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Identification of PTH-amino acids by chromatography on glass paper
>1ItCHIvES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Letter Identification
of PTH-Amino
Chromatography
112,
392-393
(1965)
to the Editors
Acids by
on Glass Paper’
For sequential analysis of amino acids in peptides, Edman’s procedure (1) has become a very useful technique. The N-terminal amino acid of a peptide is allowed to react with phenylisothio-
terminal amino acid of the original peptide is determined by the identification of the PTH-amino acid liberated after the degradation. Several methods, including chromatography on columns of Hyflo Super Cel (Z), paper (S-6), and thin layer of silica gel (7), have been reported earlier. These methods are time-consuming and in most cases the chromatograms are not well defined. In the present communication, a rapid and sensitive method of identification of PTH-amino acids on glass paper is reported. TABLE V.ZLUES
RPTIL~~,+
DIFFERENT
OF
I PTH-AMINO
SOLVENT
ACIDS
IN
SYSTEMS
R PTH-glyeine in Solvent PTH-amino
acid
Glycine Alanine Serine Cystine Threonine Methionine Valine Leucine Isoleucine Aspartic acid Glutamic acid Lysine (e-PTC) Arginine +-alanine Tyrosine Tryptophan Proline Histidinc
FIG. 1. Chromatography of PTH-amino acids on glass paper in solvent (A). The spots were localized by spraying with iodine-sodium azide reagent.
cyanate to form a PTWpeptide. The PTCpeptide, on treatment with mild acids, yields a PTH-amino acid and a residual peptide. The N1 These studies were supported by funds from the National Heart Institute of the United States Public Health Service (HE-02942) and from the Louisiana Heart Association. * The abbreviations used are: PTC-phenylthiocarbamyl; PTH-phenylthiohydantoin. 392
A
B
C
1.00
1.00
1.00
1.58 0.02 Streaked 0.05 1.87 2.21 2.70 2.71 0.00 0.01 1.23 0.00 2.01 0.18 0.86 2.76 0.00
1.08 0.44 1.01 0.78 1.07 1.08 1.11 1.09 0.47 0.69 1.00 0.03 1.11 0.80 0.99 1.10 0.07
1.16 0.39 1.24 0.77 1.17 1.21 1.21 1.21 0.00 0.02 0.6G 0.00 1.19 0.94 0.89 1.21 0.13
The PTH-amino acids used in this investigation were obtained from Mann Research Laboratories, Inc., New York, New York. Silicated glass papers were prepared as reported (8). The silicated papers were dipped in 0.5$& starch (soluble powder, J. T. Baker Chemical Co., Phillipsburg, New Jersey) solution for a minute and air-dried. The chromatograms were developed in three solvent, systems: n-heptane:chloroform, 1:l (A); n-heptane:chloroform:acetic acid, 10:9:1 (B); n-heptane: chloroform:pyridine, 10:9:1 (C). The time of development was forty minutes. The PTH-amino acid
LETTER
TO THE
spots were localized on the chromatograms by spraying with sodium azide-iodine reagent (a). An example is shown in Fig. 1. All the experiments were carried out in triplicate, and the mean RPTtI-glyeine values are reported in Table I. Since these values, although fairly reproducible, are somewhat variable due to preparation of papers, solvent system, and conditions during chromatography, known standards should be used routinely Employing the technique described above, the PTH-amino acids liberated after each Edman degradation of a glycopeptide from the major glycoprotein isolated from bovine aorta have been successfully identified. The results were verified by studying the amino acid composition of the resulting peptide after each degradation (9). It may be noted from Table I that a good resolution of most of the PTH-a,mino acids can be achieved in a short time. PTH-cystine resulted in streaking in solvent system (A). B. Radhakrishnamurthy J. Rosenberg
393
EDITORS 1). Sutton
G. S. BERENSON Departments of Medicine and Biochemistry Louisiana State University School of Medicine New Orleans, Louisiana Received August 26, 1965 REFERENCES 1. EDMAN, P.-, Acta Chem. &and. 4,283 (1950). 2. SJOQWST, J., Arkiv. Kemi. 11, 151 (1957). 3. SJOQUIST, J., Acta Chem. &and. 7,447 (1953). 4. LANDMANN, W. A., DRAKE, M. P., AND DILLAHA, J., J. Am. Chem. Sot. 76, 3638 (1953). 5. EDMAN, P., AND SJOQUIST, J., Acta Chem. &and. 10, 1507 (1956). J., Biochim. Biophys. Acta 41, 20 6. SJOQUIST, (1960). 7. BRENNER, M., NIEDERWIESER, A., AND PATAKI, G., Ezperientia 17,145 (1961). 8. BERENSON, G. S., AND DALFERES, E. It., JR., Biochim. Biophys. Acta 68, 34 (1962). B., ANDBERENSON,G. 9. RADHAKRISHNAMURTHY, S., Submitted for publication.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Letter Identification
of PTH-Amino
Chromatography
112,
392-393
(1965)
to the Editors
Acids by
on Glass Paper’
For sequential analysis of amino acids in peptides, Edman’s procedure (1) has become a very useful technique. The N-terminal amino acid of a peptide is allowed to react with phenylisothio-
terminal amino acid of the original peptide is determined by the identification of the PTH-amino acid liberated after the degradation. Several methods, including chromatography on columns of Hyflo Super Cel (Z), paper (S-6), and thin layer of silica gel (7), have been reported earlier. These methods are time-consuming and in most cases the chromatograms are not well defined. In the present communication, a rapid and sensitive method of identification of PTH-amino acids on glass paper is reported. TABLE V.ZLUES
RPTIL~~,+
DIFFERENT
OF
I PTH-AMINO
SOLVENT
ACIDS
IN
SYSTEMS
R PTH-glyeine in Solvent PTH-amino
acid
Glycine Alanine Serine Cystine Threonine Methionine Valine Leucine Isoleucine Aspartic acid Glutamic acid Lysine (e-PTC) Arginine +-alanine Tyrosine Tryptophan Proline Histidinc
FIG. 1. Chromatography of PTH-amino acids on glass paper in solvent (A). The spots were localized by spraying with iodine-sodium azide reagent.
cyanate to form a PTWpeptide. The PTCpeptide, on treatment with mild acids, yields a PTH-amino acid and a residual peptide. The N1 These studies were supported by funds from the National Heart Institute of the United States Public Health Service (HE-02942) and from the Louisiana Heart Association. * The abbreviations used are: PTC-phenylthiocarbamyl; PTH-phenylthiohydantoin. 392
A
B
C
1.00
1.00
1.00
1.58 0.02 Streaked 0.05 1.87 2.21 2.70 2.71 0.00 0.01 1.23 0.00 2.01 0.18 0.86 2.76 0.00
1.08 0.44 1.01 0.78 1.07 1.08 1.11 1.09 0.47 0.69 1.00 0.03 1.11 0.80 0.99 1.10 0.07
1.16 0.39 1.24 0.77 1.17 1.21 1.21 1.21 0.00 0.02 0.6G 0.00 1.19 0.94 0.89 1.21 0.13
The PTH-amino acids used in this investigation were obtained from Mann Research Laboratories, Inc., New York, New York. Silicated glass papers were prepared as reported (8). The silicated papers were dipped in 0.5$& starch (soluble powder, J. T. Baker Chemical Co., Phillipsburg, New Jersey) solution for a minute and air-dried. The chromatograms were developed in three solvent, systems: n-heptane:chloroform, 1:l (A); n-heptane:chloroform:acetic acid, 10:9:1 (B); n-heptane: chloroform:pyridine, 10:9:1 (C). The time of development was forty minutes. The PTH-amino acid
LETTER
TO THE
spots were localized on the chromatograms by spraying with sodium azide-iodine reagent (a). An example is shown in Fig. 1. All the experiments were carried out in triplicate, and the mean RPTtI-glyeine values are reported in Table I. Since these values, although fairly reproducible, are somewhat variable due to preparation of papers, solvent system, and conditions during chromatography, known standards should be used routinely Employing the technique described above, the PTH-amino acids liberated after each Edman degradation of a glycopeptide from the major glycoprotein isolated from bovine aorta have been successfully identified. The results were verified by studying the amino acid composition of the resulting peptide after each degradation (9). It may be noted from Table I that a good resolution of most of the PTH-a,mino acids can be achieved in a short time. PTH-cystine resulted in streaking in solvent system (A). B. Radhakrishnamurthy J. Rosenberg
393
EDITORS 1). Sutton
G. S. BERENSON Departments of Medicine and Biochemistry Louisiana State University School of Medicine New Orleans, Louisiana Received August 26, 1965 REFERENCES 1. EDMAN, P.-, Acta Chem. &and. 4,283 (1950). 2. SJOQWST, J., Arkiv. Kemi. 11, 151 (1957). 3. SJOQUIST, J., Acta Chem. &and. 7,447 (1953). 4. LANDMANN, W. A., DRAKE, M. P., AND DILLAHA, J., J. Am. Chem. Sot. 76, 3638 (1953). 5. EDMAN, P., AND SJOQUIST, J., Acta Chem. &and. 10, 1507 (1956). J., Biochim. Biophys. Acta 41, 20 6. SJOQUIST, (1960). 7. BRENNER, M., NIEDERWIESER, A., AND PATAKI, G., Ezperientia 17,145 (1961). 8. BERENSON, G. S., AND DALFERES, E. It., JR., Biochim. Biophys. Acta 68, 34 (1962). B., ANDBERENSON,G. 9. RADHAKRISHNAMURTHY, S., Submitted for publication.