Covalent structure of collagen: amino acid sequence of cyanogen bromide peptides from the amino-terminal segment of type III collagen of human liver

Human liver type III collagen was prepared by limited pepsin digestion, differential salt precipitation, and carboxymethylcellulose chromatography. Cyanogen bromide digestion of purified type III collagen chains yielded nine distinct peptides. Three peptides, alpha1(III)-CB3, alpha1(III)-CB7, and alpha1(III)-CB6, were isolated by carboxymethylcellulose chromatography and Sephadex G-50 SF gel filtration. Automated Edman degradation together with selective hydroxylamine cleavage and chymotrypsin and trypsin digestion enabled determination of their complete amino acid sequence. Compared with type I collagen, the data show tentative homology of alpha1(III)-CB3 with alpha1(I)-CB1, alpha1(I)-CB2, and alpha1(I)-CB4; alpha1(III)-CB7 with alpha1(I)-CB5; and alpha1(III)-CB6 with the amino-terminal portion of alpha1(I)-CB8. Close interspecies homology was found between the sequences presented here with 90 residues of alpha1(III)-CB3 and 26 of alpha1(III)-CB8 of calf aorta. The present study establishes the amino acid sequence of 229 residues near the amino terminus or nearly one-quarter of the type III collagen chains. The disaccharide, Glc-Gal, was convalently bound to hydroxylysine at a position corresponding to the same location in the alpha1(I) chain.     

Covalent structure of collagen: amino acid sequence of alpha 1 (III)-CB5 from type III collagen of human liver

Type III collagen was prepared from human liver by limited pepsin digestion, differential salt precipitation, and carboxymethylcellulose chromatography. Ten distinct peptides were obtained by cyanogen bromide digestion. The peptide alpha 1 (III)-CB5 was further purified by carboxymethylcellulose chromatography, and its amino acid sequence was determined. Automatic Edman degradation of intact alpha 1 (III)-CB5, tryptic and thermolytic peptides, and hydroxylamine-derived fragments was used to establish the total sequence. The mammalian collagenase site contained in the alpha 1 (III)-CB5 sequence was ascertained by digestion of native type III collagen with purified rheumatoid synovial collagenase. Collagenase cleavage occurred at a single Gly--Ile bond, one triplet before the corresponding specific cleavage site of type I collagen. The present work brings the known sequence of human liver type III collagen to include alpha 1 (III)-CB3-7-6-1-8-10-2-4-5. These correspond to the homologous region of alpha 1 (I)-CB0-1-2-4-5-8-3-7 residues 11--804.     

Covalent structure of collagen: amino acid sequence of five consecutive CNBr peptides from type III collagen of human liver

Type III collagen was solubilized from human liver by limited pepsin digestion and purified by differential salt precipitation and carboxymethylcellulose chromatography. Digestion with cyanogen bromide yielded the nine distinct peptides previously described and an additional tripeptide not recognized in earlier studies. Five of these peptides, alpha1 (III)-CB1, 2, 4, 8, and 10, were further purified by molecular sieve and/or ion exchange chromatography. They contained 12, 40, 149, 125 and 3 amino acid residues, respectively. The amino acid sequence of these peptides was determined by automated Edman degradation of tryptic (before and after maleylation), chymotryptic, thermolytic or hydroxylamine-derived peptide fragments as well as the intact peptides. The alignment of these five peptides within the collagen chain is deduced to be 1-8-10-2-4 by homology with known alpha1 (I) sequences. The known CNBr peptide alignment of the NH2-terminal portion of type III collagen so far would, therefore, be alpha1 (III)-CB3-7-6-1-8-10-2-4 and correspond to the homologous region of alpha1 (I)-CB0-1-2-4-5-8-3 or residues 11-567 of the alpha1 (III) collagen chain.     

The alpha 2 beta 1 integrin cell surface collagen receptor binds to the alpha 1 (I)-CB3 peptide of collagen

We have previously shown that platelets adhere to collagen substrates via a Mg2(+)-dependent mechanism mediated by the surface glycoprotein Ia-IIa (human leukocyte very late activation protein 2, alpha 2 beta 1 integrin) complex. The adhesion is specific for collagen and is supported by collagen types I, II, III, IV, and VI. Several other members of the integrin family of adhesive protein receptors recognize discrete linear amino acid sequences within their adhesive glycoprotein ligands. Experiments with both intact platelets and with liposomes containing the purified receptor complex indicated that the alpha 2 beta 1 receptor recognized denatured type I collagen in a Mg2(+)-dependent manner. To further localize the binding site, the alpha 1 and alpha 2 chains of type I collagen were purified by gel filtration and ion exchange chromatography and tested as adhesive substrates. Both the alpha 1(I) and alpha 2(I) chains effectively supported Mg2(+)-dependent platelet adhesion. The purified alpha 1(I) collagen chain was then subjected to cleavage with cyanogen bromide, and the resultant peptides were separated by chromatography on carboxymethylcellulose. Only the alpha 1(I)-CB3 fragment supported Mg2(+)-dependent platelet adhesion. The monoclonal antibody P1H5 which recognizes an epitope on the alpha 2 subunit of the integrin receptor and which inhibits the adhesion of both intact platelets and liposomes bearing the purified receptor to collagen also inhibited platelet adhesion to the alpha 1(I)-CB3 fragment. These results indicate that the alpha 2 beta 1 receptor recognizes a sequence of amino acids present in the alpha 1(I)-CB3 fragment of type I collagen. An identical or similar sequence likely mediates binding of the receptor to other collagen polypeptides.     

Partial covalent structure of the human alpha 2 type V collagen chain

Human cDNA libraries were screened with a cDNA fragment presumably encoding the 3' terminus of a procollagen carboxyl propeptide not identifiable as types I, II, III, or IV by protein sequence or Northern blot hybridization. One clone contained a 1350-base pair insert coding in part for 55 uninterrupted Gly-X-Y triplets. Comparison with the amino acid composition of the COOH-terminal cyanogen bromide (CB) peptides of the alpha 1 and alpha 2 type V collagen chains showed similarity only to the alpha 2(V)CB fragment. To identify the NH2 terminus of the peptide designated by methionine, an additional isolate was sequenced and found to contain a Gly-Met-Pro triplet. Thirty-one amino acids from the NH2 terminus of the alpha 2(V)CB9 fragment were then determined by Edman degradation and found to be identical to those derived from the cDNA clone. The DNA sequence encoding part of the triple helical region establishes for the first time the partial structure of a type V collagen chain. Although comparison of residues 796-1020 of the alpha 2(V) collagenous region with alpha 1 (III), alpha 1(I), and alpha 2(I) shows strong conservation of charged positions, the latter three chains appear considerably more similar to each other than to alpha 2(V). A striking feature of the alpha 2(V) sequence between 918-944 is the absence of proline residues. In the analogous region of alpha 1(I) where this amino acid is also lacking, a flexible site in the rigid triple helical structure of type I collagen has been observed (Hofmann, H., Voss, T., Kuhn, K. and Engel, J. (1984) J. Mol. Biol. 172, 325-343).     

Participation of the alpha 2(I) chain of bovine skin collagen in the formation of mature crosslinks

It is shown that regions of unreduced, insoluble cow hide collagen, represented by the peptides alpha 1(I)-CB6, alpha 2(I)-CB4 and the alpha 2(I)-CB3,5, are involved in the formation of unreducible acid-stable and mature-type crosslinks. The characteristic ratio of the CNBr peptides in soluble type I collagen was found to be changed in the insoluble collagen of cow hides. The intensity of the bands of alpha 1(I)-CB6, alpha 2(I)-CB4 and alpha 2(I)-CB3,5, shown by dodecyl sulfate polyacrylamide gel electrophoresis, is significantly reduced in such samples, which indicates an involvement of these peptides in crosslink formation. The purified highly polymeric CNBr peptide fraction was also investigated to confirm the participation of the alpha 2 chain of type I collagen in mature crosslink formation. Chymotryptic digests of such material contain peptides which originate from alpha 2(I)-CB4, alpha 2(I)-CB3,5, and alpha 1(I)-CB6. Finally, acid hydrolysates of crosslinked material were screened carefully for crosslinks down to concentrations of 1 in 1000 amino acids. Only two compounds were detected, one identified as "hydroxyaldol-histidine" and the other an as yet unknown compound. These results indicate that both the alpha 1(I) and the alpha 2(I) chains are involved in mature crosslink formation and that the polymeric CNBr peptide fraction contains components crosslinked by so far uncharacterized, nonreducible crosslinks.     

Identification of a tetrapeptide recognition sequence for the alpha 2 beta 1 integrin in collagen

The alpha 2 beta 1 integrin serves as either a specific cell surface receptor for collagen or as both a collagen and laminin receptor depending upon the cell type. Recently we established that the alpha 2 beta 1 integrin binds to a site within the alpha 1 (I)-CB3 fragment of type I collagen (Staatz, W. D., Walsh, J. J., Pexton, T., and Santoro, S. A. (1990) J. Biol. Chem. 265, 4778-4781). To define the alpha 2 beta 1 recognition sequence further we have prepared an overlapping set of synthetic peptides which completely spans the 148-amino acid alpha 1(I)-CB3 fragment and tested the peptides for ability to inhibit cell adhesion to collagen and laminin substrates. The minimal active recognition sequence defined by these experiments is a tetrapeptide of the sequence Asp-Gly-Glu-Ala (DGEA) corresponding to residues 435-438 of the type I collagen sequence. The DGEA-containing peptides effectively inhibited alpha 2 beta 1-mediated Mg2(+)-dependent adhesion of platelets, which use the alpha 2 beta 1 integrin as a collagen-specific receptor, to collagen but had no effect on alpha 5 beta 1-mediated platelet adhesion to fibronectin or alpha 6 beta 1-mediated platelet adhesion to laminin. In contrast, with T47D breast adenocarcinoma cells, which use alpha 2 beta 1 as a collagen/lamin receptor, adhesion to both collagen and laminin was inhibited by DGEA-containing peptides. Deletion of the alanine residue or substitution of alanine for either the glutamic or aspartic acid residues in DGEA-containing peptides resulted in marked loss of inhibitory activity. These results indicate that the amino acid sequence DGEA serves as a recognition site for the alpha 2 beta 1 integrin complex on platelets and other cells.     

Pinpointing the sites of hydroxylysine glycosides in peptide alpha 1-CB7 of bovine corneal collagen, and their possible role in determining fibril diameter and thus transparency

Two cyanogen bromide fragments (alpha 1-CB7 and alpha 1-CB8) of bovine corneal stromal collagen have been isolated and characterized. These added to those characterized in our previous work account for 95% of the amino acid sequence of the alpha 1(1)-chain. The hydroxylysine glycoside content of each fragment was determined and in this way the general distribution of glycoside over the entire molecule was deduced accounting for all the galactosylhydroxylysine and most of the glucosylgalactosylhydroxylysine of this heavily glycosylated type I collagen. The characterization of fragments alpha 1-CB7 and alpha 1-CB8 has enabled us to resolve the controversy over the relative mobilities of these fragments on SDS gels. Fragment alpha 1-CB7 of bovine corneal collagen was digested by trypsin and by staphylococcal proteinase V8. The resultant peptides were isolated by gel and ion-exchange chromatography and identified in relation to the known amino acid sequence of type I collagen. The hydroxylysine glycosides were determined in the relevant peptides providing a complete account of their distribution along this part of the collagen molecule. Most of the glycoside was found in the gap region of collagen especially near the edges of the axial holes where it could act as a peg to facilitate fibre formation. In addition, some glycoside was found in the overlap region where, being unable to fit into axial holes, it might impede the growth of the fibre and, with other glycoside of the overlap region, might be responsible for the narrow fibres of corneal collagen that are essential for corneal transparency. This glycoside, with that previously found in the peptide alpha 1-CB3 is the only hydroxylysine glycoside identified in the overlap region of a type I collagen.     

Covalent structure of collagen: amino acid sequence of three cyanogen bromide-derived peptides from human alpha 1(V) collagen chain

Type V collagen was prepared from human amnionic/chorionic membranes and separated into alpha 1(V) and alpha 2(V) polypeptide chains. The alpha 1(V) chain was digested with cyanogen bromide and nine peptides were obtained and purified. Three of the peptides, alpha 1(V)CB1, CB4, and CB7 having molecular weights of 5000, 8000, and 6000, respectively, were further analyzed by amino acid sequence analysis and thermolytic or tryptic digestions. CB1 contained 54 amino acids and identification of its complete sequence was aided by thermolysin digestion and isolation of two peptides, Th1 and Th2. CB4 contained 81 amino acids and sequence analysis of intact CB4 and five tryptic peptides provided us with its complete amino acid sequence. The peptide CB7 contained 67 amino acids and was cleaved into four tryptic peptides that were used for complete sequence analysis. The above results represent the first available covalent structure information on the alpha 1(V) collagen chain. These data enabled us to establish the location of these peptides within the helical structure of other collagen chains. CB4 was homologous to residues 66-145 in the collagen chain while CB1 represented residues 146-200 and CB7 was homologous with residues 201-269. This alignment was facilitated by identification of a helical collagen crossing site consisting of Hyl-Gly-His-Arg located at positions 87-90 in all collagen chains of this size thus far identified. Seventy-one percent homology (excluding Gly residues) was found between amino acids in this region of the alpha 1(XI) and of alpha 1(V) collagen chains while only 21 and 19% identity was calculated for the same region of alpha 2(V) and alpha 1(I) collagen chains, respectively.     

Failure of highly purified lysyl hydroxylase to hydroxylate lysyl residues in the non-helical regions of collagen.

The activity of highly purified lysyl hydroxylase towards lysyl residues within both the helical and the N-terminal non-helical telopeptide regions of chick type I collagen has been examined. The peptides alpha 1(I)-CB1 and alpha 2(I)-CB1, isolated from protocollagen following CNBr digestion and containing the N-terminal telopeptidyl lysyl residues, failed themselves to act as substrates. With protocollagen as substrate, analysis of products obtained following bacterial collagenase digestion of the reaction mixture showed that overall 37% hydroxylation of lysyl residues within the helical region of collagen had been obtained, which may be maximal. No hydroxylation, however, of the single lysyl residue in either alpha 1(I)-CB1 or alpha 2(I)-CB1, isolated following CNBr digestion of the reaction mixture, was observed, despite the known susceptibility of these residues to hydroxylation. These findings provide strong circumstantial evidence for the suggestion that a lysyl hydroxylase specific for the telopeptidyl residues and distinct from that active towards lysyl residues in the helical portion of the molecule may exist [Barnes, Constable, Morton & Royce (1974) Biochem. J. 139, 461-468].     

PZ-peptidase from chick embryos. Purification, properties, and action on collagen peptides.

PZ-peptidase is an endopeptidase that cleaves the synthetic substrate developed for clostridial collagenase, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (PZ-peptide). The peptidase has been purified to homogeneity from chicken embryos. The enzyme has a pH optimum of 7.5 to 8.5, and isoelectric point of 5.0, and a molecular weight of 77,000. The kinetic parameters at pH 8 and 37 degrees are: Km = 2 X 10(-4) M and Vmax = 4.2 mumol/min/mg of protein. The enzyme is inhibited by p-hydroxymercuribenzoate (100%), N-ethylmaleimide (60%), and chelating agents (40 to 60%). Maximum activity is attained in the presence of reducing agents and Ca2+, Sr2+, or Mg2+. The peptidase has no detectable action on casein, serum albumin, collagen, collagen alpha chains, various collagen peptides (alpha1)(I)-CB2, alpha1(I)-CB3, alpha1(I)-CB4), (Gly-Pro-Pro)10, or (Gly-Pro-Pro)5. It does catalyze the hydrolysis of the Hyp--Gly bond in the 17-residue collagen peptide alpha1(II)-CB6-C2 and it partially digested a mixture of collagen peptides of molecular weight 350 to 2500. A role of this peptidase in collagen breakdown appears to be restricted to a late stage when degradation products would fall in the range of 5 to 30 residues.     

Chymotryptic and tryptic peptides of fragment alpha 1-CB3 from bovine corneal collagen. Pinpointing the sites of hexose attachment.

Tryptic peptides of citraconylated fragment alpha1-CB3 and chymotryptic peptides of fragment alpha1-CB3 of bovine corneal collagen were prepared, isolated and characterized. Their amino acid compositions were consistent with the amino acid sequence of fragment alpha1-CB3 from calf skin collagen. Two glycoside sites were identified in bovine corneal fragment alpha1-CB3, one of them being the first located in the overlap region of collagen. The results are related to the uniformly narrow collagen fibres found in cornea and essential for its transparency.     

Isolation and characterization of CNBr peptides of human (alpha 1 (III) )3 collagen and tissue distribution of (alpha 1 (I) )2 alpha 2 and (alpha 1 (III) )3 collagens.

[Alpha 1(III)]3 collagen was solubilized by pepsin digestion of normal human placental membranes and was purified by differential salt precipitation and carboxymethylcellulose chromatography. This collagen was digested with CNBr, and the resultant nine peptides were isolated and characterized. The chains are cross-linked by cysteinyl residues in the COOH-terminal peptide. Isolation of peptides derived from CNBr digestion of insoluble tissues was used as an assay for the presence of [alpha 1(I)]2alpha 2 and [alpha 1(III)]3 collagens. Both types are present in human skin, intestine, liver, spleen, kidney, lung, aorta, umbilical cord, placental membranes, and myocardium. Bone and tendon contain [alpha 1(I)]2alpha 2 collagen but, unlike the other tissues, lack [alpha 1(III)]3 collagen. Both [alpha 1(I)]2alpha 2 and[alpha 1(III)]3 collagens are present in scars of human skin, myocardium, tendon, and liver and of rabbit skin. The degree of hydroxylation of proline was 4 to 5% lower in the same peptides in skin, bone, and tendon than in the other tissues. The degree of hydroxylation of lysine in the same peptides derived from different tissues varied more widely.     

The human immune response to reconstituted bovine collagen

The human immune response to bovine dermal collagen was characterized through histologic, serologic, and immunoblotting methods. Collagen-sensitive patients were identified by hypersensitivity to intradermal exposure to ZYDERM Collagen Implant--a pepsin-solubilized, reconstituted, bovine dermal collagen. Biopsies of test sites in the forearm were obtained from several collagen-sensitive patients. Histologic examination revealed an implant-associated palisading foreign body granuloma. The lesion also contained a mixed cell infiltrate of histiocytes, lymphocytes, and eosinophils. Sera were collected from patients who developed erythema or induration at intradermal test or treatment sites, and were evaluated for antibodies to bovine dermal collagen by an enzyme-linked immunosorbent assay (ELISA). Sera with anti-collagen antibodies were further characterized in this study. The circulating antibodies were reactive with both native and heat-denatured bovine dermal collagen. By using purified alpha 1(I) and alpha 2(I) polypeptides, these sera were found to have antibodies reactive with both alpha-chains. Each alpha-chain was fragmented by using cyanogen bromide (CB). The CB peptides were electrophoretically separated, and these sera were evaluated for antibodies to the major fragments by using an immunoblotting technique. Of the sera evaluated by this method, 89% (23/26) had antibodies to alpha 1-CB6; 77% (20/26) had antibodies to alpha 2-CB4; and 65% (17/26) had antibodies reactive with both CB fragments. In addition, most sera (77%) contained antibodies reactive with two or more (up to five) of the major CB peptides. The least antigenic fragment was alpha 2-CB3,5 (8%). In addition, these sera had antibody activity against both native and heat-denaturated bovine types III and II collagens. Little or no interspecies (rat or guinea pig) cross-reactivity (types I and II) was detected. Furthermore, these sera did not have antibodies against human types I, II, and III collagens.     

The isolation and characterization of the cyanogen bromide peptides from the B chain of human collagen.

Cleavage of the collagen B chain with cyanogen bromide yields nine peptides which have been isolated and characterized with regard to molecular weight and amino acid composition. The peptides are recovered in equimolar quantities and account for the full amino acid complement of the chain as isolated following limited pepsin digestion of human placental tissue. These data thus confirm the unique composition of the chain and further indicate that the chain has been isolated in essentially pure form. The total number of amino acid residues (1018) observed in the cyanogen bromide peptides of the B chain indicate that it is comparable in length to the previously characterized collagen alpha chains. Thus, the apparent larger size of the B chain noted in previous studies may possibly be attributed to the relatively large quantities of hydroxylysine-linked carbohydrate, but more likely to the increased numbers of large hydrophobic amino acids in the B chain. Although the cyanogen bromide peptide pattern obtained in studies on the B chain serves to differentiate this chain from other known chains, some possible homologies between the B chain peptides and peptides derived from the alpha chains of type I, II, and III collagens are noted.     

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.     

Intermolecular cross-linking and stereospecific molecular packing in type I collagen fibrils of the periodontal ligament

A trypsin digest of denatured NaB3H4-reduced native bovine periodontal ligament was prepared and fractionated by gel filtration and cellulose ion-exchange column chromatography. Prior to trypsin digestion, a complete acid hydrolysate was subjected to analyses for nonreducible stable and reducible intermolecular cross-links. Minute amounts of the former and significant amounts of the reduced cross-links dihydroxylysinonorleucine (1.1 mol/mol of collagen), hydroxylysinonorleucine (0.9 mol/mol of collagen), and histidinohydroxymerodesmosine (0.6 mol/mol of collagen) were found. The covalent intermolecular cross-linked two-chained peptides that were isolated were subjected to amino acid and sequence analyses. The structures for the different two-chained linked peptides were alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6-(993-22c)[Lysald-16c], alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c], alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Lysald-16c], and alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c]. The cross-link in each peptide was glycosylated. This is the first characterization by sequence analysis of a cross-link involving Hyl-87 in an alpha 2 chain in collagen. A stoichiometric conversion of residue 16c aldehyde to an intermolecular cross-link in each of the COOH-terminal nonhelical peptide regions of both alpha 1 chains in a molecule of type I collagen was found. The ratio of alpha 1 to alpha 2 intermolecularly cross-linked chains involved was 3.3:1, indicating a stereospecific three-dimensional molecular packing of type I collagen molecules in bovine periodontal ligament.     

The covalent structure of collagen. The chymotrypsin, trypsin and hydroxylamine peptides derived from alpha2-CB4 of calf-skin collagen.

The cyanogen-bromide-derived peptide alpha2-CB4 from calf skin collagen, consisting of 321 amino acid residues, has been fragmented in order to obtain peptides suitable for automated sequential analysis. Digestion with chymotrypsin liberated six unique peptides consisting of 12, 17, 19, 54, 63 and 156 amino acid residues. Treatment of alpha2-CB4 with hydroxylamine yielded four peptides with 24, 87, 96 and 114 residues. No unspecific cleavage by hydroxylamine was encountered. All of the trypsin-derived peptides of alpha2-CB4 were isolated and characterized by their amino acid compositions. Most of the peptides isolated were ordered along the peptide chain of alpha2-CB4. Ordering of the peptides was greatly assisted by the isolation of double peptides from the chymotrypsin, trypsin and hydroxylamine-derived peptide mixtures.