On the Mechanism of the Phenyl Isothiocyanate Degradation of Peptides.

Edman, Pehr; Lindberg, Bengt; Waterman, H. I.; Eliasson, N. A.; Thorell, B.

doi:10.3891/acta.chem.scand.10-0761

Cited by 328 publications

(81 citation statements)

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“…Edman Degradation (Subtractive) [12,13] Subtractive Edman degradation was the main tool used in the sequencing of peptides. Depending on the number of steps to be performed, 0.1 -1 .O pmol peptide were adjusted to pH 6-9.5 with NaOH, evaporated to dryness and dissolved in 0.4 ml 50°/, pyridine in water.…”

Section: Paper Chromatography Of Peptidesmentioning

confidence: 99%

See 1 more Smart Citation

Sequence of the Murein · Lipoprotein and the Attachment Site of the Lipid

Braun

Bosch

1972

European Journal of Biochemistry

125

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The primary structure of a covalent lipoglycoprotein (murein . lipoprotein) from the cell wall of Escherichia coli is presented. The amino acid sequence consists of57 residues. The lipid is attached to the N-terminal serine and the murein is bound to the 6-amino group of the C-terminal lysine of the lipoprotein. The repetitive design of the sequence suggcsts a very conservative evolution in which a structural gene coding for 15 amino acids was duplicated once and then only half of this gene, thus coding for 7 amino acids was fused four times with the first 29 amino acids. Some deletions but only few exchanges of amino acids apparently occurred.This structural membrane lipoprotein aggregates strongly when released from the murein by lysozyme digestion. The molecular weight was therefore determined in 2O/,, 0.5O/, and O.lo/o dodecylsulfate by polyacrylamide gel electrophoresis and gel chromatography. The molecular weight of 13000 thus found deviated from that of I0000 calculated from the sum of the amino acids, the lipid as known so far and the part of the murein remaining bound to the lipoprotein. This was not due to a different amount of dodecylsulfate binding by the lipoprotein compared with proteins containing no lipid but may be due to a more spherical shape of the small molecule in contrast to the rod-like shape of standard proteins in dodecylsulfate.The lipoprotein which is covalently attached to the murein [I] of the outer membrane of Escherichia coli [2--51 is one of thc few membrane proteins isolated so far in pure form. Even soluble lipoproteins, as they occur e.g. in the serum, are difficult to purify and only rather limited information about their structure is available today [6,7]. In addition, in the lipoprotein we are working, the lipid is very probably covalently bound [2,8] which was not found in any other lipoprotein mixture studied so far. I n order to elucidate the attachment site of the lipid and the mode of binding on the murein, we studied the amino acid sequence of the polypeptide chain. In a previous paper [Y] we discussed some features of the unusual and very interesting results especially with regard to the repetitive design of the sequence and the relevance of this study to the structure of membrane proteins and membranes in general. Now we present the experimental evidence together with a full account of the results.

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Section: Paper Chromatography Of Peptidesmentioning

confidence: 99%

“…Pure peptides were sequenced by stepwise degradation with phenylisothiocyanate [12,13]. The released N-terminal amino acid was mostly identified by analysis of an aliquot of the remaining peptide after acid hydrolysis (subtractive method).…”

Section: Sequence Determination Of the Lipoproteinmentioning

confidence: 99%

Sequence of the Murein · Lipoprotein and the Attachment Site of the Lipid

Braun

Bosch

1972

European Journal of Biochemistry

125

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“…Identification of the phenylthiohydantoin derivatives was carried out with thin layer chromatography on silica gel. The solvent systems used were chloroform-methanol (9: 1, v/v), chloroform-formic acid (100 : 5, v/v) [31] and the systems D and E of Edman and Sjoquist [32] originally devised for paper chromatography of phenylthiohydantoins. Identification made by thin layer chromatography was often checked by amino acid analysis of an alkaline hydrolysate of the phenylthiohydantoin [33] or by amino acid analysis of an acid hydrolysate of the remaining peptide.…”

Section: Amino Acid Analysismentioning

confidence: 99%

Carboxyl‐Terminal Region of Human and Bovine Erythrocyte Carbonic Anhydrases

Nyman¹,

Strid²,

Westermark³

1968

European Journal of Biochemistry

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1. Carboxyl-terminal sequences have been determined for forms B and C of the human erythrocyte carbonic anhydrase and form B of the bovine enzyme. The proteins were degraded with CNBr, and peptide fragments of 20 residues were isolated from human enzyme C and bovine enzyme B while the human B form gave a fragment consisting of only 19 residues. The sequences of these fragments were determined and by carboxypeptidase digestion of the whole proteins it was shown that they represent the carboxyl-terminal part of the proteins.2. Comparison of the sequences of the enzymes gives strong support for evolutionary homology. Greater similarities in sequence are found between bovine carbonic anhydrase and human carbonic anhydrase C than between the two forms of the human enzyme.3. All sequences cont,ain an Arg-Pro peptide bond. I n two of the sequences this bond was found to be hydrolyzable with trypsin.Several forms of carbonic anhydrase have been isolated from bovine and human erythrocytes [l -41. They have a molecular weight of about 30,000 [3,5,6] and contain in each molecule one zinc atom essential for catalytic activity [7-91. Of the bovine enzyme two electrophoretically different forms have been described. They appear to be identical in catalytic properties [l] and amino acid composition [5] and are named A and B (for nomenclature see [5]) the latter being the most abundant. Of the human carbonic anhydrases, forms B and C have becn the most studied. Form C bears resemblance to the bovine enzyme in catalytic properties [lo-121. The two human forms appear to have the same overall conformation [13,14] but differ markedly in several other respects. Besides considerable deviations in amino acid composition [3,5,15] there is evidence for structural dissimilarities in their active site regions as shown by differences in their properties as catalysts [12,16] MATERIALS AND METHODS Preparation of Carbonic AnhydrasesHuman erythrocyte carbonic anhydrases B and C were purified as described elsewhere [2]. The preparations were performed on a larger scale than earlier, starting from an amount of red cells corresponding to 20-25 liters of whole blood. The vast amount of human enzyme filtrate from the SE-Sephadex chromatography was concentrated either with Sephadex G-25 according to Flodin et al. [22] or by adsorption on a DEAE-Sephadex A-50 column in 5 mM borateNaOH buffer, pH 9.4, followed by elution with 0.25 M Tris-C1, pH 8.0, and subsequent dialysis.Bovine erythrocyte carbonic anhydrase U was prepared according to Lindskog [I] with chromatography on DEAE-cellulose as the final step. As a rule, the peak containing form B could be cut in such a way that there were no appreciable amounts of form A contaminating. Sometimes, when the separation was not satisfactory, a rechromatography on a longer DEAE-cellulose column was employed. Garboxypeptidase Digestion of Carbonic AnhydrasesPreliminary experiments showed a very slow release of ninhydrin-positive material from native human carbonic anhydrase B in the presence of carbox...

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“…As this system used the Edman degradation method, some amino acid residues, such as cysteine, were destroyed because of their susceptibility to the acidic hydrolysis and were undefined (Edman 1956). Thus several amino acid residues were not determined.…”

Section: Partial Amino Acid Sequence Of Pepmentioning

confidence: 99%