Primary structure of the alanine subunit of ricin T from seeds of the Central Asian castor oil plant. V. Peptides obtained by cyanogen bromide cleavage

The cleavage of the carboxymethylated Ala subunit of ricin T by cyanogen bromide has been performed. Four fragments have been isolated in the individual state by gel filtration and high-voltage paper electrophoresis. The sequence of the cyanogen bromide peptides in the protein chain has been established. Partial structures of three cyanogen bromide fragments have been determined.Institute of Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodynkh Soedinenii, No. 6, pp. 845–848, November–December, 1988.     

Investigation of the pyrolysis products of methionine-enkephalin-Arg-Gly-Leu using liquid chromatography-tandem mass spectrometry

Low-temperature pyrolysis of methionine-enkephalin-Arg-Gly-Leu has been carried out and the non-volatile residues have been analyzed. The fragments were separated and characterized by LC-UV/Vis-MS/MS. Two major types of pyrolysis products were identified by matching the experimental results with a theoretical list that contains the expected fragments. These products were mainly composed of cyclic oligopeptides and linear fragments produced from the peptide backbone. These fragments have preserved the sequence of amino acids in the peptide. In some cases, a complete or partial loss of an amino-acid side group was observed. Tandem mass spectrometry and cyanogen bromide cleavage experiments were used to confirm the nature of the cyclic and linear pyrolysates, in addition to chromatographic and mass spectrometric data of actual standard synthetic cyclic peptides.     

Chemical characterization of the crystalline proteins ofBac. thuringiensis. Cleavage with cyanogen bromide

The crystalline proteins fromBac. thuringiensis of the third and fourth serotypes with a molecular weight of 130,000 have been isolated and carboxymethylated, and their amino acid compositions has been determined. The CM-proteins have been found to be labile in acid media (80% CF₃COOH and 65% HCOOH). Conditions have been found for the cleavage of the CM-protein of the third serotype with cyanogen bromide. A BrCN fragment with a molecular weight of 16,000 has been isolated, the intensity of which in 5% polyacrylamide gel on scanning amounts to about half the intensity of the sum of all the fragments. The aminoacid composition of this fragment has been determined.Institute of Chemistry of the Bashkir Branch, Academy of Sciences of the USSR, Ufa. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 384–386, May–June, 1980.     

Primary structure of triacetinase — An esterase from cotton seeds IV. Peptides of the chymotryptic and thermolytic hydrolysis of cyanogen bromide fragments of triacetinase

Summary 1. Peptides from a chymotryptic hydrolyzate of cyanogen bromide fragment B-7 have been isolated and characterized. The structure of some peptides of the chymotryptic hydrolyzate have been studied and this has enabled the tryptic peptides to be linked up.2. The peptides of a thermolysin hydrolyzate of cyanogen bromide B-5 have been isolated and characterized. The study of the structure of the thermolysin peptides has permitted the linking of two tryptic peptides.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 248–253, March–April, 1978.     

Primary structure of the alanine subunit of ricin T from the seeds of the Central Asian castor oil plant VI. Tryptic peptides of the cyanogen bromide fragments B-1, B-2, and B-3

Tryptic hydrolysates of cyanogen bromide fragments B-1, B-2, and B-3 have been studied. As a results, 23 tryptic peptides have been isolated and characterized and the primary structures of 22 of them have been established. Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (371) 120 64 75. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 801–806, November–December, 1998.     

Use of disk electrophoresis for the separation of large peptide fragments of proteins

Summary The disk electrophoresis of peptide fragments from porcine pepsin obtained by cleaving the pepsin with cyanogen bromide has been performed. A method has been developed for extracting the colored peptide fractions after electrophoresis in polyacrylamide gel. An amino-acid analysis has been performed of peptide fragments B1, B2, and B4 isolated by disk electrophoresis. The results obtained have been compared with the amino-acid composition calculated from the structures of these peptides. The possibility has been shown of using the method described during structural investigations of both proteins and their low-molecular-weight fragments.The peptide preparations were supplied to us by I. Surova, V. Ostoslavskaya, L. Revina, I. Puracheva, É. Vakhitova, and G. Muratova. The amino-acid analysis was performed by E. Timokhina, A. Balduev, G. Fedyukina, O. Khodova, and N. Ivashechkina.All-Union Scientific-Research Institute of the Genetics and Breeding of Industrial Microorganisms. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 219–222, March–April, 1975.     

Purification and determination of the binding site of lactate dehydrogenase from chicken breast muscle on immobilized ferric ions.

Lactate dehydrogenase from chicken breast muscle was purified to homogeneity in one step by immobilized metal ion affinity chromatography. The purified enzyme was used to localize the binding site to immobilized Fe(III) ions. After cyanogen bromide degradation and digestion with trypsin, small enzyme fragments capable of binding to immobilized Fe(III) ions were obtained. It is proposed that several histidyl groups are involved in the binding.     

A study of the structure of lactosomatotropic hormone

Summary 1. Peptides from tryptic hydrolysis of the cyanogen bromide fragments B-1 and B-2 of LSTH have been isolated and characterized.2. The differences found in the amino-acid sequence of the individual peptides of fragment B of LSTH as compared with the corresponding sections of the O-LTH molecule are not due to species differences of the hormones.All-Union Scientific-Research Institute of the Technology of Blood Substitutes and Hormone Preparations. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 502–507, July–August, 1975.     

Semisynthesis iii - the homoserine 12,105 analogue of hen egg-white lysozyme.

The homoserine ^12,105 analogue of Hen egg-white lysozyme has been prepared by two routes. In the first approach a large excess of cyanogen bromide was used to cleave the native enzyme at methionines 12 and 105, the tertiary structure being maintained by the presence of the four disulphide bonds. Formation of the two new amide bonds was achieved using the activation of the homoserine lactone residues generated at positions 12 and 105; the reaction being most efficient when anhydrous DMSO was used as the solvent. An alternative approach using limited (two-fold excess) cyanogen bromide digestion gave the same homoserine analogue without chain fragmentation. A purified sample of the analogue showed no enzymic activity.     

Synthesis of an adsorbed reversed-phase packing material for the separation of proteins and peptides

We have described the preparation and chromatographic evaluation of an adsorbed hydrophobic stationary phase suitable for reversed-phase chromatography of proteins and peptides. The synthetic procedure involves three steps: the adsorption of a polyamine to the silica surface; crosslinking of the adsorbed polyamine layer with a bis-phenyl difunctional epoxide; and the benzoylation of the remaining accessible amino groups. Performance of this chromatographic material compared favorably with SynChropak RP-8 silica (SynChrom, Linden, IN, U.S.A.) and was stable to 40% formic acid. Good separations were obtained between the components of sample mixtures containing proteins or the cyanogen bromide fragments of sperm whale myoglobin. However, in both cases, the adsorbed hydrophobic stationary phase was less retentive. Furthermore, this medium exhibited slightly different selectivity. Whereas the heme which was present in the cyanogen bromide digest of myoglobin desorbed as the second peak from the RP-8 column, it eluted last from the adsorbed stationary phase. Comparable performance, acid stability and alternate selectivity suggest that this material is an interesting alternative to organosilane reversed-phase coatings.     

Condensed 1,3-benzothiazinones. 1. Facile synthesis of 2-amino-1,2,4-triazolo[5,1-b][1,3]-benzothiazin-9-one

Treating 2-mercaptobenzohydrazide ( 1a ) with cyanogen bromide gave 3-amino-2-imino-3,4-dihydro-2.H-1,3-benzothiazin-4-one ( 2a ). This compound underwent further cyclocondensation with a second molecule of cyanogen bromide or S-methylisothiourea sulfate to afford the biologically interesting 2-amino-1,2,4-triazolo-[5,1-b][1,3]benzothiazin-9-one ( 3c ). Compound 3c could also be prepared directly from 1a by treating with excess amount of cyanogen bromide in more satisfactory yield.     

Spectroscopic analysis and drug-binding studies of the CNBr fragments of human serum albumin

Three large fragments (A, B and C) of human serum albumin (HSA) were produced by cyanogen bromide digestion of HSA in order to investigate the specific binding sites. The fragments were isolated by use of gel filtration, followed by high performance ion exchange chromatography. The isolated fragments were examined by use of UV/Vis, steady-state fluorescence, and circular dichroism spectroscopy. The study was extended to examine the interactions of bilirubin and two anionic drugs, warfarin and naproxen, with HSA and the three fragments. The primary bilirubin binding site on HSA molecule appeared to be located between fragment A and fragment C. The results also suggest the binding sites of the two anionic drugs to mosy likely be located in fragment C of HSA molecule.     

Molecular Rydberg transitions. Intermediate coupling in simple bromides

An intermediate coupling analysis of the low-energy π → σ Rydberg transitions of hydrogen bromide, cyanogen bromide, acetylene bromide and alkyl bromides is presented. Exchange and spin-orbit coupling parameters are calculated and their variations discussed. The Br 5s orbital is delocalized in the larger alkyl bromides and Br 4p is heavily involved in π bonding in acetylene and cyanogen bromides.     

N-Cyanoimides via the Cyanation of Imides

The conversion of cyclic imides to the corresponding N-cyanoimides has been carried out using cyanogen bromide as the nitrile source. This methodology provides a convenient route for the preparation of both aromatic and aliphatic N-cyanoimides.     

Comparison of the performance of immunosorbents prepared by site-directed or random coupling of monoclonal antibodies.

The majority of methods used to prepare immunosorbents immobilize antibodies through their reactive amino acid residues. The bound antibody activity of these immunosorbents is low. Hydrazide-based matrices couple antibodies through carbohydrate chains frequently located in the Fc region. This paper reports a comparative study of the performance of immunosorbents prepared by cyanogen bromide or hydrazide immobilization methods. The experiments utilized murine monoclonal antibodies to the human plasma proteins Factor IX or Protein C. The antibodies were immobilized at low densities to beaded agarose matrices which had similar properties. The hydrazide immunosorbents had binding efficiencies which were lower (anti-Factor IX) or up to 1.6-fold higher (anti-Protein C) than comparable cyanogen bromide coupled gels. However, there was no improvement in performance due to lower recoveries of bound protein from the hydrazide gels. Control experiments demonstrated that oxidation of antibody which is required for its coupling to hydrazide gels had no effect on antibody binding to antigen. Our results indicate that, as with cyanogen bromide coupling methods, site-directed immobilization through carbohydrate residues results in a restricted ability to bind to antigen. Both monoclonals were found to contain carbohydrate in their Fab' regions through which coupling may have occurred. The frequency of carbohydrate in the Fab region and the ability to control glycosylation at these sites are factors which may impact the utility of carbohydrate-directed immobilization of antibodies.     

Separation methods for the study of collagen and treatment of collagen disorders

Liquid chromatographic and electrophoretic methods applicable to the separation of collagen and its fragments are reviewed. Special attention is paid to the separation of both stabile and labile crosslinking elements. Identification procedures exploiting the mapping of either collagen alpha-chains or of cyanogen bromide fragments are discussed. These methods can be used for diagnosing inborn errors of collagen metabolism using bioptic or necroptic samples. Analysis of urinary hydroxyproline-containing peptides or the determination of peptidically bound pyridinoline is suitable for measuring the intensity of collagen metabolism.     

Proteomics of gluten: mapping of subunit 1 Ax2* in Cheyenne cultivar by matrix-assisted laser desorption/ionization.

This paper reports results on the verification of the 1Ax2* high molecular weight glutenin subunit sequence in Cheyenne cultivar. The gene sequence of the protein is known but recently some text changes have been made, and furthermore until now no characterization of post-translational modifications has been reported. The two published sequences, named I and II, differ in four residues at positions 23, 208, 475, and 611. The first sequence contains 20 Arg and 6 Lys residues, producing 26 tryptic fragments, since the Arg(109)-Pro(110) bond is generally not cleaved by trypsin. The second sequence contains 19 Arg and 6 Lys residues, producing 25 tryptic peptides, again because of the Arg(109)-Pro(110) bond. Both sequences generate two cyanogen bromide fragments. Matrix-assisted laser desorption/ionization analysis of the tryptic digest of the high-MW glutenin subunit 1Ax2* resulted in the identification of 24 out of the 26 expected peptides for sequence I, a sequence coverage of 99.5%. These results were sufficient to rule out sequence II and any protein glycosylation and any other post-translational modifications to within the detection limits of the method. It was found that the choice of matrix considerably influenced the sequence coverage in peptide mapping.     

Cyanogen bromide cleavage generates fragments suitable for expressed protein and glycoprotein ligation

Herein cyanogen bromide is employed for the efficient production of N-terminal cysteine containing protein fragments for expressed protein ligation (EPL) from polyhistidine-tagged precursors. We provide three examples of efficient CNBr cleavage of fragments of the glycoprotein erythropoietin that can be ligated with peptides or glycopeptide mimetics potentially giving rise to semisynthetic glycoprotein therapeutics.     

2-Trimethylsilylethyl sulfides in the von Braun cyanogen bromide reaction: selective preparation of thiocyanates and application to nucleoside chemistry

Mixed 2-(trimethylsilyl)ethyl sulfides were synthesized and used in the von Braun cyanogen bromide reaction for preparing selectively thiocyanates in high yield. We show here that this cleavage reaction is highly selective in methanol in comparison with the reaction of the corresponding non-silyl sulfide analogues. This reaction was applied to the synthesis of nucleosidic thiocyanates such as the new nucleosides 14 and 18 in the search for mechanism-based inhibitors of ribonucleoside diphosphate reductase and bioactive molecules. The selective cleavage is possible for sulfides bearing hydroxyl functions and aromatic rings. The reactions of cyanogen bromide as cyanating and brominating agent were observed for the first time under the same conditions with the naphthoxyhexyl 2-trimethylsilylethyl sulfide 7, which, treated with cyanogen bromide in dichloromethane, led selectively to the p-bromonaphthoxyhexyl thiocyanate 10 in 89% yield. Another reaction induced by cyanogen bromide was observed in dichloromethane with the 2-(trimethylsilylethyl)thio nucleoside 13, which gives the corresponding symmetrical disulfide 21 in good yield.     

Cyanogen Bromide Promoted Thiol Dimerization

The synthesis of alkyl thiocyanates by the reaction of thiols with cyanogen chloride has been known,¹ although it is generally supersede by the more convenient displacement of alkyl halides with alkali thio-cyanates.² We wish to describe the behavior of sodium thiolates toward cyanogen bromide.