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An improved method for isolation of the C-terminal fragment of proteins
ANALYTICAL
BIOCHEMISTRY
An Improved ARPAD
129, 14-21 (1983)
Method for Isolation of the C-Terminal FURRA,’
GABOR
DIB~,
Fragment
of Proteins
JUDIT KOV~CS,~ AND FERENC SEBESTYBN
Department of Organic Chemistry, Eiitviis Lo&d
University, Muzeum krt 4/b, H-1088 Budapest, Hungary
Received July 2, 1982 An efficient and easily realizable method for the isolation of the C-terminal fragment is described. Proteins are ester&d by methanolic HCI and subsequently digested with pepsin. The peptide mixture is submitted to paper electrophoresis in pH 2.1 buffer. The identification of the C-terminal peptide is performed by preparing a guide peptide map, using pH 5.5 buffer in the second dimension. The C-terminal fragment appears as an on-diagonal spot. It can be isolated by a pH 5.5 run of the corresponding band from the first (pH 2.1) electrophoretogram. Since the C-terminal peptide is the fastest moving component, there is no need for its further purification. The expected yield is about 40%. -
The isolation of the C-terminal fragment facilitates the determination of the C-terminal sequence of proteins and large peptides, since the isolated C-terminal fragment can be subjected to highly effective N-terminal sequential degradation procedures. The methods used so far for the identification and isolation of the C-terminal fragment are based on the principles suggested by Naughton and Hagopian ( l), Carlton and Yanofsky (2), Matsuo and Matsubara (3), Furka et al. (4), Zabin and Fowler (5), and Horn (6). The key step in these isolation strategies is the one which allows the identification of the C-terminal fragment, i.e., in (l), the selective removal by carboxypeptidase B of Arg and Lys residues from the tryptic peptides with the C-terminal fragment remaining unchanged; in (2), the comparison of the fingerprints of the two enzymatic digests of the same protein, untreated and previously treated with carboxypeptidase; in (3), the selective radioactive labeling of the C-terminal amino acid residue of the protein, or in (6), of the non-C-terminal peptides formed by cyanogen bromide fragmentation;
in (4), the blocking of the carboxyl groups of the protein before enzymatic hydrolysis, and in (5), the blocking of amino groups before and after tryptic fragmentation. Two of the original methods (45) have been modified in some practical details (7- 11); their basic principles, however, are unchanged. The blocking of the carboxyl groups, i.e., the converting of the acidic groups of proteins into neutral or basic ones, is an essential step in our strategy for the isolation of the C-terminal fragment (4). We have also studied the possibility of other applications of the carboxy1 modification in protein chemistry (1214). Both amidation and esterification have been used for blocking with satisfactory results (4,15,16). In the course of the enzymatic hydrolysis of the modified proteins, one single a-carboxyl group is generated for each component of the peptide mixture with only the exception of the C-terminal fragment. The Cterminal peptide has no carboxyl group and, for this reason, has a unique behavior that can be exploited in a number of ways for its identification and specific isolation. For example, in contrary to the other peptides possessing a carboxyl group, the electrical charge of the C-terminal fragment and, as a consequence, the electrophoretic mobility, remains
* To whom correspondence should be addressed. * Present address: National Institute of Haematology and Blood Transfusion, Da&i u. 24, H- 1 I I3 Budapest, Hungary. 0003-2697/83/030014-08$03.00/O Copyright Q 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
14
C-TERMINAL
FRAGMENT
OF PROTEINS
15
unchanged in the entire acidic pH range.3 a stoppered flask at 4°C for 48 h, then the Furthermore, unlike the other components of excess of the reagent was evaporated in a desiccator under reduced pressure over KOH and the peptide mixture, the C-terminal fragment P205, at room temperature. To the dry resiis resistant to digestion by carboxypeptidases. due, 2 ml of 5% (v/v) formic acid and 0.2 mg Both properties have been experimentally of pepsin were added and stirred at 37°C. Afverified (4,13) and further supported (7,9). In this paper we report an improved ver- ter 8 h the solution was freeze dried. Isolation and analysis of the C-terminal sion of the original electrophoretic isolation method (4). This procedure is simpler and, at fragment. The whole peptic digest obtained the same time, gives higher yields than the from 20 mg of the starting protein was dissolved in pH 2.1 buffer and applied to a Whatearlier methods. man 3MM paper (0.7 mg/cm) together with reference markers (taurine and leucine methyl EXPERIMENTAL ester, 50 nmol/cm) on both sides. ElectroMaterials phoresis was performed on a horizontal Bovine Lu-chymotrypsin and porcine pepsin cooled-plate apparatus (Labor MIM, Hun(both of them three times recrystallized) were gary) at pH 2.1 and 32 V/cm for 130 min. purchased from Koch-Light; bovine trypsin After drying, two side strips (Fig. la) were A strip (A) (two times recrystallized) was obtained from stained with cadmium-ninhydrin. Worthington. Ovalbumin (six times recrys- was removed, stitched on to a new sheet of tallized) was prepared from hen eggs accord- Whatman 3MM paper and, again with markers on the two sides, subjected to electrophoing to Sorensen and Hoyrup ( 17). Methanolic HCl (0.1 N) was prepared by absorbing dry resis at pH 5.5 for 150 min. The two-dimenwas stained and, HCl gas in dry methanol, or by using the in- sional electrophoretogram stant methanolic HCl kit purchased from Ap- using the guidance of the markers, the diagonal was constructed as shown in Fig. 1b. The plied Science Europe B.V., Oud-Beijerland, corHolland. All other reagents and solvents were band of the pH 2.1 electrophoretogram responding to the on-diagonal spot of the twocommercial products of A.R. grade. dimensional peptide map (band B in Fig. la) Buffers employed were: pH 2.1, formic acid:acetic acidwater (1:4:45, v/v/v); pH 5.5, was cut out, stitched to a new sheet, and submitted to electrophoresis at pH 5.5 for 130 pyridine:acetic acidwater (12:5:988, v/v/v). min.4 The band containing the fastest moving component (the C-terminal fragment) was exMethods cised and the peptide was eluted from the paOvalbumin was oxidized by performic acid per by a descending wash (21). Norleucine (18). Its reduced and carboxymethylated as (200 nmol) was added as an internal standard well as reduced and aminoethylated derivaand then evaporated. tives were prepared by the methods of CanThe peptide samples containing the interfield and Anfinsen ( 19) and Raftery and Cole nal standard were hydrolyzed in 6 N HCl at (20), respectively. 110°C for 20 h in sealed, evacuated tubes. EsteriJication and enzymatic digestion. In Amino acid analyses were performed on a Jeol a typical experiment 20 mg of protein was JLCdAH analyzer. stirred with 2 ml of methanolic 0.1 N HCl in Digestion by a-chymotrypsin or trypsin was performed in 1% (w/v) ammonium bicarbon-
3 This is only true if the C-terminal fragment does not contain His. According to our observations, the mobility of the blocked His peptides is only unchanged below pH 5.5. That is why the original isolation procedure (4) has been modified (16).
4 Sometimes two or three spots appear on the diagonal, e.g., if the protein contains more than one polypeptide chain. All the corresponding bands, of course, should be processed.
16
FURICA
ET
AL.
C-TERMINAL
FRAGMENT
0
OF
0 --
I
i
I
--;.
_-_---e-w-
i Oi.90
:
~-0-Q
,,,,,.__,,,,::
yl
i
D
i
%
i : I !-i 0
Q; ---------‘“, ati
i,
-
I
y
0
I
p1 i
l-
PROTEINS
17
18
FURKA
ate (pH 7.2) at 37°C for 8 h, using an enzyme:esterified protein ratio of 1:50 (w/w). The enzyme was added in two equal portions at start and after 4 h.
ET
AL.
either above the diagonal or below it. If pH 2.1 buffer is used in the first dimension, the non-C-terminal peptides migrate to the region below the diagonal (Fig. 1b). The effect of the inverted buffer sequence is demonstrated in Fig. 2. RESULTS AND DISCUSSION The buffer sequence significantly affects the Because of its simplicity, esterification efficiency of the purification procedure. In ( 15,22) was adopted for blocking the carboxyl contrary to the original method (4), we now groups, instead of the amidation used previ- suggest the use of pH 2.1 buffer in the first ously. All operations of ester&cation and sub- electrophoretic run. This assures a higher yield sequent enzymatic digestion can be carried in fewer purification steps. In this case, the out in the same flask. Our experience shows second step (pH 5.5) is particularly efficient, that esterification is an efficient blocking pro- since the C-terminal peptide moves ahead of cedure and it can be used without risking the the impurities. The effect of the buffer seisolation of artificial products in addition to quence on the purity of the C-terminal pep tide is demonstrated in Table 1. The C-terthe C-terminal fragment. Pepsin is expected to be the best choice for minal fragment of the oxidized and esterified the enzymatic fragmentation of esterified pro- ovalbumin is isolated in two consecutive electeins, for two reasons: (a) the spontaneous hy- trophoretic steps. Using buffer sequence pH 2.1, pH 5.5, the isolated C-terminal peptide drolysis of the peptide esters at the pH optimum of pepsin is slow, and (b) the ester groups is practically pure, while applying buffer seare resistant to the action of the enzyme itself. quence pH 5.5, pH 2.1, further purification This was tested experimentally as follows: re- is necessary which subsequently lowers the duced and aminoethylated ovalbumin was yield. Although the yield of the pure peptide (4 1%) submitted to digestion by pepsin, then to esat the buffer sequence pH 2.1, pH 5.5 is conterification. The esterified peptide mixture was siderably higher than that in the previous varidivided into two parts. From the first part a ants of the method, further experiments were two-dimensional electrophoretogram was carried out to trace the cause of losses. Preprepared (pH 5.5, then pH 2.1). The peptides viously isolated C-terminal heptapeptide of appeared on a diagonal. The second part was oxidized and esterified ovalbumin was added reincubated with pepsin and then submitted in known amount to the peptic digest of reto two-dimensional electrophoresis. The elecduced, aminoethylated, and esterified ovaltrophoretic pattern proved to be the same, bumin. On processing, the recovery of the indicating the resistance of the ester groups. added heptapeptide was 58%, i.e., 42% had Qualitative tests showed that chymotrypsin been lost on the paper (guide strips, sticking, or trypsin can also be used for fragmentation of the esterified proteins at conditions de- etc.). Taking this into account, it can be deduced that in the course of the normal isoscribed under Experimental. When the enzymatic digest of an esterified lation procedure, the C-terminal heptapepprotein is submitted to two-dimensional elec- tide is present in ca. 7 1% of the amount in trophoresis by applying two different acidic the peptic digest of the oxidized and esterified buffers (pH 2.1, then pH 5.5) in the two di- ovalbumin. However, due to losses on paper, the yield is 41% after isolation. The 29% loss mensions, for reasons outlined in the introduction, the C-terminal fragment appears on of the C-terminal heptapeptide in the digest the diagonal of the electrophoretogram. The can be attributed to incomplete oxidation and/ rest of the peptides, depending on the se- or esterification as well as to incomplete or quence of the two buffers applied, are found nonspecific enzymatic fragmentation.
C-TERMINAL
FRAGMENT
OF
19
PROTEINS
.O-
-Leu-
OMe
Tau START
FIG. 2. Two-dimensional peptide maps obtained from oxidized (photo) and reduced, carboxymethylated (drawing) ovalbumin, by the use of the buffer sequence pH 5.5, pH 2.1 (other things being equal to those of Fig. I). The spots of the non-C-terminal peptides are above the diagonal.
20
FURKA TABLE
1
DEPENDENCE ON THE BUFFER SEQUENCE OF THE PURITY OF THE C-TERMINAL HEPTAPEP~IDE MLATED FROM OXIDIZED OVALBUMIN Composition Amino acid
Composition expected
C~s(0d.Q Asp Thr Ser GIU Pro GUY Ala Val Be
found”
pH 2.1, pH 5.5
pH 5.5, pH 2.1
1
1.11 0.06
1 1 I
1.11 0.06 1.00 1.19
1
0.89
1 I
0.89 0.90 41
1.19 0.25 0.06 1.09 0.43 0.9 1 1.17 0.28 1.17 0.19 0.32 0.79 0.90 45
Lell
Phe Aa Yield
(%J)~
’ The quantities of amino acids were determined relative to the internal standard (Nle) and are expressed as the number of residues. b Yields were calculated by using the mean quantities of the real components of the peptides.
With intention to introduce further simplifications, we tested the possibility of the omission of the side-chain modification of the protein (S-alkylation or oxidation) prior to esterification. Native trypsin and chymotrypsin were submitted directly to esterification. AlTABLE YIELD
2
ET
AL.
though both proteins contained disulfide bonds (23,24), the isolation of their C-terminal fragments was successful (Table 2). This simpler procedure will, however, fail if the Cterminal peptide contains half-cystine residue. A C-terminal fragment, linked by a disulfide bond to a non-C-terminal peptide, does not show the unique electrophoretic behavior mentioned above and its specific purification is impossible. H2N./CooCH3 s-s H2N -COOH Ovalbumin was used as model compound to test this effect, although it is disputed whether it has a half cystine residue near the C-terminus (25,26). Starting with directly esterified ovalbumin, the C-terminal fragment was completely missing from the diagonal of the two-dimensional electrophoretogram of the peptic digest. However, if the same digest was oxidized by pet-formic acid prior to electrophoresis, or the electrophoresis was performed in reductive medium (the buffers containing 0.2% 2-mercaptoethanol), the C-terminal fragment did appear on the diagonal and could even be isolated. On the basis of all these considerations, for the application of the method, the preparation of a preliminary two-dimensional electrophoretogram is suggested, which requires only ca. 1.5 mg digest of the esterified protein.
OF C-TERMINAL PEPTIDES ISOLATED FROM NATIVE PROTEINS
TABLE YIELD
Protein Trypsin
(22)
cY-Chymottypsin (23) A Chain B Chain total B Chain 1 B Chain 2 B Chain 3 C Chain
Isolated
Yield
peptide
OF THE C-TERMINAL PWTIDE ISOLATED DIFFERENT DERIVATIVES OF OVALBUMIN Ovalbumin
Ile-Lys-Gln-ThrBe-Ala-Ser-Asn
FROM
Yield
(%)
37
Ser-Gly-Leu Leu-Thr-Arg-Tyr Thr-Arg-Tyr Tyr Ala-Ala-Asn
(W)
3
34 22 8 9 5 43
Oxidized, then esterified Reduced, carboxymethylated, then esterified Native, esteritied, the digest oxidized Reduced, aminoethylated, then esterified Native, esterified, electrophoresis in reductive medium
41 27 26 24 22
C-TERMINAL
FRAGMENT
OF PROTEINS
21
10. Duggleby, R. G., Kaplan, H., and Visentin, L. P. (1975) Canad J. Biochem. 53.827-833. II. Hat-grave, P. A., and Fong, S.-L. (1977) J. Suprumol. Struct. 6, 559-570. 12. Furka, A., and Sebestyen, F. (1969) FEBS Lett. 4, 207-209. 13. Furka, A., and Se&sty&r, F. (1970) Acta Biochim. Biophys. Acad. Sci. Hung. 5, 131-146. 14. Furka, A. (1971) Acta Biochim. Biophys. Acad. Sci. Hung. 6, 153-155. IS. Kovacs, J., and Furka, A. (1971) Proceedings, 12th Hungarian Annual Meeting Biochem., P&x, pp. ACKNOWLEDGMENTS 383-388. The authors thank Dr. H. Medzihradszky-Schweiger 16. Furka, A., Kov&s, J., and Sebestyen,F. (1974) Abstr., for amino acid analyses as well as Mrs. I. C&z& and 2s. Commun. 9th Meet. Fed. Europ. B&hem. Sot., Gtmes for their skillful technical assistance. Budapest, p. 428. 17. Sorensen, S. P. L., and Hoyrup, H. (1915-1917) C. R. Trav. Lab. Carlsberg 29, 12-64. REFERENCES 18. Hirs, C. H. W. (1956) J. Biol. Chem. 219,61 I-612. 19. Canfield, R. E., and Anfinsen, C. B. (1963) J. Biol. 1. Naughton, M. A., and Hagopian, H. (1962) Anal. Biochem. 3, 276-284. Chem. 238,2684-2690. 2. C&ton, B. C., and Yanofsky, C. (1963) J. Biol. Chem. 20. Raftery, M. A., and Cole, R. D. (1966) J. Biol. Chem. 241, 3457-3461. 238,636-639. 3. Matsuo, H., and Matsubara, H. (1968) Proc. Sot. 21. Bennett, J. C. ( 1967) in Methods in Enzymology (Hirs, Exp. Biol. Med. 129, 564-566. C. H. W., ed.), Vol. 1 I., pp. 330-339, Academic Press, New York. 4. Furka, A., Sebestyin, F., and Karacsonyi, T. (1970) FEBS Left. 6, 34-36. 22. Chibnall, A. C., Mangan, J. L., and Rees, M. W. 5. Zabin, I., and Fowler, A. V. (1972) J. Biol. Chem. (1958) Biochem. J. 68, 114-I 18. 241, 5432-5435. 23. Walsh, K. A., and Neurath, H. (1964) Proc. Nat. 6. Horn, M. J. (1975) Anal. Biochem. 69, 583-589. Acad. Sci. USA 52, 884-889. 7. Hargrave, P. A., and Weld, F. (1973) Int. J. Peps. 24. Hartley, B. S. (1964) Nature (London) 201, 1284Protein Res. 5, 85-89. 1287. 8. Fong, S.-L., and Hatgrave. P. A. (1977) Int. J. Pept. 25. Fothergill, L. A., and Fothergill, J. E. (I 970) B&hem. Protein Rex 10, 139-145. J. 116, 555-561. 9. Duggleby, R. G., and Kaplan, H. (1975) Anal. 26. Thompson, E. 0. P., and Fischer, W. K. (1978) Aust. Biochem. 65, 346-354. J. Biol. Sci. 31, 433-442.
In those few cases when no on-diagonal pep tide appears, oxidation or S-aikylation is needed prior to esterification. Oxidation of the digest before electrophoresis or the use of reductive medium during electrophoresis are alternative possibilities. As Table 3 shows, oxidation prior to esterification seems the best choice.
BIOCHEMISTRY
An Improved ARPAD
129, 14-21 (1983)
Method for Isolation of the C-Terminal FURRA,’
GABOR
DIB~,
Fragment
of Proteins
JUDIT KOV~CS,~ AND FERENC SEBESTYBN
Department of Organic Chemistry, Eiitviis Lo&d
University, Muzeum krt 4/b, H-1088 Budapest, Hungary
Received July 2, 1982 An efficient and easily realizable method for the isolation of the C-terminal fragment is described. Proteins are ester&d by methanolic HCI and subsequently digested with pepsin. The peptide mixture is submitted to paper electrophoresis in pH 2.1 buffer. The identification of the C-terminal peptide is performed by preparing a guide peptide map, using pH 5.5 buffer in the second dimension. The C-terminal fragment appears as an on-diagonal spot. It can be isolated by a pH 5.5 run of the corresponding band from the first (pH 2.1) electrophoretogram. Since the C-terminal peptide is the fastest moving component, there is no need for its further purification. The expected yield is about 40%. -
The isolation of the C-terminal fragment facilitates the determination of the C-terminal sequence of proteins and large peptides, since the isolated C-terminal fragment can be subjected to highly effective N-terminal sequential degradation procedures. The methods used so far for the identification and isolation of the C-terminal fragment are based on the principles suggested by Naughton and Hagopian ( l), Carlton and Yanofsky (2), Matsuo and Matsubara (3), Furka et al. (4), Zabin and Fowler (5), and Horn (6). The key step in these isolation strategies is the one which allows the identification of the C-terminal fragment, i.e., in (l), the selective removal by carboxypeptidase B of Arg and Lys residues from the tryptic peptides with the C-terminal fragment remaining unchanged; in (2), the comparison of the fingerprints of the two enzymatic digests of the same protein, untreated and previously treated with carboxypeptidase; in (3), the selective radioactive labeling of the C-terminal amino acid residue of the protein, or in (6), of the non-C-terminal peptides formed by cyanogen bromide fragmentation;
in (4), the blocking of the carboxyl groups of the protein before enzymatic hydrolysis, and in (5), the blocking of amino groups before and after tryptic fragmentation. Two of the original methods (45) have been modified in some practical details (7- 11); their basic principles, however, are unchanged. The blocking of the carboxyl groups, i.e., the converting of the acidic groups of proteins into neutral or basic ones, is an essential step in our strategy for the isolation of the C-terminal fragment (4). We have also studied the possibility of other applications of the carboxy1 modification in protein chemistry (1214). Both amidation and esterification have been used for blocking with satisfactory results (4,15,16). In the course of the enzymatic hydrolysis of the modified proteins, one single a-carboxyl group is generated for each component of the peptide mixture with only the exception of the C-terminal fragment. The Cterminal peptide has no carboxyl group and, for this reason, has a unique behavior that can be exploited in a number of ways for its identification and specific isolation. For example, in contrary to the other peptides possessing a carboxyl group, the electrical charge of the C-terminal fragment and, as a consequence, the electrophoretic mobility, remains
* To whom correspondence should be addressed. * Present address: National Institute of Haematology and Blood Transfusion, Da&i u. 24, H- 1 I I3 Budapest, Hungary. 0003-2697/83/030014-08$03.00/O Copyright Q 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
14
C-TERMINAL
FRAGMENT
OF PROTEINS
15
unchanged in the entire acidic pH range.3 a stoppered flask at 4°C for 48 h, then the Furthermore, unlike the other components of excess of the reagent was evaporated in a desiccator under reduced pressure over KOH and the peptide mixture, the C-terminal fragment P205, at room temperature. To the dry resiis resistant to digestion by carboxypeptidases. due, 2 ml of 5% (v/v) formic acid and 0.2 mg Both properties have been experimentally of pepsin were added and stirred at 37°C. Afverified (4,13) and further supported (7,9). In this paper we report an improved ver- ter 8 h the solution was freeze dried. Isolation and analysis of the C-terminal sion of the original electrophoretic isolation method (4). This procedure is simpler and, at fragment. The whole peptic digest obtained the same time, gives higher yields than the from 20 mg of the starting protein was dissolved in pH 2.1 buffer and applied to a Whatearlier methods. man 3MM paper (0.7 mg/cm) together with reference markers (taurine and leucine methyl EXPERIMENTAL ester, 50 nmol/cm) on both sides. ElectroMaterials phoresis was performed on a horizontal Bovine Lu-chymotrypsin and porcine pepsin cooled-plate apparatus (Labor MIM, Hun(both of them three times recrystallized) were gary) at pH 2.1 and 32 V/cm for 130 min. purchased from Koch-Light; bovine trypsin After drying, two side strips (Fig. la) were A strip (A) (two times recrystallized) was obtained from stained with cadmium-ninhydrin. Worthington. Ovalbumin (six times recrys- was removed, stitched on to a new sheet of tallized) was prepared from hen eggs accord- Whatman 3MM paper and, again with markers on the two sides, subjected to electrophoing to Sorensen and Hoyrup ( 17). Methanolic HCl (0.1 N) was prepared by absorbing dry resis at pH 5.5 for 150 min. The two-dimenwas stained and, HCl gas in dry methanol, or by using the in- sional electrophoretogram stant methanolic HCl kit purchased from Ap- using the guidance of the markers, the diagonal was constructed as shown in Fig. 1b. The plied Science Europe B.V., Oud-Beijerland, corHolland. All other reagents and solvents were band of the pH 2.1 electrophoretogram responding to the on-diagonal spot of the twocommercial products of A.R. grade. dimensional peptide map (band B in Fig. la) Buffers employed were: pH 2.1, formic acid:acetic acidwater (1:4:45, v/v/v); pH 5.5, was cut out, stitched to a new sheet, and submitted to electrophoresis at pH 5.5 for 130 pyridine:acetic acidwater (12:5:988, v/v/v). min.4 The band containing the fastest moving component (the C-terminal fragment) was exMethods cised and the peptide was eluted from the paOvalbumin was oxidized by performic acid per by a descending wash (21). Norleucine (18). Its reduced and carboxymethylated as (200 nmol) was added as an internal standard well as reduced and aminoethylated derivaand then evaporated. tives were prepared by the methods of CanThe peptide samples containing the interfield and Anfinsen ( 19) and Raftery and Cole nal standard were hydrolyzed in 6 N HCl at (20), respectively. 110°C for 20 h in sealed, evacuated tubes. EsteriJication and enzymatic digestion. In Amino acid analyses were performed on a Jeol a typical experiment 20 mg of protein was JLCdAH analyzer. stirred with 2 ml of methanolic 0.1 N HCl in Digestion by a-chymotrypsin or trypsin was performed in 1% (w/v) ammonium bicarbon-
3 This is only true if the C-terminal fragment does not contain His. According to our observations, the mobility of the blocked His peptides is only unchanged below pH 5.5. That is why the original isolation procedure (4) has been modified (16).
4 Sometimes two or three spots appear on the diagonal, e.g., if the protein contains more than one polypeptide chain. All the corresponding bands, of course, should be processed.
16
FURICA
ET
AL.
C-TERMINAL
FRAGMENT
0
OF
0 --
I
i
I
--;.
_-_---e-w-
i Oi.90
:
~-0-Q
,,,,,.__,,,,::
yl
i
D
i
%
i : I !-i 0
Q; ---------‘“, ati
i,
-
I
y
0
I
p1 i
l-
PROTEINS
17
18
FURKA
ate (pH 7.2) at 37°C for 8 h, using an enzyme:esterified protein ratio of 1:50 (w/w). The enzyme was added in two equal portions at start and after 4 h.
ET
AL.
either above the diagonal or below it. If pH 2.1 buffer is used in the first dimension, the non-C-terminal peptides migrate to the region below the diagonal (Fig. 1b). The effect of the inverted buffer sequence is demonstrated in Fig. 2. RESULTS AND DISCUSSION The buffer sequence significantly affects the Because of its simplicity, esterification efficiency of the purification procedure. In ( 15,22) was adopted for blocking the carboxyl contrary to the original method (4), we now groups, instead of the amidation used previ- suggest the use of pH 2.1 buffer in the first ously. All operations of ester&cation and sub- electrophoretic run. This assures a higher yield sequent enzymatic digestion can be carried in fewer purification steps. In this case, the out in the same flask. Our experience shows second step (pH 5.5) is particularly efficient, that esterification is an efficient blocking pro- since the C-terminal peptide moves ahead of cedure and it can be used without risking the the impurities. The effect of the buffer seisolation of artificial products in addition to quence on the purity of the C-terminal pep tide is demonstrated in Table 1. The C-terthe C-terminal fragment. Pepsin is expected to be the best choice for minal fragment of the oxidized and esterified the enzymatic fragmentation of esterified pro- ovalbumin is isolated in two consecutive electeins, for two reasons: (a) the spontaneous hy- trophoretic steps. Using buffer sequence pH 2.1, pH 5.5, the isolated C-terminal peptide drolysis of the peptide esters at the pH optimum of pepsin is slow, and (b) the ester groups is practically pure, while applying buffer seare resistant to the action of the enzyme itself. quence pH 5.5, pH 2.1, further purification This was tested experimentally as follows: re- is necessary which subsequently lowers the duced and aminoethylated ovalbumin was yield. Although the yield of the pure peptide (4 1%) submitted to digestion by pepsin, then to esat the buffer sequence pH 2.1, pH 5.5 is conterification. The esterified peptide mixture was siderably higher than that in the previous varidivided into two parts. From the first part a ants of the method, further experiments were two-dimensional electrophoretogram was carried out to trace the cause of losses. Preprepared (pH 5.5, then pH 2.1). The peptides viously isolated C-terminal heptapeptide of appeared on a diagonal. The second part was oxidized and esterified ovalbumin was added reincubated with pepsin and then submitted in known amount to the peptic digest of reto two-dimensional electrophoresis. The elecduced, aminoethylated, and esterified ovaltrophoretic pattern proved to be the same, bumin. On processing, the recovery of the indicating the resistance of the ester groups. added heptapeptide was 58%, i.e., 42% had Qualitative tests showed that chymotrypsin been lost on the paper (guide strips, sticking, or trypsin can also be used for fragmentation of the esterified proteins at conditions de- etc.). Taking this into account, it can be deduced that in the course of the normal isoscribed under Experimental. When the enzymatic digest of an esterified lation procedure, the C-terminal heptapepprotein is submitted to two-dimensional elec- tide is present in ca. 7 1% of the amount in trophoresis by applying two different acidic the peptic digest of the oxidized and esterified buffers (pH 2.1, then pH 5.5) in the two di- ovalbumin. However, due to losses on paper, the yield is 41% after isolation. The 29% loss mensions, for reasons outlined in the introduction, the C-terminal fragment appears on of the C-terminal heptapeptide in the digest the diagonal of the electrophoretogram. The can be attributed to incomplete oxidation and/ rest of the peptides, depending on the se- or esterification as well as to incomplete or quence of the two buffers applied, are found nonspecific enzymatic fragmentation.
C-TERMINAL
FRAGMENT
OF
19
PROTEINS
.O-
-Leu-
OMe
Tau START
FIG. 2. Two-dimensional peptide maps obtained from oxidized (photo) and reduced, carboxymethylated (drawing) ovalbumin, by the use of the buffer sequence pH 5.5, pH 2.1 (other things being equal to those of Fig. I). The spots of the non-C-terminal peptides are above the diagonal.
20
FURKA TABLE
1
DEPENDENCE ON THE BUFFER SEQUENCE OF THE PURITY OF THE C-TERMINAL HEPTAPEP~IDE MLATED FROM OXIDIZED OVALBUMIN Composition Amino acid
Composition expected
C~s(0d.Q Asp Thr Ser GIU Pro GUY Ala Val Be
found”
pH 2.1, pH 5.5
pH 5.5, pH 2.1
1
1.11 0.06
1 1 I
1.11 0.06 1.00 1.19
1
0.89
1 I
0.89 0.90 41
1.19 0.25 0.06 1.09 0.43 0.9 1 1.17 0.28 1.17 0.19 0.32 0.79 0.90 45
Lell
Phe Aa Yield
(%J)~
’ The quantities of amino acids were determined relative to the internal standard (Nle) and are expressed as the number of residues. b Yields were calculated by using the mean quantities of the real components of the peptides.
With intention to introduce further simplifications, we tested the possibility of the omission of the side-chain modification of the protein (S-alkylation or oxidation) prior to esterification. Native trypsin and chymotrypsin were submitted directly to esterification. AlTABLE YIELD
2
ET
AL.
though both proteins contained disulfide bonds (23,24), the isolation of their C-terminal fragments was successful (Table 2). This simpler procedure will, however, fail if the Cterminal peptide contains half-cystine residue. A C-terminal fragment, linked by a disulfide bond to a non-C-terminal peptide, does not show the unique electrophoretic behavior mentioned above and its specific purification is impossible. H2N./CooCH3 s-s H2N -COOH Ovalbumin was used as model compound to test this effect, although it is disputed whether it has a half cystine residue near the C-terminus (25,26). Starting with directly esterified ovalbumin, the C-terminal fragment was completely missing from the diagonal of the two-dimensional electrophoretogram of the peptic digest. However, if the same digest was oxidized by pet-formic acid prior to electrophoresis, or the electrophoresis was performed in reductive medium (the buffers containing 0.2% 2-mercaptoethanol), the C-terminal fragment did appear on the diagonal and could even be isolated. On the basis of all these considerations, for the application of the method, the preparation of a preliminary two-dimensional electrophoretogram is suggested, which requires only ca. 1.5 mg digest of the esterified protein.
OF C-TERMINAL PEPTIDES ISOLATED FROM NATIVE PROTEINS
TABLE YIELD
Protein Trypsin
(22)
cY-Chymottypsin (23) A Chain B Chain total B Chain 1 B Chain 2 B Chain 3 C Chain
Isolated
Yield
peptide
OF THE C-TERMINAL PWTIDE ISOLATED DIFFERENT DERIVATIVES OF OVALBUMIN Ovalbumin
Ile-Lys-Gln-ThrBe-Ala-Ser-Asn
FROM
Yield
(%)
37
Ser-Gly-Leu Leu-Thr-Arg-Tyr Thr-Arg-Tyr Tyr Ala-Ala-Asn
(W)
3
34 22 8 9 5 43
Oxidized, then esterified Reduced, carboxymethylated, then esterified Native, esteritied, the digest oxidized Reduced, aminoethylated, then esterified Native, esterified, electrophoresis in reductive medium
41 27 26 24 22
C-TERMINAL
FRAGMENT
OF PROTEINS
21
10. Duggleby, R. G., Kaplan, H., and Visentin, L. P. (1975) Canad J. Biochem. 53.827-833. II. Hat-grave, P. A., and Fong, S.-L. (1977) J. Suprumol. Struct. 6, 559-570. 12. Furka, A., and Sebestyen, F. (1969) FEBS Lett. 4, 207-209. 13. Furka, A., and Se&sty&r, F. (1970) Acta Biochim. Biophys. Acad. Sci. Hung. 5, 131-146. 14. Furka, A. (1971) Acta Biochim. Biophys. Acad. Sci. Hung. 6, 153-155. IS. Kovacs, J., and Furka, A. (1971) Proceedings, 12th Hungarian Annual Meeting Biochem., P&x, pp. ACKNOWLEDGMENTS 383-388. The authors thank Dr. H. Medzihradszky-Schweiger 16. Furka, A., Kov&s, J., and Sebestyen,F. (1974) Abstr., for amino acid analyses as well as Mrs. I. C&z& and 2s. Commun. 9th Meet. Fed. Europ. B&hem. Sot., Gtmes for their skillful technical assistance. Budapest, p. 428. 17. Sorensen, S. P. L., and Hoyrup, H. (1915-1917) C. R. Trav. Lab. Carlsberg 29, 12-64. REFERENCES 18. Hirs, C. H. W. (1956) J. Biol. Chem. 219,61 I-612. 19. Canfield, R. E., and Anfinsen, C. B. (1963) J. Biol. 1. Naughton, M. A., and Hagopian, H. (1962) Anal. Biochem. 3, 276-284. Chem. 238,2684-2690. 2. C&ton, B. C., and Yanofsky, C. (1963) J. Biol. Chem. 20. Raftery, M. A., and Cole, R. D. (1966) J. Biol. Chem. 241, 3457-3461. 238,636-639. 3. Matsuo, H., and Matsubara, H. (1968) Proc. Sot. 21. Bennett, J. C. ( 1967) in Methods in Enzymology (Hirs, Exp. Biol. Med. 129, 564-566. C. H. W., ed.), Vol. 1 I., pp. 330-339, Academic Press, New York. 4. Furka, A., Sebestyin, F., and Karacsonyi, T. (1970) FEBS Left. 6, 34-36. 22. Chibnall, A. C., Mangan, J. L., and Rees, M. W. 5. Zabin, I., and Fowler, A. V. (1972) J. Biol. Chem. (1958) Biochem. J. 68, 114-I 18. 241, 5432-5435. 23. Walsh, K. A., and Neurath, H. (1964) Proc. Nat. 6. Horn, M. J. (1975) Anal. Biochem. 69, 583-589. Acad. Sci. USA 52, 884-889. 7. Hargrave, P. A., and Weld, F. (1973) Int. J. Peps. 24. Hartley, B. S. (1964) Nature (London) 201, 1284Protein Res. 5, 85-89. 1287. 8. Fong, S.-L., and Hatgrave. P. A. (1977) Int. J. Pept. 25. Fothergill, L. A., and Fothergill, J. E. (I 970) B&hem. Protein Rex 10, 139-145. J. 116, 555-561. 9. Duggleby, R. G., and Kaplan, H. (1975) Anal. 26. Thompson, E. 0. P., and Fischer, W. K. (1978) Aust. Biochem. 65, 346-354. J. Biol. Sci. 31, 433-442.
In those few cases when no on-diagonal pep tide appears, oxidation or S-aikylation is needed prior to esterification. Oxidation of the digest before electrophoresis or the use of reductive medium during electrophoresis are alternative possibilities. As Table 3 shows, oxidation prior to esterification seems the best choice.