Sequential Edman degredation]

Since Edman's first publication in 1950, the stepwise degradation of proteins and peptides is universally performed by protein chemists. We extensively reviewed the different manual degradations. We take two examples of manual degradation: a semi-micromethod and a micromethod in order to illustrate the evolution of manual degradation. The "dansyl-Edman" procedure proposed by Hartley in 1963 completes the manual N-terminal determination of peptides. We describe the different procedures of identification of PTH-amino acids: paper chromatography, thin layer chromatography, gas chromatography and liquid chromatography under high pressure and various modified Edman degradation procedures. Possibilities and limits of the liquid phase Sequenator of Edman reported in 1967 and the solid phase Sequencer of Laursen reported in 1971 are also considered in detail.     

Fluorescence detection of amino acids in the postcleavage conversions for manual sequencing of a peptide

A modified Edman degradation method where fluorescent derivatives of amino acids were generated from the postcleavage products of a peptide is described. In the method, the target peptide was applied onto double glass fiber membranes in a small filter disk (4 mm i.d.) and then treated with small amounts of reagents for the manual sequencing of the peptide. The anilinothiazolinone (ATZ) of N-terminus amino acid residue after the isolation from the solid-phase membranes was reacted with a primary amine, 4-(1′-cyanoisoindolyl)aniline (CIA), to form a more stable and sensitive fluorescent derivative, phenylthiocarbamoyl-CIA. An average yield of 85% was obtained in neutral pH conditions for the CIA reaction. The ATZ-CIA-amino acids were separated by reversed-phase liquid chromatography and detected by fluorometry. The lower limits of the detection for amino acids after the Edman degradation were 0.16 to 0.52 pmol (signal/noise ratio = 3) on the column. The sensitivity was approximately 10 times higher than ultraviolet absorbance detection of phenylthiohydantoin products in the conventional Edman degradation. The suitability of the method was demonstrated by the sensitive manual sequencing of insulin chain B composed of 30 amino acids.     

A simple,rapid, manual peptide microsequencing procedure

A method is described for manual Edman degradation at the nanomole level. The method is simple, requiring only two extraction steps which minimize extraction loss, time, and there is no need for a conversion step of the thiazolinones to the thiohydantoins. Two different back hydrolysis procedures are compared and their relative merits discussed. The procedure requires no specialized reagents or equipment other than an amino acid analyzer.     

Simplified methods for automated ion-exchange separation of peptides and accelerated manual Edman degradations

An apparatus for the automated separation of peptides and one for the manual Edman degradation of peptides is presented. Both are composed of relatively simple and inexpensive items and are capable of being constructed in most laboratories. Each may also serve as an important laboratory teaching and demonstration device. As many as five peptides may be simultaneously degraded with the manual Edman apparatus and as many as six degradations may be performed daily on each peptide.     

A manual method of sequential Edman degradation followed by dansylation for the determination of protein sequences

A modification of the manual method of sequential Edman degradation followed by dansylation for sequencing peptides has been developed for use on long polypeptides and intact chains of proteins. This method permits the sequence of 15 to 20 residues from the amino-terminal end of the chain to be determined.     

Identification and synthesis of a tripeptide in ECUM fluid of an upemic patient

An unidentified ninhydrin and Pauly reaction positive substance of basic nature was found in the ECUM fluid of an uremic patient. This substance was isolated from ECUM fluid by the methods of ultrafiltration method and gel-filtration, and identified as H-His-Gly-Lys-OH by amino acid analysis, manual Edman degradation method and physical constants and analytical data of synthetic tripeptide.     

Improved manual sequential analysis of peptides.

Organic solvents can affect the efficiency of peptide sequencing by the Edman degradation method by altering peptide extractive losses during manual sequence analysis. We present a modified phenylisothiocyanate procedure for the degradation of one to five peptides simultaneously with high repetitive yield (90–95%) with an average time per cycle of 75 min. Improvement in average yield per cycle (repetitive yield) varies with the choice of solvent and nature of peptide under investigation. The degree of extraction of a particular thiazolinone similarly can be improved by the selection of appropriate solvents.     

A fast and sensitive micromethod for the manual sequencing of peptides using O-phthalaldehyde as derivatizing reagent

A general procedure for the manual sequencing of peptides using the fluorogenic reagent O-phthalaldehyde (OPA) is described. The method can be applied in two different ways. One of them involves back hydrolysis of the anilinothiazolinones resulting from the Edman degradation of the peptide and subsequent detection of the free amino acids as OPA derivatives. The other is a subtractive analysis in which the amino acid composition of the remaining peptide is determined after each degradation cycle. The direct procedure can be coupled to the subtractive one in order to assure the accuracy of the sequence analysis. The method is fast and simple, and allows determination of 10 pmol of amino acid per cycle using standard reagents and instrumentation. Sensitivity can be greatly enhanced provided that ultrapure chemicals are employed. Small peptides (8-10 residues) were sequenced from 200 pmol sample, using a high-performance liquid chromatography assembly coupled to a fluorescence detector.     

The N, O-diacetylmuramidase of Chalaropsis species. V. The complete amino acid sequence.

The complete amino acid sequence of lysozyme Ch has been established by a combination of automated and manual Edman degradation and carboxypeptidase digestion.(see article)There is a single disulfide bond in the center of the molecule. The enzyme has 211 residues with a calculated molecular weight of 22,415. Lysozyme Ch has an amino acid sequence that is totally different from all other lysozymes whose sequences are known.     

Determination of Phosphorylation Sites in Peptides and Proteins Employing a Volatile Edman Reagent

A manual Edman degradation protocol has been developed that allows the identification of phosphorylation sites in ³²P-labeled peptides at the subpicomole level. By using both a volatile reagent, trifluoroethyl isothiocyanate, and volatile buffers, extraction steps are rendered unnecessary and cycle times can be reduced to 45 min. The protocol was employed to identify the site of phosphorylation in phosphoserine- and phosphotyrosine-containing peptides.     

Manual N-terminal microsequencing of proteins electroeluted from polyacrylamide gel slices

A simple and rapid procedure for preparation of proteins for manual microsequencing using sodium dodecyl sulfate gel electrophoresis is described. The procedure involves pre-electrophoretic labeling of the protein amino groups with a coloured Edman reagent, disk electrophoresis for purification or fractionation of the proteins, and reversed electrophoretic transfer of the separated protein from gel slices into a small volume of buffer (100 to 150 microliter) using a discontinuous conductivity gradient to recover the proteins. The pre-electrophoretic labeling facilitates location of the separated proteins in the gel and the monitoring of their complete electroelution. The isolated proteins are separated from excess of salts by acetone precipitation and solvent partitioning in pyridine/water (1:1) and subjected to manual DABITC/PITC degradation.     

Amino acid sequence of the Pseudomonas putida cytochrome P-450. I. Sequences of tryptic and clostripain peptides

In order to elucidate the complete amino acid sequence of Pseudomonas putida cytochrome P-450, tryptic digestion was performed on the S-carboxymethylated enzyme. Although cleavage did not occur at every lysyl and arginyl bond, 31 tryptic peptides ranging in size from 1 to 55 residues were isolated. These were sequenced by manual Edman degradation and carboxypeptidase digestion. Overlaps of some od these tryptic peptides were obtained by data obtained from partial Edman degradation and amino acid composition of the clostripain cleavage products. These results, together with data from the cyanogen bromide and acid cleavage peptides reported in the accompanying paper, established the complete amino acid sequence of P. putida cytochrome P-450.     

Purification of the STB enterotoxin of Escherichia coli and the role of selected amino acids on its secretion, stability and toxicity

The methanol-insoluble heat-stable enterotoxin of Escherichia coli (STB) was purified and characterized by automated Edman degradation and tryptic peptide analysis. The amino-terminal residue, Ser-24, confirmed that the first 23 amino acids inferred from the gene sequence were removed during translocation through the E. coli inner membrane. Tryptic peptide analysis coupled with automated Edman degradation revealed that disulphide bonds are formed between residues Cys-33 and Cys-71 and between Cys-44 and Cys-59. Oligonucleotide-directed mutagenesis performed on the STB gene demonstrated that disulphide bond formation does not precede translocation of the polypeptide through the inner membrane and that disulphide bridge formation is a periplasmic event; apparently, elimination of either of two disulphides of STB renders the molecule susceptible to periplasmic proteolysis. In addition, a loop defined by the Cys-44-Cys-59 bond contains at least two amino acids (Arg-52 and Asp-53) required for STB toxic activity.     

A revision and confirmation of the amino acid sequence of ribonuclease T1

The amino acid sequence of ribonuclease T1 was reinvestigated over the entire molecule by manual Edman degradation of performic acid-oxidized RNase T1 and some of its tryptic and chymotryptic peptides. The validity of the sequence was confirmed except for the sequence Pro-Gly-Ser at positions 71-73. This sequence should be revised to Gly-Ser-Pro.     

Isolation, structure and biological activity of the Trp-containing pentapeptide from uremic fluid.

Trp-containing pentapeptide was isolated from uremic fluid of an uremic patient by ultrafiltration with Amicon membranes followed by gel filtrations. The peptide thus obtained was identified as H-Asp-Leu-Trp-Gln-Lys-OH by amino acid analysis, manual Edman degradation method, physical constants and analytical data of synthetic pentapeptide. Structural similarity was soon realized between this peptide and pentapeptide moiety corresponding to position 123 through 127 of β-chain of fibrinogen. E-rosettes inhibition test was shown this pentapeptide to have an inhibition activity by amount more than l.Omg/ml.     

Rapid manual sequencing of multiple peptide samples in a nitrogen chamber.

Automatic protein sequenators are very expensive. Most laboratories must rely on slower manual methods. This paper describes an inexpensive nitrogen chamber in which multiple peptide samples may be sequenced manually and yet rapidly. The nitrogen environment keeps samples free from oxygen, water vapor, and dust. A complete, simple procedure for degrading peptides is also included. From six to twelve peptide samples may be degraded through one cycle of Edman degradation in a single morning. Several pieces of new equipment and procedures are described which simplify the manipulations involved in protein sequencing.     

Identification of tyrosines 154 and 307 in the extracellular domain and 653 and 766 in the intracellular domain as phosphorylation sites in the heparin-binding fibroblast growth factor receptor tyrosine kinase (flg).

Four tyrosine residues have been identified as phosphorylation sites in the tyrosine kinase isoform of the heparin-binding fibroblast growth factor receptor flg (FGF-R1). Baculoviral-insect cell-derived recombinant FGF-R1 was phosphorylated and fragmented with trypsin while immobilized on heparin-agarose beads. Phosphotyrosine peptides were purified by chromatography on immobilized anti-phosphotyrosine antibody and analyzed by Edman degradation and electrospray tandem mass spectrometry. Tyrosine residue 653, which is in a homologous spatial position to major autophosphorylation sites in the catalytic domain of the src and insulin receptor kinases, is the major intracellular FGF-R1 phosphorylation site. Residue 766 in the COOH-terminus outside the kinase domain is a secondary site. Tyrosine residues 154 and 307, which are in the extracellular domain of transmembrane receptor isoforms and are in an unusual sequence context for tyrosine phosphorylation, were also phosphorylated.     

Property and Amino Acid Sequence of a Subtilisin Inhibitor from Seeds of Beach Canavalia (Canavalia lineata)

A subtilisin inhibitor was purified from the seeds of Canavalia lineata by ammonium sulfate precipitation, ultrafiltration on a YM-30 membrane, column chromatography on DEAE-Toyopearl and SP-Toyopearl, followed by reverse-phase HPLC. The inhibitor (CLSI-I) is a low molecular weight protein (M_r about 6500) containing no half-cystine residue, and quite stable as to extreme heat and pH treatment. CLSI-I inhibited subtilisin-type serine proteases including S. griseus alkaline protease. The amino acids of CLSI-I were sequenced by manual Edman degradation after enzymatic digestion with Achromobacter lyticus lysyl endopeptidase and Staphylococcus aureus V8 protease. CLSI-I contains 65 amino acid residues and showed a high homology to potato inhibitor I family proteins.     

Current developments in stepwise edman degradation of peptides and proteins

• 1.1. Since Edman's (1950, 1956) first publications about 30 years ago, the stepwise degradation of proteins and peptides is universally performed by protein chemists. We review the mechanism of the chemical reactions, and the different special problems encountered, during degradation and different manual methods of degradation.
• 2.2. We take one example of an alternative method using DABITC manually for the degradation of peptides in order to illustrate the evolution of manual degradation techniques (Chang, 1983).
• 3.3. Possibilities and limits of the liquid phase sequenator of Edman and Begg (1967), solid phase sequencer of Laursen (1975) and gas-liquid sequenator of Hewick et al. (1981) and those of Hunkapiller et al. (1983) are considered in detail.
• 4.4. We describe different procedures for identification of PTH-AA or DABTH-AA: thin layer chromatography, gas chromatography, high performance liquid chromatography, etc., in order to illustrate the evolution of the procedures of identification.
• 5.5. We conclude by taking two manual examples and two automatic procedures of degradation to underline the progress over the last decade.

     

Duck skeletal muscle acylphosphatase: Primary structure

The complete amino acid sequence of duck skeletal muscle acylphosphatase is presented. The sequence was studied by the manual Edman degradation of the complete series of tryptic peptides and the amino acid composition of peptic peptides. The NH₂-terminus is acetylated, and the polypeptide consists of 102 amino acid residues. The sequence is compared with other known acylphosphatases from the skeletal muscle of several vertebrate species.