[37] Peptide mapping with automatic analyzers: Use of analyzers and other automatic equipment to monitor peptide separations by column methods

Hill, Robert L.; Delaney, Robert

doi:10.1016/s0076-6879(67)11039-2

Cited by 76 publications

(16 citation statements)

References 14 publications

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“…The flow rate was 20 ml/hr. Peptide elution was monitored by automatic base hydrolysis and ninhydrin analysis on 9% of the eluate (Hill and Delaney, 1967). Three peaks were observed by ninhydrin analysis and the corresponding fractions were pooled and lyophilized.…”

Section: Methodsmentioning

confidence: 99%

Carboxypeptidase inhibitor from potatoes. The effects of chemical modifications on inhibitory activity

Gm¹,

Ako²,

Dt³

et al. 1976

Biochemistry

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The carboxypeptidase inhibitor from Russet Burbank potatoes was subjected to a variety of chemical modifications and their effects on inhibitory activity toward carboxypeptidases A and B were determined. The importance of the alpha carboxylate of glycine-39 to the enzyme-inhibitor interaction was demonstrated by the observation that a derivative in which all four carboxyls were modified was inactive whereas a derivative in which only the beta carboxylates of aspartic acid residues 5, 16, and 17 were masked retained full inhibitory activity. In addition to these three aspartic acid residues, lysine residues 10 and 13, histidine residues 3 and 15, and arginine-32 were modified and residues 1-5 removed with little effect on inhibitory activity. Tryptophan residues 22 and 28 did not react with 2-hydroxy-5-nitrobenzyl bromide or o-nitrophenylsulfenyl chloride, and thus are presumed to be buried in the interior of the inhibitor molecule. Although tyrosine-37 was acetylated without affecting binding characteristics, both carboxypeptidases A and B protected against deacetylation by hydroxylamine. These studies indicate that the carboxyl terminal region of the inhibitor is in contact with enzyme in the complex. The parallel effects of modifications on inhibitory activity toward carboxypeptidases A and B support previous evidence that both enzymes utilize the same binding site on the inhibitor [C. A. Ryan (1971), Biochem. Biophys. Res. Commun. 44, 1265].

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Section: Methodsmentioning

confidence: 99%

Carboxypeptidase inhibitor from potatoes. The effects of chemical modifications on inhibitory activity

Gm¹,

Ako²,

Dt³

et al. 1976

Biochemistry

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“…The soluble tryptic (Tp) peptides were fractionated on a 0.9 X 20 cm column of Dowex 50-X8 equilibrated in 0.05 N pyridine acetate (pH 2.5) and eluted with a double linear gradient of pyridine acetate buffers as described by Bradshaw et al (1969). The separation of peptides was monitored with a Technicon Autoanalyzer equipped for automatic alkaline hydrolysis and ninhydrin analysis (Hill and Delaney, 1967). The isolated peptides were recovered after removal of the solvent by rotary evaporation.…”

Section: Methodsmentioning

confidence: 99%

Purification, characterization, and partial amino acid sequence of nerve growth factor from cobra venom

Hogue-Angeletti¹,

Frazier²,

Jacobs³

et al. 1976

Biochemistry

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The nerve growth factor (NGF) from Naja naja (cobra) venom has been purified and its structure compared to the NGF from mouse submaxillary gland. A two-step purification procedure has been devised, consisting of a gel filtration step in 1 M acetic acid followed by chromatography of the active pool on carboxymethylcellulose at pH 5. The molecular weight of the native protein was found to be 28000, and this value was reduced by approximately one-half under denaturing conditions. These values are comparable to those obtained for mouse 2.5S or betaNGF. Tryptic peptide maps of S-[14C]carboxymethyl NGF gave the number of labeled peptides expected for a structure composed of two identical or very similar subunits. Thus, the quaternary structures of mouse and cobra NGF are the same. Cyanogen bromide (CNBr) treatment of Naja naja NGF produced three fragments, of which two were purified to homogeneity. These fragments and the whole protein were analyzed in the automated protein Sequencer. The amino-terminal CNBr fragment of the protein was also subjected to digestion by thermolysin and the resultant peptides were purified and characterized. These data plus those from the characterization of the tryptic peptides provided the basis of the construction of a tentative primary structure of Naja naja NGF which is approximately 60% identical with mouse NGF.

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“…The column was developed with a four-chamber gradient containing 200 mL each of 0.05 M pyridine/acetate, pH 2.5, 0.2 M pyridine/acetate, pH 3.1, 0.5 M pyridine/acetate, pH 3.75, and 2.0 M pyridine/acetate, pH 5.0. The separation of the peptides was monitored with a Technicon autoanalyzer (Hill & Delaney, 1967). The resulting peptides were desalted with Sephadex G-25 (1 X 140 cm) in 50% acetic acid.…”

Section: Münger and Lerchmentioning

confidence: 99%

Copper metallothionein from the fungus Agaricus bisporus: chemical and spectroscopic properties

Muenger¹,

Lerch²

1985

Biochemistry

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[37] Peptide mapping with automatic analyzers: Use of analyzers and other automatic equipment to monitor peptide separations by column methods

Cited by 76 publications

References 14 publications

Carboxypeptidase inhibitor from potatoes. The effects of chemical modifications on inhibitory activity

Carboxypeptidase inhibitor from potatoes. The effects of chemical modifications on inhibitory activity

Purification, characterization, and partial amino acid sequence of nerve growth factor from cobra venom

Copper metallothionein from the fungus Agaricus bisporus: chemical and spectroscopic properties

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