Determination of the carboxyl termini of proteins with ammonium thiocyanate and acetic anhydride, with direct identification of the thiohydantoins

Cromwell, Laurence D.; Stark, George R.

doi:10.1021/bi00840a012

Cited by 61 publications

(25 citation statements)

References 19 publications

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“…The chromatograms were developed using system A of Cromwell and Stark [24]. The spots were scraped off, eluted with methanol and immediately quantified spectrophotometrically as described above and after back hydrolysis [25] to amino acids.…”

Section: Sequence Analysismentioning

confidence: 99%

See 1 more Smart Citation

The amino acid sequence of wheat histone H2A(1). A core histone with a C-terminal extension

A¹,

Wf²,

C³

1985

Eur J Biochem

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1. The complete amino acid sequence (145 residues) of histone variant H2A,,, from wheat germ Triticum 2. The sequence of histone variant H2A(,, differs from the homologous calf histone in 61 amino acid positions. aestivum cultivar T 4 has been established from Edman degradation of large overlapping fragments.These differences include an extension of H2A,,, by 19 amino acids at its carboxyl end.Histones in general are considered to play a structural rather than a regulatory role in chromatin [l], however the widespread occurrence of histone variants (isohistones), (for review see [2,31) suggests that variants may modulate the conformation of nucleosomes, and in this way modify their susceptibility to regulatory factors. We have established previously the presence of a number of isohistones of the H2A class in wheat embryos [2, 41. We are reporting now the complete structure of one of these isohistones. This structure constitutes a new histone type in that its C-terminal region is extended beyond the size hitherto considered typical for core histones of that group. MATERIALS AND METHODS Isolation of histone H2A(,,Wheat germ (Triticum aestivum) was obtained from Sasko Mills (Rondebosch, Cape). This material contained at least 80% of the cultivar T 4. Chromatin was isolated essentially according to the method of Johns and Butler [5] with some modifications as described previously [4].Exclusion chromatography of histones was performed at room temperature on a column (4.5 x 80 cm) of Bio-Gel P-60 (100-200 mesh). Approximately 200 mg of total acid-extracted (0.2 M H2S04) histone was dissolved in 10 ml of 6 M urea containing 2% (v/v) 2-mercaptoethanol, loaded onto the column and eluted with 0.02 M HCI (Fig. 1 a).Cation-exchange chromatography was carried out using LKB CM-Trisacryl M equilibrated with 0.05 M sodium acetate adjusted to pH 5.00 with HC1. A linear NaCl gradient was used to elute the H2A(,,-enriched fraction B (Fig. 1 b). Fraction B was then further purified by passage over a BioGel P-60 column equilibrated with 0.02 M HCl, 0.05 M NaCl as described previously [6]. The eluant contained in addition, 10 mM sodium bisulfite, 1 mM PhMeS02F and 1 mM Natosyllysylchloromethane, solubilized first in a small volume of propan-1-01 (Fig. 1 c). The H2A(,,-containing fraction was sometimes recycled over the P-60 column if there was still some contamination evident on gel electrophoresis. Analytical proceduresPolyacrylamide gel electrophoresis (PAGE) employing the detergent Triton X-100 was performed in glass tubes (9 x 0.5 cm) [7, 81.Histone samples were hydrolysed under vacuum for 24 h at 110°C in constant-boiling HCl containing 0.025% (w/v) phenol as antioxidant unless otherwise stated. The analyses were done with either ninhydrin methodology (Beckman 119 automatic analyser) or using hypochlorite and o-phthalaldehyde post-column fluorescence detection (Waters Associates HPLC amino acid analyser [9]).Fragmentation of H2A(,, N-Bromosuccinimide (SucNBr) cleavage [lo] was performed as described previously [ll]. The increase...

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Section: Sequence Analysismentioning

confidence: 99%

“…The combined extracts were evaporated under a stream of nitrogen at 40°C and redissolved in a small volume of methanol. The ultraviolet absorption spectrum was determined and the 2-thiohydantoin quantified assuming a molar absorption coefficient of 17500 M-' cm-' at 263 nm [24]. The cycle was then repeated.…”

Section: Sequence Analysismentioning

confidence: 99%

The amino acid sequence of wheat histone H2A(1). A core histone with a C-terminal extension

A¹,

Wf²,

C³

1985

Eur J Biochem

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“…The uv spectra of the synthetic thiohydantoins have a maximum absorption at approximately 266 nm, which is the characteristic absorption of typical thiohydantoins (5). Amino acid analysis of purified amino acid thiohydantoins after hydrolysis gave the expected phenylthiocarbomyl (PTC) amino acid derivatives.…”

Section: Derivatization Of the Activated Amino Acids By Tms-itc Ammomentioning

confidence: 99%

A Method for Preparation of Amino Acid Thiohydantoins from Free Amino Acids Activated by Acetyl Chloride for Development of Protein C-Terminal Sequencing

Liang

1997

Analytical Biochemistry

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“…At present, this procedure is limited to the sequencing of peptides containing the hydrophobic amino acids, and trp and tyr. This limitation arises because only the thiohydantoin derivatives of Gly, Val, Ala, Leu, lie, Phe, Tyr, Lys, Met, His, Trp, Asn, and Gin are readily obtainable (and unequivocally characterized as such) by the application of the original thiocyanate degradation procedure (or minor variations thereof) to the parent amino acids (6)(7)(8)21,25,26,39). Much confusion still exists as to whether or not the thiohydantoins of Asp, Glu, Ser, Thr, and Arg can similarly be prepared, and that of pro has until recently been obtained only with difficulty (40,41).…”

Section: Limitations Of the Thiocyanate Degradation Proceduresmentioning

confidence: 99%

The Protein Protocols Handbook

1996

Springer Protocols Handbooks

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hardcover) ISBN 0-89603-339-2 (combbound) PrefaceIn The Protein Protocols Handbook, I have attempted to provide a cross-section of analytical techniques commonly used for proteins and peptides, thus providing a benehtop manual and guide both for those who are new to the protein chemistry laboratory and for those more established workers who wish to use a technique for the first time.We each, of course, have our own favorite, commonly used gel system, gelstaining method, blotting method, and so on; I'm sure you will find yours here. However, I have also described a variety of altematives for many of these techniques; though they may not be superior to the methods you commonly use, they may nevertheless be more appropriate in a particular situation. Only by knowing the range of techniques that are available to you, and the strengths and limitations of these techniques, will you be able to choose the method that best suits your purpose.All chapters are written in the same format as that used in the Methods in Molecular Biology series. Each chapter opens with a description of the basic theory behind the method being described. The Materials section lists all the chemicals, reagents, buffers, and other materials necessary for carrying out the protocol. Since the principal goal of the book is to provide experimentalists with a full account of the practical steps necessary for carrying out each protocol successfully, the Methods section contains detailed step-by-step descriptions of every protocol that should result in the successful execution of each method. The Notes section complements the Methods material by indicating how best to deal with any problem or difficulty that may arise when using a given technique, and how to go about making the widest variety of modifications or alterations to the protocol.In general, I have avoided analytical techniques that require expensive specialist hardware. Such techniques are described in specialist volumes in

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Determination of the carboxyl termini of proteins with ammonium thiocyanate and acetic anhydride, with direct identification of the thiohydantoins

Cited by 61 publications

References 19 publications

The amino acid sequence of wheat histone H2A(1). A core histone with a C-terminal extension

The amino acid sequence of wheat histone H2A(1). A core histone with a C-terminal extension

A Method for Preparation of Amino Acid Thiohydantoins from Free Amino Acids Activated by Acetyl Chloride for Development of Protein C-Terminal Sequencing

The Protein Protocols Handbook

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