Specific cleavage of glucosephosphate isomerases at cysteinyl residues using 2-nitro-5-thiocyanobenzoic acid: analyses of peptides eluted from polyacrylamide gels and localization of active site histidyl and lysyl residues

Specific chemical cleavage of human placental and porcine muscle glucosephosphate isomerases at three amino peptide bonds of cysteinyl residues with 2-nitro-5-thiocyanobenzoic acid was achieved. Four primary peptides were generated from the cyanylated human glucosephosphate isomerase, indicating the quantitative cleavage of this enzyme. Four primary plus six overlap peptides were obtained from the cleavage of the swine muscle enzyme. The peptides were separated by SDS-polyacrylamide gel electrophoresis and eluted from the gels. Amino acid and carboxyl terminal analyses of the eluted peptides have permitted the alignment of these peptides with respect to the native polypeptide chain. The analysis of the enzyme which had been specifically covalently labeled at the essential lysine and histidine residues of the active center revealed that the active-site histidine and lysine residues are located on two distinct peptides with molecular weights of 27,500 and 14,000, respectively.     

The occurrence of extended acidic sequences in nonhistone chromosomal proteins

Nonhistone chromosomal proteins and soluble cytoplasmic proteins from rat liver were treated with a combination of proteases and chemical reagents which split a variety of peptide bonds but do not attack sequences consisting predominantly or exclusively of acidic amino acid residues. Analysis of the resulting digests by gel filtration chromatography and column electrophoresis demonstrated that, relative to cytoplasmic proteins, nonhistone chromosomal proteins are rich in highly charged, acidic peptides up to 12 residues in length, but rarely contain very long peptides consisting exclusively of acidic residues such as are found in the nonhistone chromosomal proteins HMG1 and HMG2.     

Specific peptide-bond cleavage by microwave irradiation in weak acid solution

A rapid and selective peptide-bond cleavage in weak acid, induced by microwave irradiation, has been developed. The specific cleavage sites of peptide bonds located only at the carboxyl-and amino-terminal ends of aspartyl residues along the peptide chain. A systematic study including the time course for the cleavage of various aspartyl-containing peptides, the effect of the acidity of the reaction solution on the completeness of peptide-bond cleavage, and the relationship between the power of microwave irradiation and the reaction time of cleavage are studied.     

Design and synthesis of new types of oligonucleopeptides

Summary Design and synthesis of oligonucleopeptides (ONPs), structural analogues of oligonucleotides, where the phosphodiester backbone is substituted by a peptide chain, are described. Oligonucleopeptides, in which the number of ordinary bonds between the nucleobases is six and the number of bonds between the backbone and nucleobase is two or four were constructed using two different approaches. The first way is based on incorporation of thyminylalanine residues into the peptide chain alternatively with glycine residues. Experimental studies of the stability of oligonucleotide-oligonucleopeptide complexes as well as model estimations of their potential surfaces indicated the low DNA binding efficiency of this type of reagents. The second approach consists of synthesis of ω-ornithine peptides followed by modification of the backbone with thyminylacetaldehyde attached to an α-amino function of ornithine residues through Schiff bases. ONPs were synthesized using the solid-phase method.     

Mass spectrometric identification of the trypsin cleavage pathway in lysyl-proline containing oligotuftsin peptides.

Trypsin cleaves specifically peptide bonds at the C-terminal side of lysine and arginine residues, except for -Arg-Pro- and -Lys-Pro- bonds which are normally resistant to proteolysis. Here we report evidence for a -Lys-Pro- tryptic cleavage in modified oligotuftsin derivatives, Ac-[TKPKG]4-NH2) (1), using high-resolution mass spectrometry and HPLC as primary methods for analysis of proteolytic reactions. The proteolytic susceptibility of -Lys-Pro- bonds was strongly dependent on flanking residues, and the flexibility of the peptide backbone might be a prerequisite for this unusual cleavage. While -Lys-Gly- bonds in 1 were rapidly cleaved, the modification of these Lys residues by the attachment of a ss-amyloid(4-10) epitope to yield -Lys(X)-Gly derivatives prevented cleavage of this bond, and provided trypsin cleavage of -Lys-Pro- bonds, the pathway of this degradation being independent on the type of Lys-N(epsilon)-side chains (acetyl group, amino acid, peptide). Substitution of the Lys residues by Ala at the P'2 positions decreased the tryptic cleavage, while replacement of the bulky side chain of Thr at the P2 positions strongly increased the cleavage of -Lys-Pro- bonds. Circular dichroism (CD) data of the modified oligotuftsin derivatives are in accord with enhanced flexibility of the peptide backbone, as a prerequisite for increased susceptibility to cleavage of -Lys-Pro- bonds. These results obtained of oligotuftsin derivatives might have implications for the proteolytic degradation of target peptides that require specific conformational preconditions.     

Labeling of cysteine-containing peptides with 2-nitro-5-thiobenzoic acid

A method for specific labeling of cysteine-containing peptides has been developed using Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Prior to cleavage with proteases or chemical reagents, proteins are reacted with DTNB, resulting in the formation of a mixed disulfide between the protein sulfhydryl group and 2-nitro-5-thiobenzoic acid (TNB). The formation of the mixed disulfide introduces a chromophore, with an absorbance peak at 328 nm. By monitoring peptide maps generated by HPLC at 210 and 328 nm, peptides containing cysteine residues are readily identified. The stability of the derivative was tested using glutathione-TNB as a model compound. Glutathione-TNB is stable to conditions used for CNBr cleavage, as well as those for tryptic cleavage. The TNB label may also increase the hydrophobicity of small peptides, which otherwise might not bind to reverse-phase matrices. This was the case for an oxidatively modified tetrapeptide isolated from Escherichia coli glutamine synthetase.     

Amino acid sequence of the Bb fragment from complement Factor B. Sequence of the major cyanogen bromide-cleavage peptide (CB-II) and completion of the sequence of the Bb fragment.

The amino acid sequence of peptide CB-II, the major product (mol.wt. 30 000) of CNBr cleavage of fragment Bb from human complement Factor B, is given. The sequence was obtained from peptides derived by trypsin cleavage of peptide CB-II and clostripain digestion of fragment Bb. Cleavage of two Asn-Gly bonds in peptide CB-II was also found useful. These results, along with those presented in the preceding paper [Gagnon & Christie (1983) Biochem. J. 209, 51-60], yield the complete sequence of the 505 amino acid residues of fragment Bb. The C-terminal half of the molecule shows strong homology of sequence with serine proteinases. Factor B has a catalytic chain (fragment Bb) with a molecular weight twice that of proteinases previously described, suggesting that it is a novel type of serine proteinase, probably with a different activation mechanism.     

Ring cleavage of 2-iminothiazolidine-4-carboxylates by catalytic reduction. A potential method for unblocking peptides formed by specific chemical cleavage at half-cystine residues

Specific chemical cleavage of proteins at S-cyanocysteine residues leads to formation of iminothiazolidinecarboxylyl peptides which, because their amino termini are blocked, are not susceptible to Edman degradation. A catalyst prepared from NiCl₂ and NaBH₄ converts 2-iminothiazolidine-4-carboxylate to alanine and iminothiazolidine carboxylylglycine to Ala-Gly in good yield. Unmodified proteins treated with this catalyst in 8M guanidinium chloride are recovered in good yield, with quantitative conversion of methionine to amino-butyrate and half-cystine to alanine by desulfuration. The catalyst also induces cleavage of a small subclass of peptide bonds, probably Phe-Thr and Phe-Ser sequences, producing discrete fragments.     

Primary structure of tyrosinase from Neurospora crassa. I. Purification and amino acid sequence of the cyanogen bromide fragments

Cyanogen bromide (CB) cleavage of Neurospora tyrosinase resulted in four major fragments, CB1 (222 residues), CB2 (82 residues), CB3 (68 residues), and CB4 (35 residues), and one minor overlap peptide CB2-4 (117 residues) due to incomplete cleavage of a methionylthreonyl bond. The sum of the amino acid residues of the four major fragments matches the total number of amino acid residues of the native protein. The amino acid sequences of the cyanogen bromide fragments CB2, CB3, and CB4 were determined by a combination of automated and manual sequence analysis on peptides derived by chemical and enzymatic cleavage of the intact and the maleylated derivatives. The peptides were the products of cleavage by mild acid hydrolysis, trypsin, pepsin, chymotrypsin, thermolysin, and Staphylococcus aureus protease V8. The cyanogen bromide fragment CB1 was found to contain two unusual amino acids whose chemical structure will be presented in the following paper.     

Synthesis of relaxin‐2 and insulin‐like peptide 5 enabled by novel tethering and traceless chemical excision

This report presents an entirely chemical, general strategy for the synthesis of relaxin‐2 and insulin‐like peptide 5. Historically, these two peptides have represented two of the more synthetically challenging members of the insulin superfamily. The key synthetic steps involve two sequential oxime ligations to covalently link the individual A‐chain and B‐chain, followed by disulfide bond formation under aqueous, redox conditions. This is followed by two chemical reactions that employ diketopiperazine cyclization‐mediated cleavage and ester hydrolysis to liberate the connecting peptide and the heterodimeric product. This approach avoids the conventional iodine‐mediated disulfide bond formation and enzyme‐assisted proteolysis to generate biologically active two‐chain peptides. This novel synthetic strategy is ideally suited for peptides such as relaxin and insulin‐like peptide 5 as they possess methionine and tryptophan that are labile under strong oxidative conditions. Additionally, these peptides possess multiple arginine and lysine residues that preclude the use of trypsin‐like enzymes to obtain biologically active hormones. This synthetic methodology is conceivably applicable to other two‐chain peptides that contain multiple disulfide bonds. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

The amino acid sequence of equine milk lysozyme

The amino acid sequence of equine milk lysozyme has been elucidated. The study involves the determination of the sequence of the N-terminal region of the whole protein, cyanogen bromide fragments, tryptic and chymotryptic peptides and fragments produced by chemical cleavage after tryptophan residues. The protein consists of a single chain of 129 amino acid residues and has a Mr of 14647. While equine milk lysozyme has the essential features of a c(chick)-type lysozyme, there is only 51% sequence homology with human milk lysozyme and 50% with domestic hen egg white lysozyme. Some of the implications of the large number of differences are discussed.     

Oxidation and oxidative cleavage of tryptophanyl peptide bonds during iodination

Tryptophan peptides are oxidized to the oxindole by various chemical and peroxidase catalyzed iodination procedures in the pH range 4.0–7.5. This oxidation results in significant cleavage of tryptophanyl peptide bonds at pH 4–5. Thus, destruction of some biologically active peptides and proteins during iodination may be due to the modification of essential tryptophan residues.     

The amino acid sequence of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B

We have determined the primary structure of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B. The enzyme is a dimeric protein of two identical subunits, each consisting of a polypeptide chain of 131 residues and a Mr = 14,536. The intact S-carboxymethyl protein was sequenced from the NH2 terminus using standard automated Edman degradation and automated Edman degradation using fluorescamine treatment at known prolines to suppress background. The isomerase was fragmented using CNBr, trypsin, iodosobenzoic acid, and acid cleavage at aspartyl-prolyl peptide bonds. The peptides resulting from each fragmentation were separated by reversed-phase high performance liquid chromatography and sequenced by automated Edman degradation. The full sequence was deduced by the overlapping of the various peptides. A search for homologous proteins was performed. Only the oxosteroid isomerase from Pseudomonas testosteroni, an expected homology, was found to be similar. Comparison of the two proteins shows that the region of strongest homology is the region containing the aspartic acid at which steroidal affinity and photoaffinity reagents have been shown to react in the P. testosteroni isomerase. The P. putida isomerase contains 3 cysteines and 2 tryptophans, whereas the P. testosteroni isomerase lacks these amino acids. The two proteins are not highly conserved.     

Synthesis of N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine: its use in peptide synthesis for placing a bromoacetyl cross-linking function at any desired sequence position.

A new amino acid derivative, N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine (BBAL), has been synthesized as a reagent to be used in solid-phase peptide synthesis for introducing a side-chain bromoacetyl group at any desired position in a peptide sequence. The bromoacetyl group subsequently serves as a sulfhydryl-selective cross-linking function for the preparation of cyclic peptides, peptide conjugates, and polymers. BBAL is synthesized by condensation of N-bromoacetyl-beta-alanine with N alpha-Boc-L-lysine and is a white powder which is readily stored, weighed, and used with a peptide synthesizer, programmed for N alpha-Boc amino acid derivatives. BBAL residues are stable to final HF deprotection/cleavage. BBAL peptides can be directly coupled to other molecules or surfaces which possess free sulfhydryl groups by forming stable thioether linkages. Peptides containing both BBAL and cysteine residues can be self-coupled to produce either cyclic molecules or linear peptide polymers, also linked through thioether bonds. Products made with BBAL peptides may be characterized by amino acid analysis of acid hydrolyzates by quantification of beta-alanine, which separates from natural amino acids in suitable analytical systems. Where sulfhydryl groups on coupling partners arise from cysteine residues, S-(carboxymethyl)cysteine in acid hydrolyzates may also be assayed for this purpose. Examples are given of the use of BBAL in preparing peptide polymers and a peptide conjugate with bovine albumin to serve as immunogens or model vaccine components.     

Complete amino acid sequence of steer liver microsomal NADH-cytochrome b5 reductase

The complete covalent structure of liver microsomal NADH-cytochrome b5 reductase from steer liver microsomes was determined. Cleavage at methionyl bonds gave 10 peptides accounting for all the residues of the protein. Acid cleavage of the reductase at the Asp-Pro bonds gave three peptides accounting for all the CNBr peptides in the molecule. Subfragmentation of these peptides by chemical and enzymatic cleavage provided overlaps which established all the fragments in an unambiguous sequence of 300 residues, corresponding to Mr 34,110. Limited tryptic digestion cleaved reductase at residues 28 and 119, yielding a preparation having two noncovalently linked peptides having a conformation which binds flavin and retains the structural features essential for NADH-cytochrome b5 activity. A model for the secondary structure of cytochrome b5 reductase is proposed that is based on computer-assisted analysis of the amino acid sequence. In this model the beta-turns are predominant and there is some 25% alpha and 30% beta structure.     

Characterization of the amino-terminal segment in type III procollagen.

Native type III collagen and procollagen were prepared from fetal bovine skin. Examination of the cleavage products produced by digestion with tadpole collagenase demonstrated that the three palpha1(III) chains of type III procollagen were linked together by disulfide bonds occurring at both the amino-terminal and carboxy-terminal portions of the molecule. Type III collagen contained interchain disulfide bonds only in the carboxy-terminal region of the molecule. After digestion of procollagen with bacterial collagenase an amino-terminal, triple-stranded peptide fragment was isolated. The reduced and alkylated chain constituents of this fragment had molecular weights of about 21 000. After digestion of procollagen with cyanogen bromide a related triple-stranded fragment was isolated. The chains of the cyanogen bromide fragment had a molecular weight of about 27 000. When the collagenase-derived peptide was fully reduced and alkylated, it became susceptible to further digestion with bacterial collagenase. This treatment released a fragment of about 97 amino acid residues which contained 12 cystein residues and had an amino acid composition typical for globular proteins. A second, non-helical fragment of about 48 amino acid residues contained three cysteines. This latter fragment is formed from sequences that overlap the amino-terminal region in the collagen alpha1(III) chain by 20 amino acids and possesses an antigenic determinant specific for the alpha1(III) chain. The collagenase-sensitive region exposed by reduction comprised about 33 amino acid residues. It was recovered as a mixture of small peptides. These results indicate that the amino-terminal region of type III procollagen has the same type of structure as the homologous region of type I procollagen. It consists of a globular, a collagen-like and a non-helical domain. Interchain disulfide bonding and the occurrence of cysteines in the non-helical domain are, however, unique for type III procollagen.     

Chemical studies on the cysteine and terminal peptides in tryptic digests of actin

1. On exhaustive digestion of carboxymethylated actin in 6m-urea solutions with carboxypeptidase A, 1 mole of phenylalanine was liberated/43000g. of protein. At a lower urea concentration and in the absence of urea, carboxymethyl-cysteine (CMCys) was also liberated. 2. Three cysteine-containing peptides were identified by the study of peptide ;maps' of tryptic digests of actin treated with thiol reagents. 3. The three peptides, each containing one residue of CMCys, were isolated from tryptic digests of carboxymethylated actin by ion-exchange chromatography. 4. One of these peptides was possibly the N-terminal peptide and contained about 17-18 residues; another was CMCys-Asp-Ile-Asp-Ile-Arg; the other, CMCys-Phe, was the C-terminal tryptic peptide. 5. The chemical evidence suggests that the actin molecule consists of a single polypeptide chain of molecular weight about 44000.     

Use of proline-specific endopeptidase in the isolation of all four "native" disulfides of hen egg white lysozyme

The disulfide peptides from the tryptic digestion of cyanogen bromide-treated hen egg white lysozyme (HEWL) were isolated by reverse phase high performance liquid chromatography (HPLC) and identified by amino acid analysis. Three peptides containing the I-VIII, II-VII, and III-V + IV-VI disulfide bonds were obtained. The two-disulfide peptide was further digested with proline-specific endopeptidase (PCE) (EC 3.4.21.26). Amino acid analysis of digest peptides separated by HPLC showed four peptides with the IV-VI disulfide bond as well as a peptide with the III-V disulfide bond. The IV-VI peptides were produced by hydrolysis of several alanine-X bonds as well as the prolyl-cystine bond. Our studies show that alanyl peptide bonds to lysyl, seryl, and leucyl residues are susceptible to hydrolysis by PCE preparations, thus substantially extending its known specificity range. The two-disulfide peptide was also digested sequentially with thermolysin and PCE; the resulting IV-VI and III-V peptides were identified by HPLC and amino acid analysis. PCE showed substantial activity at pH 5.3 as well as at pH 8.3. The lower pH is useful in studies of proteins or peptides where base-catalyzed reactions must be limited.     

Structural studies on a glycoprotein isolated from alveoli of patients with alveolar proteinosis.

A major glycoprotein 36 000 molecular weight) has been isolated from lung lavage of patients with alveolar proteinosis and found to contain five residues of hydroxyproline, fifty residues of glycine, three residues of methionine, 3 mol of sialic acid, 4.4 mol of mannose, 4.0 mol of galactose, 6.0 mol of glucosamine, and 1 mol of fucose. Cyanogen bromide (CNBr) treatment of the glycoprotein resulted, as expected, in four peptides of apparent molecular weights of 18 000, 12 000, 5000 and 1000, respectively. The chemical compositions of the CNBr peptides indicate the presence of hydroxyproline and high amounts of glycine in all but one of the peptides; two of the four CNBr peptides contain carbohydrate. Gel filtration, acrylamide gel electrophoresis and end-group analyses of the native glycoprotein and its CNBr peptides indicate that the peptides are homogeneous. End-group analyses of the CNBr cleavage products assign the 18 000 molecular weight peptide to the NH2-terminal portion and the 1000 molecular weight peptide to the COOH-terminal portion of the native glycoprotein molecule. Pronase digestion of the 36 000 molecular weight glycoprotein, followed by gel filtration and cation exchange chromatography, resulted in two fractions. One fraction was acidic and contained all the carbohydrate, a high content of aspartic acid and no hydroxyproline. The other fraction was basic and contained 8.4% hydroxyproline, 14% proline, 28% glycine and no carbohydrate, suggesting the presence of collagen-like sequence in the peptide chain. Paper electrophoresis of the basic fraction demonstrated two components, the amino acid compositions of which are identical to those of collagen. Partial amino-terminal sequence analysis of one of the CNBr peptides (18 000 molecular weight) indicated the presence of -Fly-Pro-HyP-Gly-sequence in the peptide chain, which confirms our suggestion that collagen-like regions are present in the native glycoprotein molecule. Limited acid hydrolysis of the acidic fraction and subsequent fractionation of the acid hydrolysate using Dowex column yielded a fraction which produced brown colour with ninhydrin reagent. Paper chromatography of this fraction demonstrated a large component which also stained brown with ninhydrin reagent. After acid hydrolysis, this component was found to consist of equal amounts of asparitic acid and glucosamine, indicating that the N-acetylglucosamine of the oligosaccharides is linked to the asparagine residue of the peptide. No serine or threonine linkages are present.     

Prereplicative modulation of nuclear protein kinases in the regenerating rat liver

Treatment of carboxymethylated actin with o-iodosobenzoic acid (Mahoney, W.C., and Hermodson, M.A. (1979) Biochemistry, $\text{18}$, 3810–3814) did not produce the peptide pattern expected on the basis of specific peptide bond cleavage at tryptophanyl bonds. Isolation and amino acid sequence characterization of peptides from the digest indicated that in addition to cleavage at Trp residues, cleavages occurred at Tyr-53, Tyr-198, Tyr-218, Tyr-239 and probably at Tyr-91. These results indicate that the specificity of o-iodosobenzoic acid as a reagent for peptide bond cleavage is wider than previously reported. A simple explanation for the different susceptibilities of tyrosyl-containing peptide bonds to cleavage was not apparent from inspection of the sequences adjacent to these residues.