scholarly journals A diagonal paper-electrophoretic technique for studying amino acid sequences around the cysteine and cystine residues of proteins

1967 ◽  
Vol 105 (3) ◽  
pp. 1203-1207 ◽  
Author(s):  
R N Perham
Keyword(s):  
Amino Acid ◽  
Paper Electrophoresis ◽  
Bovine Insulin ◽  
Amino Acid Sequences ◽  
Amino Group ◽  
Ammonia Treatment ◽  
Amino Groups ◽  
Ammonia Vapour ◽  
Electrophoretic Technique ◽  
Original Direction

1. A diagonal electrophoretic technique for studying the amino acid sequence around cysteine and cystine residues in proteins is described. The residues are first converted into S-aminoethylcysteine, and the protein is then treated with S-ethyl trifluorothioacetate, which trifluoroacetylates all the protein amino groups. The modified protein is digested enzymically and the resulting peptides are separated by paper electrophoresis. After exposure of the peptides on the paper to ammonia vapour, the electrophoresis is repeated, this time at right angles to the original direction. Peptides from which a trifluoroacetyl group is removed by the ammonia treatment will vacate the 45° diagonal formed by all other unaffected peptides owing to the exposure of an additional amino group and consequent increased electrophoretic mobility towards the cathode. Peptides containing lysine or S-aminoethylcysteine are readily purified by this technique. 2. The successful application of the technique to bovine insulin is described. 3. Various methods for distinguishing peptides containing lysine from those containing S-aminoethylcysteine in more complicated proteins are suggested and discussed.

scholarly journals The Possibility of Common Amino Acid Sequences in High-Sulphur Protein Fractions from Wool

1969 ◽  
Vol 22 (5) ◽  
pp. 1197 ◽  
Author(s):  
RL Darskus ◽  
JM Gillespie ◽  
H Lindley
Keyword(s):  
Amino Acid ◽  
High Voltage ◽  
Paper Electrophoresis ◽  
Structural Features ◽  
Amino Acid Sequences ◽  
Protein Fractions ◽  
Common Amino Acid ◽  
Amino Acid Compositions ◽  
Merino Wool ◽  
Derivatives Of

S-Carboxymethyl derivatives of the high-sulphur components of reduced Merino wool have been subdivided by chromatography into 17 fractions, the amino acid compositions of which are reported. Tryptic, chymotryptic, and thermolysin digests of each fraction have been studied by high-voltage paper electrophoresis at pH 3�5 and 6�5. The results suggest that the high-sulphur proteins consist of families of proteins probably containing common structural features. Evidence is presented that the heterogeneity of high-sulphur proteins is not artefactual.

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

1967 ◽  
Vol 102 (3) ◽  
pp. 801-814 ◽  
Author(s):  
M. C. Corfield ◽  
J. C. Fletcher ◽  
A. Robson
Keyword(s):  
Amino Acid ◽  
Cation Exchange ◽  
Protein Fraction ◽  
Exchange Resin ◽  
Cation Exchange Resin ◽  
Paper Electrophoresis ◽  
Amino Acid Sequences ◽  
Tryptic Digest ◽  
Parent Protein ◽  
Wool Proteins

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.

scholarly journals o-Quinones formed in plant extracts. Their reactions with amino acids and peptides

1969 ◽  
Vol 112 (5) ◽  
pp. 609-616 ◽  
Author(s):  
W. S. Pierpoint
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Plant Extracts ◽  
Thiol Group ◽  
Amino Group ◽  
Amino Groups ◽  
Terminal Amino Acid ◽  
Secondary Reactions ◽  
Terminal Amino ◽  
Group 5

1. The reactions of amino acids and peptides with the o-quinones produced by the enzymic oxidation of chlorogenic acid and caffeic acid have been studied manometrically and spectrophotometrically. 2. Amino acids, except lysine and cysteine, react primarily through their α-amino groups to give red or brown products. These reactions, which compete with the polymerization of the quinones, are followed by secondary reactions that may absorb oxygen and give products with other colours. 3. The ∈-amino group of lysine reacts with the o-quinones in a similar way. The thiol group of cysteine reacts with the quinones, without absorbing oxygen, giving colourless products. 4. Peptides containing cysteine react with the o-quinones through their thiol group. 5. Other peptides, such as glycyl-leucine and leucylglycine, react primarily through their α-amino group and the overall reaction resembles that of the N-terminal amino acid except that it is quicker. 6. With some peptides, the secondary reactions differ from those that occur between the o-quinones and the N-terminal amino acids. The colours produced from carnosine resemble those produced from histidine rather than those from β-alanine, and the reactions of prolylalanine with o-quinones are more complex than those of proline.

scholarly journals The amino acid sequences of erabutoxins, neurotoxic proteins of sea-snake (Laticauda semifasciata) venom

1971 ◽  
Vol 122 (4) ◽  
pp. 453-461 ◽  
Author(s):  
S. Sato ◽  
N. Tamiya
Keyword(s):  
Amino Acid ◽  
Paper Electrophoresis ◽  
Amino Acid Sequences ◽  
Edman Degradation ◽  
Terminal Sequence ◽  
Amino Acid Sequence Analysis ◽  
Sea Snake ◽  
Tryptic Digest ◽  
Laticauda Semifasciata ◽  
Erabutoxin B

1. Erabutoxin b was reduced, S-carboxymethylated and hydrolysed with trypsin. Seven tryptic fragments were isolated by column chromatography and paper electrophoresis. Some of the fragments were further hydrolysed with α-chymotrypsin, pepsin, Nagarse, Proctase A or Proctase B. The amino acid sequences of the fragment peptides were determined by subtractive Edman degradation. 2. From the tryptic digest of reduced, S-carboxymethylated and trifluoroacetylated erabutoxin b two fragments were isolated. From the amino acid composition of the fragments and from the terminal sequence studies on the reduced and S-carboxymethylated erabutoxin b, the sequence of the above seven tryptic fragments was elucidated. 3. The tryptic digestion of reduced and S-carboxymethylated erabutoxin a gave fragments, only one of which was different from the corresponding fragment from erabutoxin b. The amino acid sequence analysis of the fragment peptide showed that the only difference between erabutoxins a and b was that the former had asparagine and the latter had histidine at position 26.

A Diagonal Paper Electrophoretic Method for the Selective Isolation of Histidyl Peptides

1971 ◽  
Vol 49 (11) ◽  
pp. 1225-1232 ◽  
Author(s):  
W. H. Cruickshank ◽  
T. M. Radhakrishnan ◽  
H. Kaplan
Keyword(s):  
High Voltage ◽  
Positive Charge ◽  
Proteolytic Enzymes ◽  
Paper Electrophoresis ◽  
Selective Isolation ◽  
Amino Groups ◽  
Electrophoretic Method ◽  
Original Direction ◽  
Citraconic Anhydride ◽  
Net Positive Charge

Thiolysis of an imidazolyl-dinitrophenyl-histidyl peptide at either pH 3.5 or 6.5 results in an increase in the net positive charge on the peptide. It is shown that this property can be used to form the basis of a diagonal paper electrophoretic purification of histidyl peptides from proteins. The amino groups of the protein are first reacted with citraconic anhydride and then the citraconyl protein is reacted with 1-fluoro-2,4-dinitrobenzene. The dinitrophenyl-citraconyl protein is digested with pepsin in 10% formic acid and, if necessary, with other proteolytic enzymes. The enzymatic digest is subjected to high-voltage paper electrophoresis at either pH 3.5 or 6.5. A guide strip is removed, thiolyzed with 2-mercaptoethanol, and subjected to electrophoresis at the same pH at right angles to the original direction of electrophoresis. The histidyl peptides are displaced off the diagonal toward the cathode. The off-diagonal peptides are isolated from the original electrophoretogram by thiolysis and electrophoresis using the diagonal electrophoretogram to locate the positions of the dinitrophenyl-histidyl peptides.

scholarly journals Neurotoxins from the venoms of the sea snakes Hydrophis ornatus and Hydrophis lapemoides

1983 ◽  
Vol 213 (1) ◽  
pp. 31-38 ◽  
Author(s):  
N Tamiya ◽  
N Maeda ◽  
H G Cogger
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Paper Electrophoresis ◽  
Amino Acid Sequences ◽  
British Library ◽  
Amino Acid Residues ◽  
Protein Amino Acid ◽  
Tryptic Peptides ◽  
Sea Snakes ◽  
And Migration

The main neurotoxic components, toxins Hydrophis ornatus a and Hydrophis lapemoides a, were isolated from the venoms of the sea snakes Hydrophis ornatus and Hydrophis lapemoides respectively. The amino acid sequence of toxin Hydrophis ornatus a was deduced to be identical with that of toxin Astrotia stokesii a [Maeda & Tamiya (1978) Biochem. J. 175, 507-517] on the basis of identity of the tryptic peptide ‘map’ and the amino acid composition of each peptide. The amino acid sequence of toxin Hydrophis lapemoides a was determined mainly on the basis of identity of the amino acid compositions, mobilities on paper electrophoresis and migration positions on paper chromatography of the tryptic peptides with those of other sea-snake toxins whose sequences are known. Both toxins Hydrophis ornatus a and Hydrophis lapemoides a consisted of 60 amino acid residues and there were six amino acid replacements between them. The taxonomy of sea snakes in the Hydrophis ornatus complex has long been confused, and the above snakes were originally assigned to taxa that proved to be inconsistent with the relationships indicated by the neurotoxin amino acid sequences obtained. A subsequent re-examination of the specimens revealed an error in the original identifications and demonstrated the value of the protein amino acid sequences in systematic and phylogenetic studies. The isolation procedure and results of amino acid analysis of the tryptic peptides have been deposited as Supplementary Publication SUP 50121 (8 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1983) 209, 5.

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

1969 ◽  
Vol 115 (2) ◽  
pp. 323-334 ◽  
Author(s):  
M. C. Corfield ◽  
J. C. Fletcher
Keyword(s):  
Amino Acid ◽  
Cation Exchange ◽  
Protein Fraction ◽  
Exchange Resin ◽  
Cation Exchange Resin ◽  
Paper Electrophoresis ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Soluble Protein Fraction ◽  
Peptide Fractions

1. A chymotryptic digest of the protein fraction U.S.3. from oxidized wool was separated into 51 peptide fractions by chromatography on a column of cation-exchange resin. 2. The less acidic fractions were separated into their component peptides by a combination of cation-exchange-resin chromatography, paper chromatography and paper electrophoresis. 3. The amino acid sequences of 34 of these peptides were elucidated, and those of 14 others partially determined. 4. Overlaps between the tryptic and chymotryptic peptides from fraction U.S.3 have enabled ten extended amino acid sequences to be deduced, the longest containing 20 amino acid residues. 5. The relevance of the results to the structures of the helical and non-helical regions of wool is discussed.

scholarly journals Amino acid sequences of α-helical segments from S-carboxymethylkerateine-A. Tryptic and chymotryptic peptides from a type-II segment

1978 ◽  
Vol 173 (2) ◽  
pp. 353-363 ◽  
Author(s):  
D M Hogg ◽  
L M Dowling ◽  
W G Crewther
Keyword(s):  
Amino Acid ◽  
Deae Cellulose ◽  
Paper Electrophoresis ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Helical Conformation ◽  
Cationic Residues ◽  
Different Origins ◽  
Large Peptide

1. Amino acid-sequence studies were done on a peptide of mol.wt. approx. 12500 that was isolated from the highly helical fragments obtained by partial chymotryptic digestion of the low-sulphur proteins (S-carboxymethylkerateine-A) from wool. 2. The peptides obtained by tryptic and chymotryptic digestion of this large peptide were separated by ion-exchange chromatography on DEAE-cellulose at pH8.5 with an (NH4)(2)CO(3) concentration gradient and, where necessary, purified further by paper electrophoresis. 3. Determination of the sequences of many of these peptides showed that a high proportion of the cationic residues occurs in pairs. 4. Although two of the four S-carboxymethylcysteine residues are located in what appears to be a non-helical region near the N-terminus the other two S-carboxymethylcysteine residues occur in or near sequences suggesting a helical conformation. 5. Some peptides were obtained, in low yields, that appeared to be homologues of more major ones. These suggest either homologies in the helical portions of the low-sulphur proteins or the presence of closely related amino acid sequences in helical regions of completely different origins. 6. A partial sequence of the complete peptide is proposed.

scholarly journals N-Terminal sequences of α-crystallin

1969 ◽  
Vol 115 (4) ◽  
pp. 789-796 ◽  
Author(s):  
P. H. Corran ◽  
S. G. Waley
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Lens Protein ◽  
Amino Group ◽  
Tryptic Digest ◽  
Methionine Residue ◽  
Common Precursor ◽  
A Chain

The amino acid sequences at the N-terminal ends of the chains of the lens protein, α-crystallin, were studied. Both the main kinds of chain in bovine α-crystallin (A chains and B chains) have an N-terminal methionine residue, and the amino group is acetylated. Selective purification of the peptides in a tryptic digest of bovine α-crystallin gave a preparation consisting largely of the N-terminal peptide from the A chains, and the sequence of this peptide was elucidated. Subsequently, the N-terminal peptides were prepared from separated A and B chains. The proposed sequences are: A chain, acetyl-Met-Asp-Ile-Ala-Ile-Gln-His-Pro-Trp-Phe-Lys; B chain, acetyl-Met-Asp-Ile-Ala-Ile-His-(Pro,Trp)-Ile-Arg. The similarity between the sequences supports the hypothesis that the A and B chains are derived evolutionarily from a common precursor.

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