N-epsilon-acetylation of porcine mature erythrocytes ubiquitin

Highly purified of porcine mature erythrocytes ubiquitin were obtained according to the experimental procedure reported by Jabusch and Deutsch (1983). N-epsilon-acetylation in vitro of internal lysyl residues of ubiquitin by p-nitro-phenyl-acetate at pH 8.0 was performed. The extent of acetylation of ubiquitin was determined: about 4-5 residues (4.5 residues) of N-epsilon-lysine groups of ubiquitin were acetylated. We have assigned by Edman degradation the sites of acetylation and the sites of remaining free internal N-epsilon-lysine residues in the sequence: fully acetylated: Lys-6, Lys-11 and Lys-33. Partially free N-epsilon-lysine: Lys-27 and Lys-29 and probably Lys-48 and Lys-63. 50 cycles Edman degradation were performed on porcine ubiquitin and the first 45 N-terminal residues were identified. We have partially determined that the molecular conservation of 45 amino acid sequence of ubiquitin between cattle, man and swine since the 45 amino acid sequence out of 76 residues are identical. The amino acid composition between human and porcine ubiquitin are also identical.     

Studies on the high-sulphur proteins of reduced mohair. The isolation and amino acid sequence of protein scmkb-m1.2.

The complete amino acid sequence of mohair protein, SCMKB-M1.2 (97 residues), was determined. The protein was isolated from reduced and carboxymethylated mohair by chromatography on DEAE-cellulose phosphate. Peptides for sequence determination were obtained by digestion with trypsin, pepsin, chymotrypsin, thermolysin and papain, and were fractionated by DEAE-cellulose chromatography, paper chromatography and electrophoresis. The sequence of the peptides were determined by the Edman degradation method (by use of both the Beckman Sequence and a non-automatic procedure), and by partial acid hydrolysis. The protein is closely homologous to wool protein SCMKB-IIIB2, and also contains acetylated alanine as N-terminal amino acid.     

Studies on the methods for the determination of phosphorylation sites in highly phosphorylated peptides or proteins: phosphorylation sites of hen egg white riboflavin binding protein

To determine the phosphate binding sites in hen egg white riboflavin binding protein (RBP), a highly phosphorylated peptide, which consisted of 23 amino acid residues including eight phosphoserines, was isolated from the tryptic digest of reduced and carboxymethylated RBP. The conditions of the beta-elimination-addition reaction to convert phosphoserine residues in the peptide to cysteic acids, S-methylcysteines, alanines, and beta-methylaminoalanines (DL-alpha-amino-beta-methylamino propionic acid) were examined. These converted peptides were purified by HPLC and subjected to Edman degradation. The results of Edman degradation indicated that the S-methylcysteine derivative of the peptide gave the most satisfactory result for determining the phosphate binding sites in the peptide. The phosphorylation sites of the peptide determined by the method mentioned above are as follows: His182-Leu-Leu-Ser185-Glu-Ser(P)-Ser(P)-Glu-Glu190-Ser (P)-Ser(P)-Ser(P)-Met-Ser195(P)-Ser(P)-Ser(P)-Glu-Glu-. These studies indicated that the conversion of phosphoserines in phosphoproteins to S-methylcysteines followed by Edman analysis was a useful method for the elucidation of the phosphorylation sites in phosphopeptides.     

Salivary cystatin SA-III, a potential precursor of the acquired enamel pellicle, is phosphorylated at both its amino- and carboxyl-terminal regions

Cystatin SA-III was purified from human submandibular/sublingual glandular secretions by adsorption to hydroxyapatite, gel filtration chromatography, and reversed-phase HPLC. The amino acid sequence of its amino-terminus was deduced by sequential Edman degradation and found to be identical to the first 10 residues of cystatin HSP-12. The purified protein was digested with endoproteinase Asp-N and the digestion products were subjected to fast atom bombardment mass spectroscopy. m/z values corresponding to 12 peptides were aligned to the sequence of cystatin S preceded by the eight-residue amino-terminal peptide detected in HSP-12. This process resulted in the assignment of peptides corresponding with 118 out of the 121 amino acid residues predicted from the nucleotide sequence for cystatin SA-III. In order to align several peptides, it was necessary to substitute four residues of phosphoserine for four residues of serine. Fast atom bombardment mass spectrometry and additional Edman degradation procedures localized the phosphate moieties to Ser-3, Ser-99, Ser-112, and Ser-116. This is the first report of the structure of cystatin SA-III deduced by amino acid sequencing techniques and indicates the sites of phosphoserine within the molecule. Based on these assignments, cystatin SA-III is unique among salivary proteins in that it possesses phosphate groups at its amino-terminus as well as its carboxyl-terminus.     

The primary structure of rat liver ribosomal protein L39

The covalent structure of the rat liver 60 S ribosomal subunit protein L39 was determined. Fourteen tryptic peptides were purified, and the sequence of each was established by a micromanual procedure; they accounted for all 50 residues of L39. The sequence of the NH2-terminal 32 residues of L39, obtained by automated Edman degradation of the intact protein, provided the alignment of the first seven tryptic peptides. Two peptides, CNI (28 residues) and CNII (22 residues), were produced by cleavage of protein L39 with cyanogen bromide and the sequence of CNII was determined by automated Edman degradation. This sequence established the order of tryptic peptides T8 through T14. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. Protein L39 contains 50 amino acids and has a molecular weight of 7308. There are indications that a portion of rat L39 is related to a fragment of Escherichia coli ribosomal protein S1.     

Sequence of residues 400--403 of bovine serum albumin.

A large tryptic peptide of bovine serum albumin (residues 377--582) was subjected to 32 cycles of Edman degradation to determine the sequence of the last remaining unknown segment of this protein. Residues 400--403 were identified as gly-Phe-Gln-Asn. Amide assignments were also made at positions 388 (glutamine), 389 (asparagine), 391 (aspartic acid) and 392 (glutamine).     

Covalent structural analysis of yeast inorganic pyrophosphatase: Location of groups implicated in the catalytic function; placement of the NH2-terminal 100 residues in the subunit

Covalent structural analysis of two of the three cyanogen bromide fragments from yeast inorganic pyrophosphatase (EC 3.6.1.1, pyrophosphate phosphohydrolase) was undertaken by a strategy involving both automated Edman degradation and conventional sequence analysis. Automated degradation of intact, reduced and carboxymethylated pyrophosphatase provided the sequence of the first 34 residues in the NH₂-terminal 45-residue peptide, CNBr VI, in addition to a partial sequence through 50 cycles which confirmed the overlap into the internal fragment, CNBr III. The sequence of CNBr VI was completed through analysis of peptides derived from hydrolysis of the fragment with trypsin and chymotrypsin. Structural analysis of CNBr III has provided the sequence of the first 55 amino acids in this 103-residue fragment. The sequence was established by conventional and automated procedures applied to the analysis of tryptic peptides generated from the citraconylated fragment. These findings constitute the sequence of the first 100 residues in the pyrophosphatase subunit and, together with structural information obtained earlier, define over half of the covalent structure of the molecule. Moreover, the sequence derived thus far permits the placement of a number of amino acids that are of importance relative to studies of the enzyme mechanism, and with regard to analysis of its three-dimensional structure.     

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.     

A strategy for the rapid identification of phosphorylation sites in the phosphoproteome

Edman phosphate ((32)P) release sequencing provides a high sensitivity means of identifying phosphorylation sites in proteins that complements mass spectrometry techniques. We have developed a bioinformatic assessment tool, the cleavage of radiolabeled protein (CRP) program, which enables experimental identification of phosphorylation sites via (32)P labeling and Edman degradation of cleaved proteins obtained at femtomole levels. By observing the Edman cycle(s) in which radioactivity is found, candidate phosphorylation sites are identified by determining which residues occur at the observed number of cycles downstream from a peptide cleavage site. In cases where more than one residue could be responsible for the observed radioactivity, additional experiments with cleavage reagents having alternative specificities may resolve the ambiguity. Given a protein sequence and a cleavage site, CRP performs these experiments in silico, identifying resolved sites based on user-supplied experimental data, as well as suggesting combinations of reagents for additional analyses. Analysis of the PhosphoBase protein sequence database suggests that CRP data from two cleavage experiments can be used to identify unambiguously 60% of known phosphorylation sites. Data from additional cleavage experiments may increase the overall coverage to 70% of known sites. By comparing theoretical data obtained from the CRP program with (32)P release data obtained from an Edman sequencer, a known phosphorylation site was identified unambiguously and correctly. In addition, our results show that in vivo phosphorylation sites can be determined routinely by differential proteolysis analysis and Edman cycling with less than 1 fmol of protein and 1000 cpm.     

Optimization of the Oxidative Folding Reaction and Disulfide Structure Determination of Human α-Defensin 1, 2, 3 and 5

The optimal conditions were determined for oxidative folding of the reduced human α-defensins, HNP1, HNP2, HNP3 and HD5, preferentially into their native disulfide structures. Since the human α-defensin-molecule in both reduced and oxidized forms raised a solubility problem arising from its basic and hydrophobic compositions, buffer concentration had to be lowered and cosolvent, such as CH₃CN, had to be added to the folding medium in the presence of reduced and oxidized gluthathione (GSH/GSSG) to prevent aggregation and also to realize predominant formation of the native conformer. The four synthetic human α-defensins of high homogeneity were confirmed to exhibit the same antimicrobial potencies against E. coli as those reported for the natural products. All these peptides were shown to possess the native disulfide structure by sequence analyses and mass measurements with cystine segments obtained by enzymatic digestion. Edman degradation allowed for disulfide assignment of cystine segments involving adjacent Cys residues composed of three peptide chains, for which two possible disulfide modes could be considered, with the guidance of the cycles detecting diPTH cystine. As for HNP1, HNP2 and HNP3, however, diPTH cystine was expected at the same cycles in both structures, which would have resulted in not being able to distinguish between the two alternative modes. To avoid this, it was necessary to provide an acetyl tag for the specific peptide chain originating from the N-terminus. Edman degradation of cystine segments tagged with the acetyl group would be a practical procedure for analyzing disulfide structures involving adjacent Cys residues.     

In situ cyanogen bromide cleavage of N-terminally blocked proteins in a gas-phase sequencer

Herein we describe a procedure for the in situ cyanogen bromide cleavage of N-terminally blocked proteins which have been immobilised onto the glass fiber sample disk of the gas-phase sequencer. In this manner, new amino terminii suitable for automated Edman degradation can be generated. Cytochrome C was cleaved using this method on the carboxyl side of methionine residues 65 and 80. This allowed sequence analysis to begin simultaneously at residues 66 and 81 (Table 1). This procedure offers an alternative sequencing tactic for methionine-containing proteins which are N-terminally blocked.     

A manual sequencing method for identification of phosphorylated amino acids in phosphopeptides

We describe a simple and inexpensive method for determining the location of phosphoamino acids in an isolated phosphopeptide. Phosphopeptides are immobilized on arylamine membrane discs using water-soluble carbodiimide, and the immobilized peptides are subjected to manual Edman degradation. The use of a membrane disc resulted in excellent recovery (65 to 80%) of radiolabeled phosphate following Edman degradation. Furthermore, interference from carryover and peptide washout is reduced to a minimum. The methodology should therefore be able to generate reliable results when used to analyze phosphopeptides that contain multiple phosphorylation sites. In addition to its advantage in sensitivity, the manual sequencing method is easy to perform and does not entail any sophisticated instrumentation.     

D-L amino acid analysis using automated precolumn derivatization with 1-fluoro-2,4-dinitrophenyl-5-alanine amide

Summary An automated procedure for the precolumn derivatization of enantiomeric amino acid mixtures with 1-fluoro-2,4-dinitrophenyl-5-alanine amide and a liquid chromatographic method for the separation of the derivatives with UV detection are reported. The system described allows to perform routine analyses using microbore columns with a sensitivity at the picomol level. Improvements for the use of this reagent in the protocol of a subtractive Edman degradation procedure of peptides to determine the sequence position of amino acid residues with concomitant identification of their chirality are also described.     

New assay for enzymatic phosphate release: application to aspartate transcarbamylase and other enzymes

The colorimetric method for phosphate determination described in the preceding paper is adapted for the assay of orthophosphate liberated in the aspartate transcarbamylase reaction. The method provides for simple, accurate, and sensitive measurement of enzyme activity. The assay uses ammonium molybdate and zinc acetate to form a colored complex with the enzymatically released phosphate; mild conditions which minimize the nonenzymatic background degradation of the substrate, carbamoyl phosphate, are used. Since the assay procedure is relatively rapid, it is especially attractive in situations where results are desired immediately. The method can be used for the assay of any enzyme which releases inorganic phosphate, even in the presence of labile organophosphate compounds.     

CRP: Cleavage of Radiolabeled Phosphoproteins

The CRP (Cleavage of Radiolabeled Phosphoproteins) program guides the design and interpretation of experiments to identify protein phosphorylation sites by Edman sequencing of unseparated peptides. Traditionally, phosphorylation sites are determined by cleaving the phosphoprotein and separating the peptides for Edman 32P-phosphate release sequencing. CRP analysis of a phosphoprotein's sequence accelerates this process by omitting the separation step: given a protein sequence of interest, the CRP program performs an in silico proteolytic cleavage of the sequence and reports the predicted Edman cycles in which radioactivity would be observed if a given serine, threonine or tyrosine were phosphorylated. Experimentally observed cycles containing 32P can be compared with CRP predictions to confirm candidate sites and/or explore the ability of additional cleavage experiments to resolve remaining ambiguities. To reduce ambiguity, the phosphorylated residue (P-Tyr, P-Ser or P-Thr) can be determined experimentally, and CRP will ignore sites with alternative residues. CRP also provides simple predictions of likely phosphorylation sites using known kinase recognition motifs. The CRP interface is available at http://fasta.bioch.virginia.edu/crp.     

The use of radiolabelled T1 ribonuclease to monitor the efficiency of Edman degradations (Short Communication)

A simple radiolabel tracer method is presented for monitoring the efficiency of the first ten cycles of an Edman degradation. This method consists of adding a very small amount of radiolabelled S-carboxymethylated T₁ ribonuclease to the protein to be sequenced, and counting the radioactivity of a small sample from each of the degradative cycles.     

Site of phosphorylation of SpoIIAA, the anti-anti-sigma factor for sporulation-specific sigma F of Bacillus subtilis.

Sigma F is regulated by an anti-sigma factor, SpoIIAB, and an anti-anti-sigma factor, SpoIIAA. SpoIIAB also functions as a phosphokinase which transfers phosphate from ATP to SpoIIAA; this phosphorylation is thought to be involved in the regulatory mechanism. By using [gamma-32P]ATP to phosphorylate SpoIIAA, cleaving the protein proteolytically, and analyzing the one resulting radiolabelled peptide by the Edman degradation procedure, we show that the site of phosphorylation in SpoIIAA is Ser-58.     

Complete amino acid sequence of amelogenin in developing bovine enamel

Pure amelogenin protein in developing bovine incisor enamel was isolated and its primary structure was investigated by sequencing the peptides obtained after clostripain and chymotrypsin digestions and CNBr degradation with an automated Edman sequencer. The enamel protein was found to be composed of 170 amino acid residues with one phosphate having a molecular weight of 19,350 and its complete amino acid sequence was elucidated. This protein has no sequence homology with any other tissue or secretory protein of known structure.     

The covalent structure of calf skin type III collagen. VI. The amino acid sequence of the carboxyterminal cyanogen bromide peptide alpha 1(III)CB9B (position 928--1028).

The C-terminal cyanogen bromide peptide alpha 1(III)CB9B is 101 amino acid residues in length and occupies position 928--1028 along the alpha 1(III) chain. For sequence analysis, alpha 1(III)CB9B was fragmented with trypsin and chymotrypsin. The peptides obtained were separated using molecular sieve and ion exchange chromatography and sequenced using the automated Edman degradation procedure.     

Amino acid sequence of the 'b5-like' heme-binding domain from chicken sulfite oxidase

We present in this paper the sequence of the heme-binding domain of chicken sulfite oxidase which can be obtained by chymotryptic digestion of the native enzyme. The results of an automatic degradation have been reported previously. In the present work peptides were obtained from the heme-binding domain by digestion with trypsin, chymotrypsin and Staphylococcus aureus V8 protease; they were manually sequenced by the dansyl/Edman procedure. The evidence thus obtained is sufficient to completely establish the order of the 97 residues. In addition, two rounds of Edman degradation on sulfite oxidase itself allowed us to identify the same two residues, H-Ala-Pro, present at the N-terminus of the heme-binding domain; this result suggests that the latter constitutes the amino-terminal end of the sulfite oxidase peptide chain. The data presented here confirm the strong similarity between sulfite oxidase and microsomal cytochrome b5 already suggested by our first results. A sequence alignment is proposed for the two proteins. Inspection of the calf liver cytochrome b5 three-dimensional model together with the alignment suggests a similar overall structure for sulfite oxidase core with a limited number of backbone modifications. Our results point to a common evolutionary origin for sulfite oxidase core and microsomal cytochrome b5.