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PubMed1985

Quantification and specific detection of collagenous proteins using an enzyme-linked immunosorbent assay and an immunoblotting for cyanogen bromide peptides.

A method for the detection of collagenous proteins within cyanogen bromide digests of tissues has been devised. The peptides produced by digestion with cyanogen bromide were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter. They were stained on the filter by incubation first with antibodies to collagen and then with a second antibody covalently linked to horseradish peroxidase, 4-chloro-1-naphthol was added, and the bound enzyme was assayed. This procedure is useful for the identification and characterization of collagens of types I, III, IV, and V in tissues. In addition, we have developed a sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) which is convenient for quantifying collagens (types I, III, and IV) in tissues. In this kind of assay, soluble cyanogen bromide peptides compete with cyanogen bromide peptides adsorbed onto a solid-phase support for rabbit anti-collagen antibodies. We determined the amount of bound antibody by using goat anti-rabbit immunoglobulin G covalently conjugated to horseradish peroxidase and then provided a substrate for the enzymatic reaction. The sensitivity range of the ELISA is 0.09 micrograms/ml in the region of 90 to 10% binding.

Animals↗
PubMed1975

Structural studies on rabbit skeletal muscle actin. Ordering of the peptides produced by cleavage with cyanogen bromide.

The 17 peptides produced by cleavage of actin with cyanogen bromide have been ordered with regard to their sequence in the actin molecule. Tryptic digestion of actin followed by isolation of the methionine-containing "overlap" peptides permitted the unique alignment of most, but not all of the cyanogen bromide peptides. However, maleylation of the actin molecule followed by tryptic digestion and isolation of methionine-containing peptides from maleylated actin permitted the proper placement of the remaining cyanogen bromide peptides. The ordering of cyanogen bromide peptides, together with the amino acid sequence of the individual peptides, constitutes the entire amino acid sequence of rabbit skeletal muscle actin (ELZINGA, M., COLLINS, J. H., KUEHL, W. M., and ADENLSTEIN, R. S. (1973) Proc. Natl. Acad. Sci. U. S. A. 70,2687-2691).

Actins↗
PubMed1997

Derivatization and template-guided ligation of oligodeoxyribonucleotides using cyanogen bromide and N-substituted morpholines.

Cyanogen bromide has been found to induce the template-guided condensation of oligonucleotides only in the presence of N-substituted morpholines. Based on 31P, 1H and 13C NMR spectroscopy data, the mechanism of the phosphomonoester group activation by cyanogen bromide in N-substituted morpholine buffers is suggested. It has also been shown that BrCN can be used for the synthesis of oligonucleotide derivatives in aqueous solution.

Buffers↗
PubMed1984

Quantitation of types I and III collagens in human tissue samples and cell culture by cyanogen bromide peptide analysis.

A procedure for the quantitation of types I and III collagens by cyanogen bromide peptide analysis was developed with the aim of eliminating certain problems associated with this method. Ion-exchange chromatography reduced high background levels on gel scans used to quantitate the peptides; reduction with beta-mercaptoethanol substantially increased the efficiency of the cyanogen bromide cleavage; use of a concave gradient in acrylamide from 8 to 20% improved the resolution of cyanogen bromide peptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and a normalization procedure eliminated variations due to differences in the amount of material loaded on the gel system. This method of quantitation was applied to human aorta samples and to collagen secreted by human skin fibroblasts. Metachromasy of type I and type III collagen cyanogen bromide peptides stained with Coomassie blue R-250 was established and this was used as an index of the purity of the cyanogen bromide peptide preparations. Type I and III collagens were prepared from human placental tissue, and these purified collagens were used to construct calibration curves to determine the relationship between the quantity of diagnostic cyanogen bromide peptides present and the composition of the sample in terms of types I and III collagens.

Aged↗
PubMed1979

Antigenic determinants of influenza virus hemagglutinin. II. Antigenic reactivity of the isolated N-terminal cyanogen bromide peptide of A/Memphis/72 hemagglutinin heavy chain.

Gel filtration of a cyanogen bromide digest of pure intact hemagglutinin from A/Memphis/102/72 influenza virus allowed the isolation of a variety of fragments. One of these fragments consists of three cyanogen bromide peptides (CNl and CN3 from HA1, and CNl from HA2) which remain linked together by disulphide bonds. This fragment was found to be antigenically active, as it was able to form antigen-antibody complexes (detected by affinity chromatography of radioiodinated peptide-IgG mixtures on protein A-Sepharose) with IgG directed against the protein moiety of viral hemagglutinin. The three cyanogen bromide peptides present in this disulphide-linked fragment were separated by gel filtration, carried out under reducing conditions, and tested for antigenic activity after controlled reoxidation of the individual peptides. Only one cyanogen bromide peptide, CNl from HA1, showed significant binding to antibody. The results indicate that antigenic activity of A/Mem/1O2/72 hemagglutinin resides within the N-terminal 170 amino acid residues of the hemagglutinin heavy chain.

Antibodies, Viral↗
PubMed1990

Formylated peptides from cyanogen bromide digests identified by fast atom bombardment mass spectrometry.

Exposure of proteins to 70% formic acid during cyanogen bromide digestion can result in formation of artifact peaks during subsequent purification by HPLC and detection of [M + H + 28]+ ions during analysis by fast atom bombardment (FAB) mass spectrometry. Following cyanogen bromide digestion, peptides from equine heart cytochrome c, bacteriorhodopsin, bovine adrenal medulla dodecapeptide, and bovine adrenal peptide E were analyzed by positive ion FAB mass spectrometry. The cyanogen bromide peptides of cytochrome c and bacteriorhodopsin showed mixtures of formylated ions, [M + H + 28]+, and nonformylated ions, [M + H]+. Bovine adrenal medulla dodecapeptide and bovine adrenal peptide E were not formylated during digestion. Formylated peptides could be resolved from the corresponding nonformylated peptides using reversed-phase HPLC. Instability of the formylated peptides prevented localization of the adduct by Edman degradation. However, B/E-linked scanning during FAB mass spectrometric analysis with collisional activation of the [M + H + 28]+ ion of a cyanogen bromide peptide from cytochrome c suggested that formylation occurred at a threonine residue. On the basis of stability measurements in aqueous solution and analysis by FAB mass spectrometry, it was determined that serine and threonine residues are the most likely sites of esterification by formic acid during cyanogen bromide digestion of proteins. Furthermore, substitution of 70% trifluoroacetic acid for formic acid during cyanogen bromide digestion eliminated formylation and generated little or no trifluoroacetylation.

Adrenal Medulla↗
PubMed1982

ATP-independent proteolysis of globin cyanogen bromide peptides in rabbit reticulocyte cell-free extracts.

The catabolism of two rabbit globin cyanogen bromide peptides in cell-free extracts of ATP-depleted rabbit reticulocytes has been studied. Proteolysis of the peptides (3533 and 5957 molecular weight) proceeded rapidly in the absence of ATP, had a pH optimum of approximately 7.8, and was inhibited by omicron-phenanthroline, N-ethylmaleimide, cystamine zinc and cobalt ions, and puromycin peptide high-molecular-weight aggregates. Proteolysis of puromycin peptides was inhibited by the rabbit globin cyanogen bromide peptides. The ability of cell-free extracts of degrade the globin cyanogen bromide peptides decreased with the reticulocyte maturity. Blocking the globin cyanogen bromide peptide amino groups by succinic and maleic anhydride treatment decreased susceptibility to degradation. It is suggested that the globin cyanogen bromide peptides might provide model substrates, replacing puromycin peptides, for the investigation of ATP-independent proteolytic events.

Adenosine Triphosphate↗
PubMed1983

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. II) The primary structure of thermophilic lactate dehydrogenase from Bacillus stearothermophilus. Cyanogen bromide fragments and partial sequence.

The polypeptide chain of thermophilic lactate dehydrogenase from Bacillus stearothermophilus was split with cyanogen bromide. The 6 cyanogen bromide fragments were then separated and isolated by gel filtration (Bio-Gel P 10, Sephadex G-75) and ionic exchange chromatography (Biorex 70), respectively. Peptide fractionation was performed in 50% formic acid. Fragment yield varied between 30 and 75%. About 75% of the amino-acid sequence was determined by the automatic N-terminal sequence analysis (amino-acid sequenator) of the cyanogen bromide fragments (41-57 cycles degraded) and N-terminal region of lactate dehydrogenase (74 cycles degraded). Typical structure differences between thermophilic and mesophilic lactate dehydrogenases are already indicated by the comparison of the amino-acid composition of the thermophilic enzyme from B. stearothermophilus with the mesophilic from bacilli and higher organisms. Comparison of the N-terminal sequence reveals that sequence homology is higher (83-98%) between the thermophilic lactate dehydrogenases from B. stearothermophilus, B. caldotenax and B. caldolyticus than between the mesophilic lactate dehydrogenases of bacilli among each other or between thermophilic and mesophilic lactate dehydrogenases (about 60%). High temperature would appear to limit variation in structure.

Amino Acid Sequence↗
PubMed1981

Primary structure of 3-phosphoglycerate kinase from horse muscle. II. Amino acid sequence of cyanogen bromide peptides CB1-CB4 and CB6-CB14, sequence of methionine-containing regions, and complete sequence of the enzyme.

The amino acid sequences of 13 of the 14 cyanogen bromide peptides of horse muscle 3-phosphoglycerate kinase have been determined. These peptides together constitute 75% of the structure of the enzyme. Except for the smallest peptides, automated sequence analysis of the parent peptide was employed together with the automated or manual (5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Edman method) sequencing of relevant peptides obtained by proteolytic digestion. In the case of the five smallest peptides, all between 6 and 11 residues in length, sequences were derived from analyses of the intact peptides. CB14a, a peptide produced in low yield during the preparation of the cyanogen bromide fragments, was helpful in the sequence analysis of CB14. This peptide appeared to have arisen as the result of a cyanogen bromide cleavage on the carboxyl side of a tryptophanyl residue. The sequences of these 13 cyanogen bromide peptides, together with that of CB1, reported previously (Hardy, G. W., Darbre, A., and Merrett, M. (1981) J. Biol. Chem. 256, 10284-10292), have been combined with data obtained from the sequence analysis of tryptic peptides derived from the methionine-containing regions of the enzyme to determine the complete sequence. Isolation of these overlap peptides was facilitated by the labeling of methionyl residues with iodo[2-14C]acetic acid prior to tryptic digestion. Tryptophan-containing tryptic peptides were also isolated, and their sequence analysis enabled the positions of the four tryptophanyl residues to be established. In this way, the problem caused by tryptophan degradation during the preparation of the cyanogen bromide fragments was overcome. The NH2 terminus of CB1, the NH2-terminal cyanogen bromide peptide, was identified as N-acetyl serine by mass spectrometry. The alignment of the cyanogen bromide fragments of horse muscle 3-phosphoglycerate kinase described here is in complete agreement with that based on X-ray analysis of the enzyme reported previously (Banks, R. D., Blake, C. C. F., Evans, P. R., Haser, R., Rice, D. W., Hardy, G. W., Merrett, M., and Phillips, A. W. (1979) Nature 279, 773-777). The complete structure is comprised of 416 residues and has a molecular weight of 44,519.

Amino Acid Sequence↗
PubMed1975

Primary structure of porcine pepsin. I. Purification and placement of cyanogen bromide fragments and the amino acid sequence of fragment CB5.

Fragments resulting from the cyanogen bromide cleavage of reduced and aminoethylated porcine pepsin were purified. Only four of the five fragments theoretically present could be accounted for in major yield when the cyanogen bromide reaction was carried out at room temperature. The NH2-terminal fragment, CB2, contained an internal homoserine which was not cleaved to any significant extent. The amino acid sequence around this internal homoserine was determined by isolating and partially determining the sequence of an alpha-chymotryptic peptide. Cleavage at this methionine was increased by 50% when the cyanogen bromide reaction was carried out at 37 degrees. The NH2- and COOH-terminal sequences of five major fragments were determined. The placement of these fragments in the native pepsin molecule was demonstrated. The amino acid sequence of one of the fragments, CB5, was determined. This fragment contains 44 residues with an internal disulfide bridge. The COOH-terminal methionine of this fragment was connected to another 37-residue cyanogen bromide fragment of known sequence. Together these two fragments formed the COOH-terminal 81 residues of porcine pepsin.

Amino Acid Sequence↗
PubMed1977

Amino acid sequence studies on the alpha chain of human fibrinogen. Isolation and characterization of two linked alpha-chained cyanogen bromide fragments from fully cross-linked fibrin.

Fully cross-linked human fibrin was digested with cyanogen bromide and the resulting fragments were characterized and compared with the fragments produced upon cyanogen bromide treatment of fibrinogen alpha chains. The largest molecular-weight fraction isolated by gel filtration on Sephadex G-150 was reduced and alkylated, and upon rechromatography on Sephadex G-150 it eluted at the same place as the original material. This large molecular weight fraction was subjected to amino acid analysis and the amino-terminal sequences of its constituent chains were determined by both dimethylaminonaphthyl sulfonation (Dns) and thioacetylation procedures. The identified sequences corresponded to two cyanogen bromide fragments previously found in alpha chains isolated from fibrinogen, one of which has a molecular weight of about 30 000 and the other 6000. The latter is thought to be the carboxy-terminal penultimate cyanogen bromide fragment of the alpha chain.

Amino Acid Sequence↗
PubMed1986

Amino acid sequence of cyanogen bromide fragments of potato phosphorylase.

Amino acid sequence analysis of the cyanogen bromide peptides of potato alpha-glucan phosphorylase was undertaken for comparison with rabbit muscle glycogen phosphorylase and for elucidation of the structural bases for the differences in the catalytic and regulatory properties between the animal and plant enzymes. The potato enzyme was carboxymethylated and cleaved with cyanogen bromide. The 17 distinct fragments produced were isolated by a combination of gel filtration, sulfopropyl ion exchange chromatography, and high performance liquid chromatography. The molecular weights of these fragments are distributed in a range of 300 to 30,000. Fragment CI has a blocked amino terminus, and has the same amino acid sequence as CII, which has been assigned as the amino-terminal fragment of potato phosphorylase. The blocking group was deduced to be an acetyl group from the results of fast atom bombardment mass spectrometry of an amino-terminal pentapeptide. This paper describes the sequence determination of all the cyanogen bromide fragments of potato phosphorylase. The complete structure is presented in the following paper (Nakano, K., and Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236).

Animals↗
PubMed1976

Primary structure of streptococcal proteinase. II. Isolation, composition, and amino acid sequences of the tryptic and chymotryptic peptides of cyanogen bromide fragment 5.

The amino acid sequence of the cyanogen bromide fragment 5 of streptococcal proteinase has been determined. This fragment comprises residues 130 to 253 of the proteinase chain. Six tryptic peptides were isolated from maleylated cyanogen bromide fragment 5, and their alignment was obtained by the overlap of chymotryptic peptides. Sequence analysis of tryptic, chymotryptic, and thermolysin peptides was performed by the 5-deimethylaminoaphthalene-1-sulfonyl technique and carboxypeptidases digestion.

Amino Acid Sequence↗
PubMed1983

[Primary structure of the elongation factor G from Escherichia coli. VI. Structure of peptides of cyanogen bromide cleavage of the G-factor molecule].

Peptides obtained as a result of cyanogen bromide cleavage of the G-factor have been studied. All 12 peptides embracing the whole structure of fragment T4 have been isolated. For their amino acid sequence determination, cyanogen bromide peptides have been further cleaved with trypsin, chymotrypsin, thermolysin, staphylococcal glutamic protease and BNPS-skatole. The complete primary structure of 9 from 12 cyanogen bromide peptides has been determined.

Amino Acid Sequence↗
PubMed1984

Primary structure of flavocytochrome b2 from baker's yeast. Purification by reverse-phase high-pressure liquid chromatography and sequencing of fragment alpha cyanogen bromide peptides.

Reverse-phase high-pressure liquid chromatography has been used for the purification of some large cyanogen bromide peptides from flavocytochrome b2 fragment alpha. Acetonitrile gradients at acid and/or neutral pH using mu Bondapak C18 columns were useful for the smaller peptides (43 and 67 residues). The two larger ones, alpha CB1 and alpha CB2, could only be separated from each other by trifluoroacetic acid/1-propanol gradients on mu Bondapak-CN columns. The various systems tested are presented and compared. The elucidation of the amino acid sequence of alpha CB2 (95 residues), alpha CB3 (67 residues) and alpha CB4 (43 residues) is described. The fragments were digested with trypsin, chymotrypsin and Staphylococcus aureus V8 protease as necessary. Fragment alpha CB2 was also cleaved at the unique tryptophanyl bond with cyanogen bromide. Peptides were fractionated by Sephadex chromatography, thin-layer finger-printing and/or high-pressure liquid chromatography. Peptides were sequenced mostly in the liquid phase sequenator. The cyanogen bromide peptides could be ordered using information obtained previously, as well as additional data obtained in this work. Together with the previous elucidation of cytochrome b2 core sequence and of the hinge region [Guiard, B. and Lederer, F. (1976) Biochimie (Paris) 58, 305--316; Ghrir, R. and Lederer, F. (1981) Eur. J. Biochem. 120, 279--287], the present results enable us to present the complete sequence of fragment alpha (314 residues) with only three overlaps missing between cyanogen bromide peptides. Sequence comparisons with other known flavoproteins do not indicate any noticeable similarity. Structural predictions indicate an alteration of alpha helices and beta structure. The possibility that the non-heme-binding portion of fragment alpha could constitute a flavin-binding domain is discussed.

Amino Acid Sequence↗
PubMed1985

Cyanogen bromide cleaves Fc fragments of pooled human IgG at both methionine and tryptophan residues.

An attempt was made to obtain fragments containing the C gamma 2 region by selectively cleaving human Fc fragments prepared from pooled IgG at Met residues using cyanogen bromide. Based on the known locations of Met residues in the Fc regions of human IgG subclasses, fragments between Met 252 and 358, comprising the C gamma 2 domains, were expected from IgGl Gm -1, IgG2, IgG3 and IgG4. Greater fragmentation of the Fc fragments occurred, however, than was predicted. Automated N-terminal sequencing identified five major points (Trp 381, 313 and 277, and Met 397 and 252) and two minor points of cleavage (Met 428 and Trp 417). The majority of cleavage points occurred at Trp rather than Met. Furthermore, cleavage at Met 358, necessary to produce C gamma 2 domains, was not detected. Control experiments verified the integrity of the Fc fragments handled in exactly the same manner without cyanogen bromide exposure and the ability of the same cyanogen bromide preparation to produce the expected cleavages at Met of sperm whale apomyoglobin without fragmentation at Trp. Cleavage at Met 358 did not occur presumably because of the difficulty associated with cyanogen bromide cleavage at Met-Thr peptide bonds. Cleavage at Trp probably occurred by way of halogen promoted oxidation of the indole nucleus with resultant peptide bond fissure. These observations show that cyanogen bromide cleavage of pooled human Fc fragments is not selective for Met, but also cleaves at Trp residues. The resultant fragmentation of the C gamma 2 region coupled with the inability to make the required cleavage at the 358-359 Met-Thr bond resulted in the inability to produce fragments comprising the C gamma 2 domains. The reasons for the selective cleavage at Met in some proteins and the cleavage at both Trp and Met in others are not known.

Chemical Phenomena↗
PubMed1975

Amino-acid sequence of lac repressor from Escherichia coli. Isolation, sequence analysis and sequence assembly of tryptic peptides and cyanogen-bromide fragments.

The lac repressor from Escherichia coli, composed of four identical subunits with a molecular weight of 37160, was carboxymethylated and fragmented by tryptic digestion and cyanogen bromide treatment. Using ion-exchange chromatography, gel filtration and preparative thin-layer electrophoresis and chromatography 29 of the 30 tryptic peptides were isolated in pure form. Direct Edman degradation and the dansyl-Edman technique were used to determine the sequence of the small tryptic peptides. Special emphasis was put on the sequence determination of the six large tryptic fragments which together account for 177 residues, corresponding to 51% of the repressor subunit with its 347 residues. The large tryptic fragments were analyzed after fragmentation with chymotrypsin, thermolysin and dipeptidyl aminopeptidase I. Thus the sequence of all 30 tryptic peptides could be deduced. The complete sequences of all cyanogen bromide fragments were deduced from peptides obtained by tryptic, chymotryptic and thermolytic digestion of the individual fragments and by automated stepwise Edman degradation of lac repressor and of the large cyanogen bromide fragments. The order of the cyanogen bromide fragments was given by overlapping tryptic peptides. The resulting amino acid composition of the monomer is Asp15, Asn11, Thr18, Ser30, Glu14, Gln27, Pro13, Gly22, Ala44, Cys3, Val33, Met9, Ile17, Leu40, Tyr8, Phe4, Trp2, Lys11, His7, Arg19. The sequence of lac repressor shows no similarities with that of other proteins known to bind to DNA or RNA. The N-terminal 55 residues contain two homologous regions. This part of the sequence which is involved in lac operator binding might have been formed by gene duplication.

Amino Acid Sequence↗