 70% (A) and 90% (B) MSA at 25 °C

June 2006
831
Fig. 9. HPLC Profiles (A) and Time Courses (B) for NT and Its Hydrolysates during Incubation in 70% MSA at 25 °C
TM
HPLC conditions: column, Puresil C18 (4.6ꢀ250 mm); elution, linear 40 min gradient elution from 16 to 24% MeCN in 0.1% TFA; flow, 1.0 ml/min; detection, 210 nm.
References
of the internal peptide bonds or the deamidation at the side
chain of Asn at the position 5 in 70% MSA, resulting in the
appearance of numerous peaks on HPLC chromatogram (Fig.
1
)
Hashimoto T., Ohki K., Sakura N., Chem. Pharm. Bull., 43, 2068—
074 (1995).
Saito S., Ohki K., Sakura N., Hashimoto T., Biol. Pharm. Bull., 19,
68—770 (1996).
3) Hashimoto T., Saito S., Ohki K., Sakura N., Chem. Pharm. Bull., 44,
77—879 (1996).
Hashimoto T., Ohki K., Sakura N., Chem. Pharm. Bull., 44, 2033—
036 (1996).
2
2)
9A), with the peak of NT (2—13) (5a) decidedly the largest.
7
When the hydrolysis was carried out in 90% MSA, the un-
known peaks eluted earlier (tR ꢅ35 min) on HPLC chro-
matogram became larger than peak a (data not shown).
8
4
)
2
The half-life period (t ) of Gn-RH (1) in 90% MSA at
1/2
5) Ohki K., Sakura N., Hashimoto T., Chem. Pharm. Bull., 45 194—197
(1997).
25 °C was 1.4 h (Fig. 4). Previously we reported a highly se-
lective cleavage of thyrotropin releasing hormone (TRH,
6) Matsuo H., Baba Y., Nair R. M. G., Arimura A., Schally A. V.,
Biochem. Biophys. Res. Commun., 43, 1334—1339 (1971).
7) O’Harte F., Bockman C. S., Abel P. W., Conlon J. M., Peptides, 12,
pGlu-His-Pro-NH ) under similar conditions, where more
2
5)
than 50% of TRH remained intact after 72 h. Thus, there
was a large difference in the susceptibility of pGlu-His pep-
tide bonds in Gn-RH (1) and TRH under similar conditions.
In each pGlu-X-peptide, the cleavage reaction of a pGlu-His
peptide bond seemed to be greatly affected by the amino acid
sequence that followed it. The t1/2 values of the other biologi-
cally active peptides were quite varied at 72 h for d-NMU-8
1
1—15 (1991).
8)
Bernardi L., Bosisio G., Goffredo O., de Castiglione R., Experientia,
20, 490— 492 (1964).
9) Ferreira S. H., Bartelt D. C., Greene L. J., Biochemistry, 9, 2583—
593 (1970).
2
1
1
0) Carraway R., Leeman S. E., J. Biol. Chem., 250, 1907—1911 (1975).
1) Sakura N., Kurosawa K., Hashimoto T., Chem. Pharm. Bull., 43,
1
148—1153 (1995).
(
2) (in 90% MSA), 15 h for PH (3) (in 70% MSA), 20 h for 12) Sakura N., Ohta S., Uchida Y., Kurosawa K., Okimura K., Hashimoto
T., Chem. Pharm. Bull., 39, 2016—2020 (1991).
BPP-5a (4) (in 70% MSA), and 15 h for NT (5) (in 90%
MSA). Thus, the susceptibility of various peptide bonds in-
cluding the pGlu-peptide bonds was dependent on the pri-
mary structures of these peptides.
13) Kurosawa K., Sakura N., Hashimoto T., “Peptide Chemistry 1994,” ed.
by Ohno M., Protein Research Foundation Press, Osaka, 1995, pp.
3
25—328.
14) Narita K., Titani K., J. Biochem. (Tokyo), 63, 226—241 (1968).
Present study demonstrated that the selective removal of 15) Kawai T., Shibata A., Kurosawa K., Sato Y., Kato S. Ohki K.,
Hashimoto T., Sakura N., Chem. Pharm. Bull., 54, 659—664 (2006).
6) Piszkiewicz D., Landon M., Smith E. L., Biochem. Biophys. Res. Com-
mun., 40, 1173—1178 (1970).
pGlu residue from various biologically active peptides could
1
generally be attained by means of high concentration of
MSA at low temperature, which may be applicable to struc-
tural examination of pGlu-peptides.
1
7) Landon M., Methods Enzymol., 47, 145—149 (1977).
18) Segalas I., Thai R., Menez R., Vita C., FEBS Lett., 371, 171—175
1995).
(
Acknowledgements The authors thank Miss Mayumi Kashihara of this
university for measuring mass spectra.