The primary structure of Lactobacillus casei thymidylate synthetase. I. The isolation of cyanogen bromide peptides 1 through 5 and the complete amino acid sequence of CNBr 1, 2, 3, and 5.

Thymidylate synthetase from Lactobacillus casei was S-carboxymethylated and degraded by treatment with cyanogen bromide. Although the protein contains 6 methionine residues, only 5 cyanogen bromide peptides were obtained due to the presence of 1 methionine on the NH2 terminus and another adjacent to a threonine residue which was resistant to cleavage. The peptides were isolated by differential extraction, first with ammonium acetate, then pyridine acetate, and finally the residue was solubilized with 50% acetic acid. Each peptide was further purified to homogeneity by Bio-Gel chromatography. The size of the peptides from the amino to carboxyl end of the enzyme subunit was CNBr 1, 4,100; CNBr 2, 10,300; CNBr 3, 8,100; CNBr 4, 11,800; CNBr 5, 2,200. The sum of the amino acid residues of the peptides is equal to the sum of the residues in an enzyme subunit, indicating that all of the CNBr peptides have been isolated. The CNBr-resistant methionine was located in CNBr 2 and the 5-fluoro-2'-deoxyuridine 5'-monophosphate binding site in CNBr 4. The holoenzyme molecular weight, based on the residue weights of the amino acids in the two equivalent subunits, is equal to 73,176. The complete sequence of each of the CNBr peptides, except for CNBr 4, which is presented in the following paper, is described.     

Acanthamoeba actin and profilin can be cross-linked between glutamic acid 364 of actin and lysine 115 of profilin

Acanthamoeba profilin was cross-linked to actin via a zero-length isopeptide bond using carbodiimide. The covalently linked 1:1 complex was purified and treated with cyanogen bromide. This cleaves actin into small cyanogen bromide (CNBr) peptides and leaves the profilin intact owing to its lack of methionine. Profilin with one covalently attached actin CNBr peptide was purified by gel filtration followed by gel electrophoresis and electroblotting on polybase-coated glass-fiber membranes. Since the NH2 terminus of profilin is blocked, Edman degradation gave only the sequence of the conjugated actin CNBr fragment beginning with Trp-356. The profilin-actin CNBr peptide conjugate was digested further with trypsin and the cross-linked peptide identified by comparison with the tryptic peptide pattern obtained from carbodiimide-treated profilin. Amino-acid sequence analysis of the cross-linked tryptic peptides produced two residues at each cycle. Their order corresponds to actin starting at Trp-356 and profilin starting at Ala-94. From the absence of the phenylthiohydantoin-amino acid residues in specific cycles, we conclude that actin Glu-364 is linked to Lys-115 in profilin. Experiments with the isoforms of profilin I and profilin II gave identical results. The cross-linked region in profilin is homologous with sequences in the larger actin filament capping proteins fragmin and gelsolin.     

Photoaffinity modification of Escherichia coli ribosomes by fMet-tRNAf Met derivatives in the 70S initiation complex

Photoaffinity labeling of E. coli ribosomes within the 70S initiation complex was studied by using photoreactive derivatives of fMet-tRNAfMet bearing arylazidogroups scattered statistically over guanosine residues. It is shown that fMet-azido-tRNAfMet-II bearing 2 moles of the reagent residues per mole of tRNA (modified in the conditions of stability of tRNA tertiary structure) is fully active in aminoacylation and in the factor-dependent binding with ribosomes to form the 70S initiation complex. Functional activity of fMet-azido-tRNAfMet-I bearing also 2 moles of the reagent residues per mole of tRNA (but modified in conditions of lability of tRNA tertiary structure) decreases up to approximately 45% in aminoacylation and up to 70% in IF-2 X GTP-dependent binding to the ribosomes. Irradiation of complexes 70S ribosome-MS2-RNA-fMet-azido-tRNAfMet results in covalent linking of the tRNA derivative to the ribosomes. Both subunits are labeled, the 30S to a larger extent than 50S. It is shown that fMet-azido-tRNAfMet-II labels proteins S1, S7, S9, L27 whereas fMet-azido-tRNAfMet-1--proteins S1, S3, S5, S9, S14, L1, L2, L7/L12.     

The adenine pocket of a single catalytic site is derivatized when the bovine heart mitochondrial F1-ATPase is photoinactivated with 4-amino-1-octylquinaldinium

The bovine heart mitochondrial F₁-ATPase (MF₁) is reversibly inhibited in the dark by 4-amino-1-octylquinaldinium (AOQ) with an I_0.5 value of 48 μM. When irradiated in the presence of AOQ, MF₁ is photoinactivated with an apparent K_d of 12 μM. About 1.1 mol of [³H]AOQ were incorporated per mol of MF₁ on complete photoinactivation. Fractionation of a cyanogen bromide digest of MF₁ photolabeled with [³H]AOQ followed by fractionation of peptic digests of partially purified cyanogen bromide fragments led to isolation of two CNBr/peptic fragments labeled with³H. Sequence analysis of the labeled peptides revealed that one contained residues 423–441 of the β subunit. A gap in position 2 of the sequence indicates that βPhe424 is derivatized. The phenyl side-chain of this residue is part of a pocket that binds the adenine moiety of ATP or ADP at catalytic sites. The other peptide, which was labeled to a greater extent, contained residues 342–358 of the β subunit, but in this case, no gap was found in the sequence indicating that the derivatized amino-acid side-chain might not have survived the conditions of automatic Edman degradation. This peptide contains βTyr345, the side-chain of which is also a component of the pocket that binds the adenine moiety of ATP or ADP to catalytic sites. However, for the reason stated, there is no direct evidence that βTyr345 is labeled in this peptide.     

Thiosulfate Reductase of Desulfovibrio vulgaris

The thiosulfate reductase of Desulfovibrio vulgaris has been purified and some of its properties have been determined. Only one protein component was detected when the purified enzyme was subjected to polyacrylamide gel electrophoresis at pH values of 8.9, 8.0, and 7.6. In the presence of H(2), the enzyme, when coupled to hydrogenase and with methyl viologen as an electron carrier, catalyzed the reduction of thiosulfate to hydogen sulfide. The use of specifically labeled (35)S-thiosulfate revealed that the outer sulfur atom was reduced to sulfide and the inner sulfur atom was released as sulfite. Thus, the enzyme catalyzes the reductive dismutation of thiosulfate to sulfide and sulfite. The molecular weight of the enzyme was determined by sedimentation equilibrium (16,300) and amino acid analysis (15,500). The enzyme sedimented as a single, symmetrical component with a calculated sedimentation coefficient of 2.21S. Amino acid analysis revealed the presence of two half-cystine residues per mole of enzyme and a total of 128 amino acid residues. Carbohydrate and organic phosphorus analyses revealed the presence of 9.2 moles of carbohydrate and 4.8 moles of phosphate per mole of enzyme. The substrate specificity of the enzyme was studied.     

Human complement component C1s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation

Human C1s proenzyme (Mr 83 000) was isolated by a rapid two-stage method involving affinity chromatography of C1 on IgG-Sepharose and isolation of subcomponent C1s by ion-exchange chromatography on DEAE-Sephacel. Single-chain C1s proenzyme was activated to two-chain C1s with self-activated C1r. After reduction and S-carboxamidomethylation the heavy chain of C1s (Mr 57 000) was isolated by ion exchange chromatography on DEAE-Sephacel. Cleavage of C1s heavy chain with CNBr yielded five fragments whose N-terminal sequences were determined. The alignment of the fragments within the heavy chain was established by tryptic peptides containing methionine. C1s heavy chain comprises about 470 amino acid residues and 42% of its sequence was determined. An intrachain sequence homology and a homology to the alpha 2 chain of human haptoglobin were identified. The C-terminal CNBr fragment comprising 44 amino acid residues was completely sequenced. From BNPS-skatole cleavage of reduced and alkylated C1s proenzyme a fragment was isolated which overlaps the C1s heavy and light chain parts and which contains the peptide bond cleaved during activation. The results show that this is an Arg-Ile bond and that under standard conditions of activation no peptide material is liberated from this portion of the molecule. The sequence data and homology to two-chain serine proteases indicate a single interchain disulfide bond in C1s.     

Identification of an essential arginine residue in the beta subunit of the chloroplast ATPase

The ATPase activity of soluble chloroplast coupling factor (CF1) was irreversibly inactivated by phenylglyoxal, an arginine reagent. Under the conditions of inactivation, 2.48 mol of [14C]phenylglyoxal were incorporated per 400,000 g of enzyme when the ATPase was inactivated 50% by the reagent. Isolation of the component polypeptide subunits of the [14C]phenylglyoxal-modified enzyme revealed that the distribution of moles of labeled reagent/mol of subunit was the following: alpha, 0.37; beta, 0.40; gamma, 0.08; delta, none; epsilon, 0.03. CNBr treatment of the isolated alpha and beta subunits and fractionation of the peptides by gel electrophoresis revealed that the radioactivity bound to the alpha subunit was nonspecifically associated with several peptides, while a single peptide derived from the beta subunit contained the majority of the radioactivity associated with this subunit. After treating the isolated beta subunit with trypsin and Staphylococcus aureus protease, a major radioactive peptide was isolated with a sequence Arg-Ile-Thr-Ser-Ile-Lys. This sequence, when compared with the primary structure of the CF1 beta subunit as translated from the gene (Zurawski, G., Bottomley, W., and Whitfeld, P. R. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6260-6264) indicated that the arginine marked with the asterisk, the predominant residue modified by phenylglyoxal when the ATPase activity of CF1 is inactivated by the reagent, is Arg 312.     

Amino acid sequence of a cyanogen bromide fragment containing the two tryptophanyl residues of lobster arginine kinase (Homarus vulgaris).

Lobster arginine kinase [EC 2.7.3.3] contains 2 tryptophanyl residues and 9 methionyl residues. The whole carboxymethylated protein was first subjected to CNBr cleavage and the resulting fragments were isolated by gel filtration and other experimental approaches. One fragment, CB5, which contains 60 residues including the two tryptophanyl residues and two of the five cysteinyl residues of the protein, was characterized and the results are reported inthis paper. The overall strategy for the establishment of the complete sequence of this fragment was based on the use of three types of peptides: (a) whole cyanogen bromide peptide CB5 which was partially characterized by automatic Edman degradation using a sequencer: 42 steps were performed out of 60 residues, (b) tryptic peptides of CB5, (c) peptides formed by cleavage of S-carboxymethylated arginine kinase (whole protein) at the two tryptophanyl residues with BNPS-skatole. The complete amino acid sequence of the CNBr polypeptide (CB5) which contains the two tryptophanyl residues of the whole protein was established.     

Quantitative evaluation of congo red binding to amyloid-like proteins with a beta-pleated sheet conformation

The binding of Congo red to several purified amyloid-like peptides having a beta-pleated sheet conformation was quantitatively examined. Congo red binds preferentially to the beta-pleated sheet conformation of both insulin fibrils and poly-L-lysine. Congo red does not bind nearly so well to poly-L-serine or polyglycine, despite the fact that these peptides also have a beta-pleated sheet conformation. Binding to insulin fibrils was saturable with an apparent Bmax of 2 moles of Congo red per mole of insulin fibrils and an apparent KD of 1.75 x 10(-7) M. Binding to beta-poly-L-lysine was similar but had a much higher apparent Bmax of 43. Binding of Congo red to beta-poly-L-lysine was pH dependent and appeared to be determined by the number of protonated lysine residues in the 250 amino acid peptide. We present a new hypothesis in which Congo red binds to amyloid-like proteins via bonds between the two negatively charged sulfonic acid groups of Congo red and two positively charged amino acid residues of two separate protein molecules which are properly oriented by virtue of the beta-pleated sheet conformation of the peptide backbone.     

Identification and amino acid sequence of the deoxynucleoside triphosphate binding site in Escherichia coli DNA polymerase I

We have labeled the large fragment of Escherichia coli DNA polymerase I (Pol I) with pyridoxal 5'-phosphate, a substrate binding site directed reagent for DNA polymerases [Modak, M. J. (1976) Biochemistry 15, 3620-3626]. A covalent attachment of pyridoxal phosphate to Pol I results in the loss of substrate binding as well as the polymerase activity. The inactivation was found to be strictly dependent on the presence of a divalent metal ion. Four moles of pyridoxal phosphate was found to react per mole of the enzyme, while in the presence of substrate deoxynucleoside triphosphate only 3 mol of pyridoxal phosphate was bound. To identify the substrate-protected site on the enzyme, tryptic peptides from enzyme labeled with pyridoxal phosphate and tritiated borohydride, in the presence and absence of substrate, were resolved on a C-18 reverse-phase column. A single peptide containing the substrate-protected site was identified and further purified. The amino acid composition and sequence analysis of this peptide revealed it to span residues 756-775 in the primary acid sequence of Pol I. Lys-758 of this sequence was found to be the site of the pyridoxal phosphate reaction. It is therefore concluded that Lys-758 is the site of binding for the metal chelate form of nucleotide substrates in E. coli DNA polymerase I.     

Cross-linking of rabbit skeletal muscle troponin subunits: labeling of cysteine-98 of troponin C with 4-maleimidobenzophenone and analysis of products formed in the binary complex with troponin T and the ternary complex with troponins I and T

The sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) was used to study the interaction of rabbit skeletal muscle troponin subunits TnC, TnT, and TnI. TnC was labeled at Cys-98 by the maleimide moiety of BPMal and then mixed with either TnT alone or TnI plus TnT, in the presence of Ca2+. Upon photolysis, TnI and/or TnT formed covalent cross-links with TnC. The cross-linked TnC-TnT heterodimer obtained from the binary complex was digested into progressively smaller cross-linked peptides that were purified by HPLC and then characterized by amino acid analysis and sequencing. An initial cross-linked CNBr fraction contained the expected peptide CB9 (residues 84-135) of TnC, plus CNBr peptides spanning residues 152-230 of TnT. Results from a peptic digest of the CNBr cross-linked fraction permitted the identification of residues 159-197 as the most highly cross-linked region in TnT. A final subtilisin digest yielded a heterogeneous cross-linked fraction, which suggested that an especially high degree of cross-links was formed in the vicinity of residues 175-178 (Met-Lys-Lys-Lys) of TnT. Although this region of TnT had previously been implicated in binding, we show here for the first time that it is close to Cys-98 of TnC. In an analogous study on the binary complex of TnC and TnI [Leszyk, J., Collins, J. H., Leavis, P. C., & Tao, T. (1987) Biochemistry 26, 7042-7047], we previously showed that Cys-98 of TnC was cross-linked mainly to CN4, the "inhibitory region", of TnI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a region of the primary sequence of troponin C involved in calcium ion-dependent interaction with troponin I.

1. The CNBr digest of troponin C from rabbit fast skeletal muscle was shown to possess many of the functional properties of the whole troponin C molecule. 2. A peptide corresponding to residues 83-134 was isolated, which forms a Ca(2+-dependent complex with troponin I and neutralizes the inhibition by troponin I of the Mg(2+-stimulated adenosine triphosphatase of desensitized actomyosin. 3. The peptide inhibits the phosphorylation of fast-skeletal-muscle, but not cardiac-muscle, troponin I, by 3' :5'-cyclic AMP-dependent protein kinase. In this property it was as effective as whole skeletal-muscle troponin C when compared on a molar basis. 4. Biological activity was also present in other fractions obtained from the CNBr digest. 5. By gel filtration and affinity chromatography of the whole CNBr digest of troponin C, two peptides, one of which was identified as representing residues 83-134, were shown to form Ca(2+-dependent complexes with troponin I. 6. The significance of these findings for the mechanism of interaction of troponin C and troponin I is discussed.     

Contribution to Catalysis and to Stability of the Essential Cysteine Residue at the Active Site of d-Glucosaminate Dehydratase from Pseudomonas fluorescens

Chemical modification of purified d-glucosaminate dehydratase (GADH) apoenzyme by N-ethyl-maleimide (NEM) and by 7-chloro-4-aminobenzo-2-oxa-1,3-diazole (NBDC1) resulted in the time- and concentration-dependent inactivation of the enzyme in each case. The inactivation followed pseudo-first-order kinetics and a double-logarithmic plot of the observed pseudo-first-order rate constant against reagent concentration proved evidence for an approximately first-order reaction, suggesting that the modification of a single cysteine residue per mole of enzyme resulted in inactivation. Amino acid analysis of the NEM-inactivated enzyme showed that three moles of cysteine residues among six moles per mole of subunit were modified under these conditions, therefore one of the three cysteine residues modified by NEM may be essential for activity. Pyridoxal 5′-phosphate (PLP) and D-glucosaminate (GlcNA) protected the enzyme against inactivation by NEM and NBDCI. The apoenzyme was inactivated by EDTA and activity of enzyme was restored by incubation with Mn²⁺ in the presence of PLP. Incubation of the EDTA-treated enzyme with NEM inhibited the restoration of activity. These results suggest that one of the cysteine residues of GADH may be chelated to a Mn²⁺ at or near the active site of GADH, contributing to formation of the active enzyme.     

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

A soluble glycoprotein of Mr = 80,000 has been isolated from lung lavage of patients with alveolar proteinosis and found to contain 5 residues of hydroxyproline, 91 residues of glycine, 3 residues of methionine, 3.8 molecules of sialic acid, 6 molecules of mannose, 5.9 molecules of galactose, 1 molecule of fucose, and 9.1 molecules of glucosamine. Cyanogen bromide (CNBr) treatment of the glycoprotein resulted in four peptides with molecular weights of 36,000, 27,000, 12,000, and 5,000. The chemical compositions of the CNBr peptides indicated the presence of hydroxyproline and high amounts of glycine in all but one of the peptides; two of the four CNBr peptides contained carbohydrate. Limited trypsin digestion of the glycoprotein of Mr = 80,000 resulted in four peptides with molecular weights of 62,000, 36,000, 26,000 and 18,000, the latter being the NH2-terminal peptide of the native glycoprotein molecule. The peptide of Mr = 26,000 was found to be the COOH-terminal peptide.     

The structure of a glycopeptide (GP-II) isolated from Rhizopus saccharogenic amylase.

Mild alkaline treatment of glycopeptide (GP-II) resulted in the loss of 1 mole of serine and 5 moles of threonine per mole of GP-II, suggesting the presence of O-glycosyl bonds between 1 serine and 5 threonine residues and carbohydrate chains. Treatment of GP-II with alkaline borohydride released only disaccharide. Methylation studies of the carbohydrate moiety gave 2,3,4,6-tetra-O-methyl and 2,4,6-tri-O-methyl derivatives of mannose in a ratio of approximately 1:1. In addition, one step of Smith degradation resulted in the loss of about 6 residues of mannose per mole of GP-II. Moreover, alpha-mannosidase [EC 3.2.1.24] liberated about 6 residles of mannose per mole of GP-II. On the basis of these data, the structure of the carbohydrate moiety of GP-II was confirmed to be 3-O-alpha-mannosylmannose. The amino- and carboxyl-terminal amino acids of GP-II were determined to be threonine and serine, respectively. On reductive cleavage of N-proline bonds with metallic sodium in liquid ammonia, 2 moles of alanine per mole of GP-II were lost. From the compositions of three fragments isolated from the reductive cleavage products, the amino acid sequence of the peptide portion of GP-II was determined. Based on these data, a probable structure was proposed for GP-II.     

Amino acid sequences of portions of the alpha and beta chains of bovine fibrinogen.

The N-terminal portions of the Aα and Bβ chains of bovine fibrinogen (CNBr Aα and Bβ), each of which contains an ArgGly bond that is hydrolyzed by thrombin, have been isolated by cyanogen bromide cleavage of fibrinogen and column chromatography of the resulting material. These peptides were digested with thrombin, releasing fibrinopeptide A and GlyProArg from CNBr Aα, and fibrinopeptide B from CNBr Bβ. The C-terminal peptides produced by digestion with thrombin (CNBr α and CNBr β) were purified, and the amino acid sequences of portions of these peptides (30 residues from the N-terminus of CNBr α and 32 residues from the N-terminus of CNBr β) were determined with an automatic sequenator using the Edman degradation.     

Bovine Peripheral Nervous System Myelin P2 Protein: Chemical and Immunological Characterization of the Cyanogen Bromide Peptides

Cleavage of bovine P2 protein by cyanogen bromide (CNBr) produced peptide fractions CN1, CN2, and CN3 which were isolated by gel filtration chromatography. CN2 was found to contain two NH2-terminals (lysine and valine) and accounted for both of the cysteine residues of P2. When reduced carboxymethylated P2 (RCM-P2) was digested with CNBr, peptides CN1 and CN3 were obtained as were (1) a peptide with NH2-terminal lysine (Lys) that contained no homoserine and only one cysteine residue and (2) a peptide with NH2-terminal valine (Val) that was co-eluted with CN3. These data and the chemical characterization of all the CNBr peptides obtained from P2 and RCM-P2 suggest that isolated P2 protein has a structure composed of the CNBr peptides in the order CN3-CN1-CN2(Val)-CN2(Lys) with an intrachain disulfide bond between the cysteine residues located in the two constituent peptides of CN2, CN2(Lys) and CN2(Val). To locate the neuritogenic region(s) within the P2 protein structure, CN1, CN2, and CN3 were tested for the ability to induced experimental allergic neuritis (EAN) in Lewis rats. The disease-inducing sites of P2 protein were found only in CN1; neither CN2 nor CN3 produced disease. EAN induced by CN1 was comparable to that induced with P2 protein as determined by disease onset, clinical symptoms, and histologic lesions.     

Identification and characterization of an NADPH-cytochrome P450 reductase derived peptide involved in binding to cytochrome P450

The amino acids of cytochrome P450 reductase involved in the interaction with cytochrome P450 were identified with a differential labeling technique. The water-soluble carbodiimide EDC (1-ethyl-3-[3- (dimethylamino)propyl]-carbodiimide) was used with the nucleophile methylamine to modify carboxyl residues. When the modification was performed in the presence of cytochrome P450, there was no inhibition in the ability of the modified reductase to bind to cytochrome P450. However, subsequent modification of the reductase in the absence of cytochrome P450 caused a fourfold increase in the Km and an 80% decrease in kcat/Km (relative to the reductase modified in the first step), for the interaction with cytochrome P450. These effects are attributed to the modification of approximately 3.2 mol of carboxyl residues per mole of reductase. Tryptic peptides generated from the modified reductase were purified by reverse phase high-performance liquid chromatography and characterized. Amino acid sequencing and analysis suggest that the peptide which contains approximately 40% of the labeled carboxyl residues corresponds to amino acid residues 109-130 of rat liver NADPH-cytochrome P450 reductase. One or more of the seven carboxyl containing amino acids within this peptide is presumably involved in the interaction with cytochrome P450.     

Complete amino acid sequence and location of the five disulfide bridges in porcine pancreatic alpha-amylase

Porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase EC 3.2.1.1), a single polypeptide chain, contains nine residues of methionine. Eight different fragments resulting from cleavage of this molecule by cyanogen bromide were characterized. The sequences of six of them have previously been reported. Two missing fragments, CN2 (82 residues) and CN3b1 (76 residues) were purified after breaking of the interpeptidic disulfide bridge and their complete sequence as well as that of the previously purified CN1 peptide (102 residues) are reported here. The location of the three disulfide bridges present in these peptides was determined. Ordering of the carboxymethylated cyanogen bromide fragments was carried out by pulse labeling the amylase chain in vivo. The complete sequence of the porcine pancreatic amylase chain (496 residues) and the location of its five disulfide bridges is presented. Comparison with human and mouse pancreatic and salivary alpha-amylases and with rat pancreatic amylase obtained from the corresponding cDNA nucleotidic sequences shows a high degree of homology between mammalian alpha-amylases.     

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.