The Use of Acrylonitrile for the Subtractive Identification of the <i>N</i>-Terminal Residues of Small Peptides

Fletcher, J. C.

doi:10.1042/bj0980034c

Cited by 20 publications

(7 citation statements)

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“…1), namely Ser-Hyp4 and Ser-Hyp-Lys (Table 1). Fletcher's (1966) subtractive method showed that both peptide fragments contained N-terminal serine. Ser-Hyp-Lys was degraded further by partial acid hydrolysis (6M-HCI, 15min at 100°C) to yield Hyp-Lys and Ser-Hyp.…”

Section: Resultsmentioning

confidence: 99%

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Galactosylserine in extensin

Lamport¹,

Katona²,

Roerig³

1973

Biochemical Journal

154

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Cell walls obtained from tomato suspension cultures were treated at pH1 for 1h at 100 degrees C to remove arabinose oligosaccharide substituents from the hydroxyproline residues of extensin. Tryptic attack of these acid-stripped walls yielded glycopeptides containing galactose. When one of these glycopeptides (designated S(2)A(6); sequence NH(2)-Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Lys-CO(2)H) was treated with (a) NaOH-NaBH(4) or (b) NaOH-Na(2)SO(3) some of the serine was converted into (a) alanine or (b) cysteic acid, and the peptide lost galactose. Maleylation or 3-carboxypropionylation of N-terminal serine was necessary for conversion of this residue and for complete loss of galactose. These results indicate that a single galactose residue is attached O-glycosidically to each of the two serine residues. Hydrazinolysis of peptide S(2)A(6) or of isolated cell walls also led to destruction of serine. In control experiments non-glycosylated serine was not destroyed during hydrazinolysis. Thus the galactosylserine linkage is sensitive to N(2)H(4).

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Section: Resultsmentioning

confidence: 99%

“…Cyanoethylation with acrylonitrile was used for subtractive N-terminus identification (Fletcher, 1966). Amino groups were also maleylated (Butler et al, 1969) and 3-carboxypropionylated (Klotz, 1967 Tube no.…”

Section: Reaction Ofamino Groupsmentioning

confidence: 99%

Galactosylserine in extensin

Lamport¹,

Katona²,

Roerig³

1973

Biochemical Journal

154

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“…in 5 q-HCI, DNS-amino acids were separated by paper electrophoresis at pH4-4 (Gray & Hartley, 1963b), or by thin-layer chromatography on silica gel (Cole, Fletcher & Robson, 1965). N-Terminal residues of peptides were also identified by reaction with [131I]pipsyl chloride (Fletcher, 1967) or by reaction with acrylonitrile (Fletcher, 1966). I-Fluoro-2,4-dinitrobenzene (Sanger, 1945) was occasionally used (DNP method), but the amount of material available was generally insufficient.…”

Section: Separation Of Peptidesmentioning

confidence: 99%

Amino acid sequences of peptides from a tryptic digest of a urea-soluble protein fraction (U.S.3) from oxidized wool

Corfield

Fletcher

1967

Biochemical Journal

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1. A tryptic digest of the protein fraction U.S.3 from oxidized wool has been separated into 32 peptide fractions by cation-exchange resin chromatography. 2. Most of these fractions have been resolved into their component peptides by a combination of the techniques of cation-exchange resin chromatography, paper chromatography and paper electrophoresis. 3. The amino acid compositions of 58 of the peptides in the digest present in the largest amounts have been determined. 4. The amino acid sequences of 38 of these have been completely elucidated and those of six others partially derived. 5. These findings indicate that the parent protein in wool from which the protein fraction U.S.3 is derived has a minimum molecular weight of 74000. 6. The structures of wool proteins are discussed in the light of the peptide sequences determined, and, in particular, of those sequences in fraction U.S.3 that could not be elucidated.

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“…By the acrylonitrile technique [7] the serine was found to be N-terminal and by the acetic anhydridelpyridine technique [ 121 the threonine was C-terminal. The peptide was subjected to two cycles of Edman degradation and the residue chromatographed on paper in the WaleyWatson system.…”

Section: Efficiency Of the Fractionation Proceduresmentioning

confidence: 99%

“…N-terminal amino acids were determined by the acrylonitrile technique of Fletcher [7], or the phenylisothiocyanate method of Edman 181. Amino acid sequence analysis by the latter method was carried out either by the variant entailing separation of the intermediate thiazolinone [9] or as a substractive procedure [lo].…”

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Structure of Porcine Secretin

Mutt

Jorpes

Magnusson

1970

European Journal of Biochemistry

183

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Porcine secretin is a heptacosapeptide with the amino acid sequence His-Ser-Asp-Gly-ThrPhe -Thr-Ser -G h -Leu -Ser -Arg -Leu -Arg -Asp-Ser -Ala -Arg -Leu -Gln -Arg -Leu -Leu-Gln-Gly-LeuValNH,. Fourteen of its twenty-seven amino acids occur in the same position as they do in porcine glucagon.I n a previous paper [11 we reported that porcine secretin could be degraded by trypsin into five peptides. The amino acid compositions of the peptides and their positions in the intact secretin molecule were given. Since their N-terminal amino acids were also determined, and since the arginines could be expectcd to occur C-terminally, the amino acid sequence in two of them was obvious: peptide Tr-2 = Leu-Arg and peptide Tr-4 = Leu-Gln-Arg (Tr = tryptic. The peptides are numbered from the N-terminus). Peptides Tr-1, Tr-2, Tr-3, Tr-4 and Tr-5 were referred to as b, d, a, c, and e respectively in the previous paper [l]. Clearly determination of the amino acid sequences in peptides Tr-1, Tr-3 and Tr-5 was all that remained necessary for establishing the primary structure of secretin. This proved to be unevenful, and the sequences of the tryptio peptides were communicated at the 4th International Symposium of Natural Products, organized by the International Union of Pure and Applied Chemistry, IUPAC, in 1966 [2].The tryptic peptides are: Tr-1 = His-Ser-AspGly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg ; Tr-2 = LeuArg; Tr-3 = Asp-Ser-Ala-Arg; Tr-4 = Leu-Gln-Arg; Tr-5 = Leu-Leu-Gin-Gly-Leu-ValNH,.Consequently it is evident that starting from the N-terminal histidine no less than fourteen amino acids occupy the same position in porcine secretin as they do in porcine glucagon [3].

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The Use of Acrylonitrile for the Subtractive Identification of the N-Terminal Residues of Small Peptides

Cited by 20 publications

References 8 publications

Galactosylserine in extensin

Galactosylserine in extensin

Amino acid sequences of peptides from a tryptic digest of a urea-soluble protein fraction (U.S.3) from oxidized wool

Structure of Porcine Secretin

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