Comparative studies on partial microsequence-analysis of Bence-Jones proteins

1. Sequence analyses of Bence-Jones proteins up to 15 amino acids from the N-terminus provide decision of subgroups. 2. Investigation of primary structure of 3 Bence-Jones proteins, monomer and dimer of a kappa type, is limited at position 9-10 using DABITC reagent, whereas DABITC/PITC double coupling method allows sequencing up to 20-21 amino acids. 3. Sequencing of tryptic peptides allows only determination of 6-9 amino acids. 4. Microsequencing of tryptic peptide T13a shows the allotypic variant, inv b+, of Bence-Jones proteins TRA and GAN.     

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.     

The N-terminal amino acid sequence of the beta-subunit of the legumin-like protein from seeds of Ginkgo biloba.

The sequence of the first 52 amino acids at the N-terminus of the beta-subunit of a legumin-like protein from seeds of the gymnosperm Ginkgo biloba were determined by automated sequencing and DABITC/PITC microsequence analyses of peptides derived from the protein by enzymatic digestions and chemical cleavage with CNBr. The protein from Ginkgo exhibits sequence homologies (32-49% identities) with the 11S globulins and legume-like proteins from seeds of various angiosperm monocotyledons and dicotyledons.     

The primary structure of initiation factor IF3 from Bacillus stearothermophilus

The complete amino acid sequence of the initiation factor IF3 from Bacillus stearothermophilus has been elucidated. This was achieved by splitting the protein with trypsin, Staphylococcus protease or cyanogen bromide. The amino acid sequence was determined by manual Edman degradation, using the DABITC/PITC double-coupling method. The IF3 molecule contains 171 amino acids and has an M_r of 19 677. The sequence was compared to the homologous molecule from Escherichia coli; about 50% of the amino acid residues were found to be identical.     

Contents to volume 167

The chemical synthesis of 4-(N-tertbutyloxycarbonylaminomethyl)-phenylisothiocyanate starting from 4-nitrobenzylamine is described. This derivative represents an Edman-type reagent with a masked amino group which renders the thiohydantoin upon deblocking susceptible to fluorogenic detection. The coupling efficiency is determined in comparison to degradations with PITC, DABITC and FITC. The detection sensitivity on thin layer chromatograms is compared to the thiohydantoins derived from DABITC.     

大肠杆菌热稳定性肠毒素的分离纯化和氨基酸顺序测定

上海市儿童医院婴儿腹泻病患中分离到产肠毒素的菌株,该菌株只产热稳定性肠毒素,经CAD-44树脂吸附,SephadexG-25凝胶过泸,DEAE-Seharose CL-6B离子交换层析,反相高压液相层析分离纯化后,用手工固相DABITC/PITC双偶合Edman降解法测定其氨基酸顺序为:Asn·Ser·Ser·Asn·Tyr·Cys·Cys·Gly·Leu·Cys·Cys·Asn·Pro·Ala·Cys·Thr·Gly·Cys·Tyr。     

Solid-phase sequence analysis of proteins electroblotted or spotted onto polyvinylidene difluoride membranes

Electroblotted proteins noncovalently bound to polyvinylidene difluoride (PVDF) membranes are typically sequenced using adsorptive sequencer protocols (gas-phase or pulsed-liquid) that do not require a covalent linkage between protein and surface. We have developed simple chemical protocols where proteins are first electroblotted onto unmodified PVDF membranes, visualized with common protein stains, and then immobilized for solid-phase sequence analysis. Adsorbed, stained proteins are first treated with phenylisothiocyanate (PITC) to modify alpha and epsilon amines. The protein is then overlayed with a solution of 1,4-phenylene di-isothiocyanate (DITC), followed by a few microliters of a basic solution containing a poly(alkylamine). As the polymer dries onto the surface both polymer and remaining protein amino groups are crosslinked by DITC. The protein is thus immobilized to the membrane surface by entrapment in a thin polymer coating. The coating is transparent to the degradation chemistry, and extensive enough to remain immobilized even in the absence of any covalent link between polymer and surface. Partial modification with PITC allows for identification of N-terminal and internal lysine residues during sequencing. The process was tested with a variety of poly(alkylamines), linear and branched, with molecular weights ranging from 600 to over 100,000. Proteins bound in this manner were successfully sequenced using covalent (solid-phase) sequencer protocols with cycle times as short as 26 min.     

Comparison of the amino acid sequences of the lectins from seeds of Dioclea lehmanni and Canavalia maritima.

The amino acid sequences of the major lectins from the seeds of Dioclea lehmanni and Canavalia maritima were determined by DABITC/PITC microsequence analysis of peptides derived from the proteins by enzymatic digestions with trypsin, chymotrypsin and the protease from S. aureus V8. These sequences were found to be very similar to those of the lectins from Dioclea grandiflora and Canavalia ensiformis (Con A). The D. lehmanni lectin was unusual amongst legume lectins in that it contained a single Cys.     

Isolation of Protein Inhibitors of Papain,Trypsin, and α-Amylase in the Grain of Foxtail Millet

The amino acid sequence of Indian peafowl egg-white lysozyme has been identified. The reduced and carboxymethylated lysozyme was digested with trypsin followed by purification of the resulting peptides by reverse-phase HPLC. The tryptic peptides obtained were sequenced using the DABITC/PITC double coupling manual sequencing method. The alignment of the tryptic peptides were deduced by comparison with corresponding peptides of hen egg-white lysozyme. This protein proved to consist of 129 amino acid residues, and a relative molecular mass of 14423 Da was calculated. Amino acid sequence comparison of peafowl lysozyme and other phasianoid bird lysozymes revealed a maximum homology ratio of 98% with turkey lysozyme.     

Isolation and properties of a natural inhibitor of the chloroplast adenosine triphosphatase

The complete sequence of protein L17 which is a component of the large subunit of the E. coli ribosome has been determined. Peptides deriving from enzymatic hydrolysis with trypsin, thermolysin, chymotrypsin and S. aureus and A. mellea protease were isolated and sequenced by the DABITC/PITC double coupling method. Some overlapping peptides were obtained after mild acid cleavage of the protein. According to the amino acid sequence protein L17 contains 127 residues and has a molecular mass of 14 365. The primary structure of protein L17 agrees well with the amino acid analysis of the intact protein and its N-terminal sequence as derived from automatic sequencing in an improved Beckman sequencer. Secondary predictions and a search for homologous sequence stretches to other ribosomal proteins were made.     

Covalent immobilization of proteins for high-sensitivity sequence analysis: electroblotting onto chemically activated glass from sodium dodecyl sulfate-polyacrylamide gels

We report a new method for the preparation of proteins in a form suitable for high-sensitivity N-terminal amino acid sequence analysis. Proteins separated by polyacrylamide gel electrophoresis were electrophoretically transferred onto glass fiber filter paper chemically activated by the introduction of phenyl isothiocyanate functional groups. The proteins became covalently coupled to the matrix during the electrotransfer process. Bands containing transferred proteins were detected by fluorescent staining or autoradiography, cut out from the glass fiber filter, and directly loaded into the cartridge of a gas-phase sequenator. The covalent nature of the interactions between protein and glass fiber support permitted the use of more vigorous solid-phase sequencing protocols and of alternative sequencing reagents. This high-efficiency isolation and covalent coupling method provides the essential first step toward enhanced-sensitivity protein sequence analysis. The method has been successfully applied to the isolation of a wide variety of proteins from SDS-polyacrylamide gels, and was shown to be compatible with both the standard Edman reagent phenyl isothiocyanate and alternative sequencing reagents such as 4-(N,N'-dimethylamino)azobenzene-4'-isothiocyanate (DABITC).     

A new sensitive Edman-type reagent: 4-(N-1-dimethylaminonaphthalene-5-sulfonylamino)phenyl isothiocyanate. Its synthesis and application for micro-sequencing of polypeptides

A new fluorescent PITC homologue Edman-type reagent, 4-(N-1-dimethylaminonaphthalene-5-sulfonylamino)phenyl isothiocyanate (DNSAPITC), was synthesized following a simple three-step synthetic route. The reagent was crystallized and characterized by thin-layer chromatography, IR and electron impact mass spectrometry. Reference DNSAPTH-amino acid derivatives were prepared and a two-dimensional chromatography system on micro-polyamide sheets was developed for separating this mixture. On these sheets the sensitivity was 1-5 pmol, by exposure at 366 nm. Model peptides and proteins were subjected to Edman degradations with this new reagent. A similar coupling efficiency and repetitive degradation yield to those of PITC were found with this reagent. The advantages and limitations of this reagent for sensitive microsequencing are discussed.     

615小鼠血红蛋白珠蛋白α链的分离纯化及氨基酸组成

用CM－cellulose-23柱层析分离纯化了615小鼠珠蛋白α链,测定其N－末端氨基酸为缬氨酸。615小鼠珠蛋白α链含有141个氨基酸残基,其中含19个亮氨酸残基,10个组氨酸残基,9个缬氨酸残基,上述三种氨基酸残基的数目与文献中新本不同。     

The complete amino-acid sequence of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.)

After reduction and alkylation of the disulfide bonds of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.) followed by CNBr cleavage three peptide fragments with 68, 62 and 11 amino-acid residues could be separated on DEAE-Sepharose CL-6B. The peptides or the inhibitor itself were further specifically cleaved either by trypsin or by the mixture of (CH3)2SO/HCl/HBr at the arginyl- and the tryptophyl-peptide bond, respectively. The complete amino-acid sequences of the peptides were determined by manual solid phase DABITC/PITC double coupling micro-method and the primary structure of the arrowhead inhibitor B consisting of 141 amino-acid residues was then elucidated. Twenty pairs of amino-acid residues are repeated in the molecule of this inhibitor, three of these pairs even occur three times. The possible locations of the reactive sites are discussed. On the basis of sequence comparisons between this inhibitor and all other serine proteinase inhibitors the arrowhead inhibitor may belong to a new family.     

Identification of outer membrane proteins from an Antarctic bacterium Pseudomonas syringae Lz4W

Subcellular fractionation of proteins is a preferred method of choice for detection and identification of proteins from complex mixtures such as bacterial cells. To characterize the membrane proteins of the Antarctic bacterium Pseudomonas syringae Lz4W, the membrane fractions were prepared using three different methods, namely Triton X-100 solubilization, sucrose density gradient, and carbonate extraction methods. The proteins were separated on one-dimensional polyacrylamide gels and analyzed using a combination of liquid chromatography-coupled electrospray ionization-MS. The membrane proteins that were prepared by carbonate extraction were separated on two-dimensional PAGE in different pI ranges using the detergent 2% amidosulfobetaine (ASB). The proteins were then subjected to matrix-assisted laser desorption ionization-time-of-flight/time-of-flight for analysis and identification. Because the genome sequence of P. syringae Lz4W is not known, the proteins were identified by using the relevant sequence databases of the Pseudomonas sp available at National Centre for Biotechnology Information (NCBI). The sequence identification of some tryptic peptides were validated by de novo sequencing and others by chemical modification and mass spectrometry. The peptide sequences of P. syringae Lz4W were then matched with the sequences of the peptides from different Pseudomonas sp. by similarity search of the proteins from different species using clustal W2 program. Thus by using a combination of the methods, we have been able to identify large number of proteins of this bacterial strain, which include most of the outer membrane proteins.     

The amino acid sequence of a 20 kDa bifunctional subtilisin/alpha-amylase inhibitor from bran [correction of brain] of rice (Oryza sativa L.) seeds.

A 20 kDa bifunctional inhibitor of the microbial proteinase, subtilisin, and the alpha-amylase from the larvae of the red flour beetle (Tribolium castaneum) was purified from bran of rice seeds by saline extraction, precipitation with ammonium sulphate, ion-exchange chromatography on DEAE-Cellulose and Toyopearl CM-650, and preparative HPLC on Vydac C18. The complete primary structure was determined by automatic degradation of the intact, reduced and S-alkylated protein, and by manual DABITC/PITC micro-sequencing of peptides obtained from the protein following separate enzymic digestions with trypsin, pepsin, chymotrypsin, elastase and the protease from S. aureus V8. The protein sequence, which contained 176 residues, showed strong homology with similar bifunctional inhibitors previously isolated from wheat and barley which are related to the Kunitz family of proteinase inhibitors from legume seeds.     

The purification and amino acid sequence of the lethal neurotoxin Tx1 from the venom of the Brazilian 'armed' spider Phoneutria nigriventer

A lethal neurotoxic polypeptide of Mr 8 kDa was purified from the venom of the South American 'armed' or wandering spider Phoneutria nigriventer by centrifugation, gel filtration on Superose 12, and reverse phase FPLC on columns of Pharmacia PepRPC and ProRPC. The purified neurotoxin Tx1 had an LD50 of 0.05 mg/kg in mice following intracerebroventricular injection. The complete amino acid sequence of the neurotoxin was determined by automated Edman degradation of the native and S-carboxymethylated protein in pulsed liquid and dual phase sequencers, and by the manual DABITC/PITC double coupling method applied to fragments obtained after digestions with the S. aureus V8 protease and trypsin. The neurotoxin Tx1 consists of a single chain of 77 amino acid residues, which contains a high proportion of cysteine. The primary structure showed no homology to other identified spider toxins.     

Improvements in the application of the 4,4-N,N-dimethylaminoazobenzene-4'-isothiocyanate micromethod to the sequence analysis of proteins

Different experimental conditions have been tested to improve the sequence determination of peptides and proteins by the DABITC (4,4-N,N-dimethylaminoazobenzene-4'-isothiocyanate) method and to facilitate automation in the analysis of the released 4,4-N,N-dimethylaminoazobenzene-4'-thiohydantoin derivatives (DABTHs). Conditions for a complete and rapid separation of all amino acid derivatives have been optimized by using different reversed-phase columns. The stability of the DABTHs in several water-organic solvent mixtures was determined by quantitative analysis and permitted the selection of the appropriate solvents for use in autosamplers. Also the amino acid side-products specific to individual residues which may be observed during thin-layer chromatography of DABTHs can be completely resolved by HPLC and are helpful for a safe assignment of the amino acid residues. The analytical procedures developed have been used to examine the influence of oxygen and detergents on the efficiency of the application of the DABITC manual micromethod on proteins. In the presence of oxygen the recovery of DABTHs is lower in most cases than when the operation is carried out in an inert atmosphere. The presence of a limited amount of detergents does not interfere in the HPLC analysis of DABTHs and, moreover, can increase the efficiency of the sequence analysis of proteins depending on their nature and concentration. In particular, it has been observed that sodium dodecyl sulfate at a concentration of 0.1% can in some cases produce a threefold increase in the recovery of DABTHs.     

615小鼠血红蛋白α链的氨基酸组成及个别肽段的氨基酸序列

用CM-Cellulose-23柱层析分离纯化了615小鼠珠蛋白α链,测定其N端氨基酸残基为缬氨酸.615小鼠珠蛋白α链含有141个氨基酸残基,其中19个亮氨酸残基,10个组氨酸残基,9个缬氨酸残基,上述氨基酸残基的数目与文献中其亲本C₅₇BL不同.用胰蛋白酶水解615小鼠珠蛋白α链,发现有不溶性的‘核心’和可溶性的酶解片段.其中一个酶解肽段从N端数第8位氨基酸残基发生了突变,由亲本的缬氨酸变为亮氨酸.     

The complete amino acid sequence of the major Kunitz trypsin inhibitor from the seeds of Prosopsis juliflora.

The major inhibitor of trypsin in seeds of Prosopsis juliflora was purified by precipitation with ammonium sulphate, ion-exchange column chromatography on DEAE- and CM-Sepharose and preparative reverse phase HPLC on a Vydac C-18 column. The protein inhibited trypsin in the stoichiometric ratio of 1:1, but had only weak activity against chymotrypsin and did not inhibit human salivary or porcine pancreatic alpha-amylases. SDS-PAGE indicated that the inhibitor has a Mr of ca 20,000, and IEF-PAGE showed that the pI is 8.8. The complete amino acid sequence was determined by automatic degradation, and by DABITC/PITC microsequence analysis of peptides obtained from enzyme digestions of the reduced and S-carboxymethylated protein with trypsin, chymotrypsin, elastase, the Glu-specific protease from S. aureus and the Lys-specific protease from Lysobacter enzymogenes. The inhibitor consisted of two polypeptide chains, of 137 residues (alpha chain) and 38 residues (beta chain) linked together by a single disulphide bond. The amino acid sequence of the protein exhibited homology with a number of Kunitz proteinase inhibitors from other legume seeds, the bifunctional subtilisin/alpha-amylase inhibitors from cereals and the taste-modifying protein miraculin.