A method for the quantitative determination of <i>N</i>-terminal amino acids based on radioisotope dilution

Callewaert, George L.; Vernon, C. A.

doi:10.1042/bj1070728

Cited by 6 publications

(2 citation statements)

References 4 publications

(4 reference statements)

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“…I n terms of man-hours the most time-consuming opera- Analysis of Phenylthiohydantoins The succew of protein and peptide sequencers depends on the availability of reliable methods for identifying phenylthiohydantoins, and for this reason, much effort has gone into the development of new analytical methods recently [9,. Our choice of the isotope dilution method [9] was dictated by the instrumentation available and the fact that it can, in principle, be made quantitative [21]. An advantage of the procedure is that only compounds derived from 3H-labelled phenyl isothiocyanate can be detected.…”

Section: The Reaction Columnmentioning

confidence: 99%

Solid‐Phase Edman Degradation

Laursen

1971

European Journal of Biochemistry

415

156

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A modification of the Edman degradation is described whereby peptides are degraded sequentially on a solid support by a machine, the solid-phase peptide sequencer. Reagents are pumped through a column containing the resin-bound peptide, and the amino acid thiazolinones released after each degradative cycle are collected on a fraction collector for subsequent analysis. A complete cycle requires 2 h, and all operations are controlled automatically by a programmer. An improved peptide resin is described, to which peptides can be attached in yields ranging from about 7001, for the 30 amino acid insulin B-chain to nearly lOOO/, for smaller peptides. Of the 18 amino acids examined so far, glycine, alanine, phenylalanine, tyrosine, valine, leucine, isoleucine, proline, methionine, asparagine, glutamine, histidine, lysine, threonine, serine and cysteic acid are degraded normally ; the phenylthiohydantoin of threonine is partially dehydrated and that of serine is almost completely destroyed by trifluoroacetic acid, however. The phenylthiohydantoin of glutamic acid is not detected, because it remains bound by its y-carboxyl group to the resin. When aspartic acid is encountered in peptides, further degradation is completely blocked, presumably as a result of the fi-carboxyl group having formed a cyclic h i d e . Using the sequencer and unpurified reagents, it was possible to degrade entirely a 0.24 pmole sample of the 21 amino acid insulin A-chain (oxidized) and to analyze the products within a 3-day period. Degradative yields averaged 94O/, per cycle. Similarly, 0.1 pmole of the insulin B-chain was degraded through 18 cycles before identification of the phenylthiohydantoins became ambiguous. The sequencer is simple in design and operation, and is inexpensive to construct. The scope and limitations of the method are discussed.The Edman degradation [l], long one of the most useful methods for amino acid sequence determination, has undergone almost continual modification and improvement over the years, culminating in the protein sequenator [2], which is capable of performing as many as 60 degradative cycles automatically. Concurrently with Edman and Begg's report [2], several laboratories [3-51 demonstrated the feasibility of degrading peptides attached to an insoluble resin, an extension of the solid-phase peptide synthesis of Merrifield [6]: The usefulness of the solid-phase method lies in its applicability to smaller peptides not adequately handled by the protein sequenator [2] and its potential for being automated.Our objective in developing the solid-phase Edman degradation has been to provide a rapid, simple and economical method for sequencing peptides containing up to about 15-20 amino acids, the size range normally found in enzymic digests of proteins. Our progress towards this goal is described here.A preliminary report of this work has been published [7].

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Section: The Reaction Columnmentioning

confidence: 99%

Solid‐Phase Edman Degradation

Laursen

1971

European Journal of Biochemistry

415

156

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“…We report the full experimental details of a method based on quantitative N-terminal analysis using radioisotope dilution. The method has been described in outline previously [8].…”

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An Assessment of Some of the Methods Available for the Determination of Molecular Weights of Proteins as Applied to Aspartate Aminotransferase from Pig Heart

Banks¹,

Doonan²,

Flögel³

et al. 1976

European Journal of Biochemistry

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The isopotential specific volume of cytoplasmic aspartate aminotransferase from pig heart was found to be 0.763 ml g‐1 whereas the value of the apparent specific volume obtained by summation of contributions from each type of amino acid in the protein is 0.735 ml g‐1. Use of the experimentally determined isopotential specific volume largely abolishes the discrepancy between a previously reported value of the molecular weight of the native (dimeric) enzyme and that of the enzyme subunit obtained from its primary structure (46300). A new non‐empirical method based on quantitative N‐terminal analysis involving radioisotope dilution is described for the determination of subunit molecular weight of proteins. The method is capable of considerable accuracy and sensitivity. Some of the methods available for the determination of molecular weights and subunit compositions of proteins are discussed.

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Labeled proteins, their preparation and applications

Káš

Rauch

1983

Topics in Current Chemistry

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A method for the quantitative determination of N-terminal amino acids based on radioisotope dilution

Cited by 6 publications

References 4 publications

Solid‐Phase Edman Degradation

Solid‐Phase Edman Degradation

An Assessment of Some of the Methods Available for the Determination of Molecular Weights of Proteins as Applied to Aspartate Aminotransferase from Pig Heart

Labeled proteins, their preparation and applications

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