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.

Diagonal Chromatography for the Selective Purification of Tyrosyl Peptides

1974 ◽  
Vol 52 (11) ◽  
pp. 1013-1017 ◽  
Author(s):  
William H. Cruickshank ◽  
Barry L. Malchy ◽  
Harvey Kaplan
Keyword(s):  
Acetic Acid ◽  
Stationary Phase ◽  
Enzymatic Digestion ◽  
Acid Water ◽  
Chromatographic Purification ◽  
Amino Groups ◽  
Chromatographic System ◽  
Original Direction ◽  
Acetic Acid Water ◽  
Citraconic Anhydride

Thiolysis of an O-dinitrophenyl-tyrosyl peptide results in an increased solubility in the stationary phase of a n-butanol – acetic acid – water – pyridine (15:3:12:10) (BAWP) paper chromatographic system. It is shown that this property can be used to form the basis of a diagonal paper chromatographic purification of tyrosyl peptides from enzymatic digests of 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 subjected to enzymatic digestion, applied to a strip of Whatman 3 MM paper, thiolyzed with 5% 2-mercaptoethanol in acetone, and subjected to chromatography using BAWP as solvent. A guide strip is removed, thiolyzed with 5% 2-mercaptoethanol in 25% pyridine, and resubjected to chromatography in BAWP at right angles to the original direction of chromatography. The tyrosyl peptides are displaced off the diagonal towards the origin. The off-diagonal peptides are isolated from the original chromatogram by thiolysis and chromatography using the diagonal chromatogram to locate the positions of the dinitrophenyl-tyrosyl peptides.

scholarly journals A CYTOCHEMICAL STUDY OF CYTOPLASMIC BASIC PROTEINS IN THE ASCIDIAN OOCYTE

1965 ◽  
Vol 25 (2) ◽  
pp. 319-326 ◽  
Author(s):  
Richard Davenport ◽  
Janice C. Davenport
Keyword(s):  
Positive Charge ◽  
Amino Groups ◽  
Acid Dyes ◽  
Fast Green ◽  
Cytochemical Study ◽  
Basic Proteins ◽  
Protein Molecules ◽  
Nucleoprotein Complex ◽  
High Concentrations ◽  
Net Positive Charge

The cytoplasm of young oocytes of the ascidians contains high concentrations of proteins which are stainable with alkaline fast green at pH 8.1 and above. These proteins cannot be stained even with acid dyes at low pH unless RNA is removed. Deamination and formalin blockage of amino groups is incapable of destroying the net positive charge on these protein molecules in the presence of RNA, but these treatments destroy the charge if RNA is removed. It is therefore concluded that basic proteins and RNA exist as a nucleoprotein complex in the ribosomes of these young oocytes. The detectable RNA of the mature oocytes and unfertilized eggs shows no evidence of being associated with basic proteins.

Morphine-like activity of sheep β-lipotropin and of its tryptic fragments

1977 ◽  
Vol 55 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Nabil G. Seidah ◽  
Martin Lis ◽  
Christina Gianoulakis ◽  
Richard Routhier ◽  
Suzanne Benjannet ◽  
...  
Keyword(s):  
High Voltage ◽  
Vas Deferens ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Amino Groups ◽  
Mouse Vas Deferens ◽  
Active Peptides ◽  
Active Core ◽  
Citraconic Anhydride ◽  
High Voltage Electrophoresis

Sheep β-lipotropin (β-LPH) (sequence 1–91) was selectively cleaved with trypsin after blocking the ε-amino groups of lysine with citraconic anhydride. The resulting peptides were purified by a combination of cation-exchange chromatography and high-voltage electrophoresis. The purified fragments were then tested for their morphine-like activity in the mouse vas deferens bioassay. The active peptides were 61–91 and 61–80. All other regions of the molecule investigated were not active. Moreover, the peptides 61–91 and 61–80 were about as active as the synthetic methionine-enkephalin, and in turn these were about 100 times more active than β-LPH itself. The inhibition of electrically stimulated mouse vas deferens by these peptides is reversed by naloxone, and suggests a competitive character of interaction. It is thus concluded that the active core for the morphine-like activity in the mouse vas deferens bioassay is the fragment 61–65 of β-LPH.

scholarly journals The amino acid sequence of pike-whale (lesser-rorqual) pancreatic ribonuclease

1976 ◽  
Vol 157 (2) ◽  
pp. 317-323 ◽  
Author(s):  
M Emmens ◽  
G W Welling ◽  
J J Beintema
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
High Voltage ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Paper Electrophoresis ◽  
British Library ◽  
Balaenoptera Acutorostrata ◽  
Peptide Bonds ◽  
Pancreatic Ribonuclease

Pancreatic RNAase (ribonuclease) from the pike whale (lesser rorqual, Balaenoptera acutorostrata) was isolated by affinity chromatography. The protein was digested with different proteolytic enzymes. Peptides were isolated by gel filtration, preparative high-voltage paper electrophoresis and paper chromatography. The amino acid sequence of peptides was determined by the dansyl-Edman method. Although we do not have an amino acid composition for the whole protein, all peptide bonds were overlapped by one or more peptides. Residues 85-96 are bridged by a peptide of unstaisfactory composition and the sequence here depends, at least in part, on homology for its confirmation. Another region in which a similar situation obtains is residues 39-40. This pancreatic RNAase differs at 24-33% of the positions from all other mammalian pancreatic RNAases sequenced to date, except for pig RNAase, from which it differs by 19%. This indicates that whale RNAase has evolved independently during the larger part of the evolution of the mammals. Lesser-rorqual pancreatic RNAase is partially glycosidated (30%) at asparagine-76 in an Asn-Ser-Thr sequence (residues 76-78). Pig RNAase also has carbohydrate attached to asparagine-76 and is identical with lesser-rorqual RNAase in residues 76-98. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50066 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms ginen in Biochem. J. (1976) 135, 5.

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.

The Action of Thrombin on Modified Fibrinogen

1983 ◽  
Vol 49 (03) ◽  
pp. 208-213
Author(s):  
A J Osbahr
Keyword(s):  
Physical Properties ◽  
Negative Charge ◽  
Lysine Residue ◽  
Fibrinopeptide A ◽  
Amino Groups ◽  
Lysine Residues ◽  
Citraconic Anhydride

SummaryThe modification of canine fibrinogen with citraconic anhydride modified the ε-amino groups of the fibrinogen and at the same time generated additional negative charges into the protein. The addition of thrombin to the modified fibrinogen did not induce polymerization; however, the fibrinopeptide was released at a faster rate than from the unmodified fibrinogen. The physical properties of the citraconylated fibrinogen were markedly altered by the modification of 50-60 lysine residues in one hour. A modified fibrinopeptide-A was released by thrombin from the modified fibrinogen and was electrophoretically more anionic than the unmodified fibrinopeptide-A. Edman analysis confirmed the modification of the lysine residue present in the peptide. The rate of removal of citraconylated fibrinopeptide-A from modified fibrinogen by thrombin was 30 to 40 percent greater than the cleavage of unmodified fibrinopeptide-A from unmodified fibrinogen. However, the modification of 60 or more lysine residues in the fibrinogen produced a decrease in the rate of cleavage of citraconylated fibrinopeptide-A. The results suggest that additional negative charge in the vicinity of the attachment of fibrinopeptide-A to canine fibrinogen aids in the removal of the peptide by thrombin.

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