CNBr Cleavage Research Articles

Multiple Distinct Membrane Heparan Sulfate Proteoglycans in Human Lung Fibroblasts

Heparitinase digestion of the hydrophobic membrane-associated heparan sulfate proteoglycans (HSPG) of fetal human lung fibroblasts yields core proteins of various sizes: i.e. monomeric core proteins of 125, 90, 64, 48, and 35 kDa and a disulfide-linked dimeric core protein composed of approximately 35-kDa subunits. By immunizing BALB/c mice with liposome-incorporated HSPG, we have obtained a total of five anti-HSPG monoclonal antibodies (Mabs, i.e. Mabs S1, 1C7, 2E9, 6G12, and 10H4) with different specificities. Polyacrylamide gel electrophoresis of 125I-labeled membrane HSPG immunoprecipitated with these Mabs revealed that Mabs 1C7 and 2E9 bind only membrane HSPG which yield a 125-kDa core protein after heparitinase digestion, whereas Mab S1-bound HSPG yield a 64-kDa core protein, and Mabs 6G12 and 10H4 retain membrane HSPG with a 48-kDa core protein. Western blotting of the heparitinase-digested proteoglycans and immunostaining with the Mabs confirmed this pattern of reactivity. However, in this assay, Mabs 6G12 and 10H4 also detected a minor approximately 90-kDa core protein in addition to the 48-kDa core protein. Except perhaps for the 10H4 epitope, the epitopes recognized by these Mabs appear to be part of the peptide moieties as they resisted complete deglycosylation of the HSPG with trifluoromethanesulfonic acid. Since these data were inconsistent with a direct relationship between the major core proteins, the 48-, 64-, and 125-kDa core proteins were immunopurified and further compared by peptide mapping with Staphylococcus aureus protease V8, trypsin, and CNBr cleavage. Clearly distinct peptide patterns were obtained for the three different core proteins. These results imply that the 48-, 64-, and the 125-kDa membrane HSPG core proteins of human lung fibroblasts are derived from distinct proteoglycans.

Glycation of calmodulin: chemistry and structural and functional consequences

In the presence of Ca2+ and glucose, calmodulin incorporates 2.5 mol of glucose/mol of protein. In the absence of Ca2+, only 1.5 mol of glucose is incorporated per mole of calmodulin. Glycation of calmodulin is associated with variable reductions in its capacity to activate three Ca2+/calmodulin-dependent brain target enzyme systems, including adenylyl cyclase, phosphodiesterase, and protein kinase. In addition, glycated calmodulin exhibits a 54% reduction in its Ca2+ binding capacity. Isolated CNBr cleavage fragments of glycated calmodulin suggest that glycation follows a nonspecific pattern in that each of seven available lysines is susceptible to modification. A limit observed on the extent of glycation appears related to the accompanying increase in negative charge on the protein. Glycation results in minimal structural rearrangements in calmodulin, and the Ca2+-induced increase in alpha-helix content and radius of gyration is the same for glycated and unmodified calmodulin. Since glycated calmodulin's Ca2+ binding capacity is reduced, this implies that the Ca2+-induced conformational changes in calmodulin do not require all four Ca2+ binding sites to be occupied. Examination of the lysine positions in calmodulin suggests that Ca2+ binding to domains II and IV is sufficient to induce these changes. The functional consequences of calmodulin glycation therefore cannot be attributed to inhibition of these conformational changes. An alternative explanation is that the inhibition arises from interference at the target enzyme binding site by bound glucose. While glycation shows minimal structural effects, a large pH dependence is observed for the alpha-helix content of unmodified calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)

The isolation and reconstitution of the ADP/ATP carrier from wild-type Saccharomyces cerevisiae Identification of primarily one type (AAC-2)

Methods for isolation of the ADP/ATP carrier (AAC) from yeast ( Saccharomyces cerevisiae) are described which allow separation of the carrier from the initially copurified porin which poses a specific problem in yeast. The procedure varies according to whether one wishes to obtain a stable CAT-AAC complex, the free and active AAC for reconstitution, or the SDS-denatured pure AAC peptide. CNBr cleavage of AAC enabled us to differentiate clearly between isogenes AAC-1 and AAC-2 recently found in yeast, due to the exclusive occurrence of a methionine (M-115) residue at the end of the first domain in AAC-2. Thus the AAC isolated from wild-type yeast is primarily or exclusively AAC-2. The isolated AAC is active in ADP/ATP exchange in reconsitituted liposomes with a V max of 1100 μmol/min per g protein and K m = 15 μM for ADP, and a V max of 900 μmol/min per g protein and K m = 9 μM for ATP.

Characterization of mammalian type IX collagen fragments from limited pepsin digests of a transplantable swarm rat chondrosarcoma

Collagenous fragments from type IX molecules have been solubilized by limited pepsin proteolysis of a transplantable rat chondrosarcoma and isolated by selective salt precipitation. Chromatography of the solubilized precipitate on CM-cellulose under nondenaturing conditions yielded three fractions. When examined by polarimetry, the material in all three fractions revealed native collagen helical structure with melting points which ranged from 31-37°C. When the fractions were denatured and rechromatographed on a column of agarose beads, the most acidic fraction eluted as 13-kDa polypeptides with and without prior reduction and alkylation. In contrast, the second and third fractions eluted as 100-kDa and 30-kDa polypeptides prior to reduction, but on reduction and alkylation produced reducible products of 34 kDa and 10 kDa, respectively. The general compositional features of the three fractions closely resemble comparable collagenous fragments of type IX collagen from other species. The denaturation products of the 13-kDa nonreducible, the 30-kDa reducible, and the 100-kDa reducible fractions were sequentially purified by CM-cellulose and reversed-phase chromatography to resolve the chain constituents. The isolated 10-kDa,13-kDa, and 34-kDa chains were cleaved with CNBr, and the cleavage products identified by gel-permeation chromatography. Two 13kDa polypeptides, 13K2 and 13K3, which did not contain any methionyl residues and were not cleaved with CNBr, were digested with trypsin, and the peptide digests were resolved by reversed-phase chromatography. Comparisons of the CNBr and tryptic cleavage products demonstrate that the three major collagenous fragments are composed of three unique polypeptides. A partial amino acid sequence of an 8-kDa CNBr peptide derived from a purified 10-kDa peptide (10K1) matches identically the amino acid sequence derived from a cDNA sequence in the rat α1(IX) chain (Kimura et al., 1989). These studies, then, present convenient procedures useful in the isolation of mammalian type IX collagen fragments and describe features of the rat molecule, indicating that it is similar to the avian counterpart with respect to chain composition and general molecular structure.

Molecular differences between rat-liver and rat-kidney biliverdin reductase. Implications for their in vivo regulation.

Rat-liver biliverdin reductase exists in two molecular forms. The major form 1 has a molecular mass of 34 kDa, while the minor form 2 has a molecular mass of 56 kDa. Form 1 was converted into a second major form (form 3) with a molecular mass of 68 kDa by a NAD+-dependent peroxisomal dehydrogenase which was induced under conditions of oxidative stress [Frydman, R. B., Tomaro, M. L., Awruch, J. & Frydman, B. (1984) Biochem. Biophys. Res. Commun. 121, 249]. Molecular form 1 from rat kidney was not affected by the dehydrogenase, and a structural explanation for this difference was therefore sought. Both form 1 biliverdin reductases, isolated from rat liver and kidney, were purified to homogeneity using affinity chromatography, FPLC and HPLC techniques. The homogeneous enzymes were found to be identical when compared by their HPLC retention times, amino acid compositions and electrophoretic behaviour on polyacrylamide gels under non-denaturing conditions and on SDS/polyacrylamide gels. On HPLC analysis the peptides resulting from the CNBr cleavage were found to be the same for both enzymes, when either the native enzymes or their thioethylpyridine derivatives were compared. When the HPLC fingerprints of the tryptic digests were compared, they were found to be very similar, except for a peptide eluting at 31.60 min in the liver digest and at 23.60 min in the kidney digest. When the enzyme from both origins was alkylated with 4-dimethylaminoazobenzene-4'-iodoacetamide and then digested with trypsin, the HPLC fingerprints of the alkylated cysteine-carrying peptides were almost identical, except for a peptide with a retention time of 19.03 min in the liver digest and of 18.19 min in the kidney digest. The liver reductase was not amenable to Edman degradation suggesting a block at the NH2-terminus; in the kidney enzyme, however, it was free and an NH2-terminal sequence of 12 amino acids could be determined. The liver enzyme was found to be more sensitive toward p-hydroxymercuriphenyl sulfonate than the kidney enzyme.

Localization of γ-Glutamyl-phosphate residues to the α2CB3–5Peptide of type I Chicken bone collagen

alpha 2(I) chains of chicken bone collagen were isolated and purified. Analyses confirmed previous studies that the alpha 2(I) chains contained gamma-glutamyl phosphate residues. Sodium borohydride reduction of the gamma-glutamyl phosphate residues of the CaCl2 extracted and purified alpha 2(I) chains and subsequent CNBr cleavage showed that most of the gamma-glutamyl phosphate groups were located on the alpha 2CB3-5 peptides of the type I collagen molecule, suggesting a specific function for these phosphorylated residues. The reported linkage of the Ser(P) containing phosphoproteins of dentin to the alpha 2CB4 regions of collagen may indicate different biological functions for these two different, protein-bound organic phosphate residues.

Identification and localization of allergenic determinants on grass group I antigens using monoclonal antibodies.

Epitopes recognized by five mAb which block the binding of human IgE antibodies to grass group I (GpI) Ag were characterized and partially mapped. Site specificity studies defined four apparently non-overlapping blocking antibody binding sites on the meadow fescue GpI molecule, Fes e I. One of these sites (site A) was localized to a 14,000 m.w. fragment designated P3 generated by CNBr cleavage of purified Fes e I. The P3 peptide possessed human IgE binding sites as well as other epitopes (non-site A) defined by 19 other anti-GpI mAb. All of the P3 reactive antibodies recognized cross-reactive determinants found on GpI Ag isolated from five different grasses suggesting that P3 is a conserved portion of grass GpI molecules. The P3 fragment from Fes e I was used to immunize mice and induced antibodies which reacted with intact GpI Ag from all 5 different grasses currently being studied in this laboratory.

The Myeloid Blood Cell Differentiation-Inducing Protein MGI-2A Is Interleukin-6

The mouse myeloid blood cell differentiation-inducing protein, macrophage and granulocyte inducer, type 2A (MGI-2A), was purified, and the amino acid sequence of a CNBr cleavage peptide (22 residues) was determined. This amino acid sequence is identical to the sequence found in positions 73 to 94 of mouse interleukin-6 (IL-6). Recombinant mouse IL-6 protein induces differentiation of mouse myeloid leukemic cells that are induced to differentiation by MGI-2, and monoclonal antimouse-MGI-2 antibody, which neutralizes MGI-2, also completely neutralizes this IL-6-induced differentiation. These results show that the major type of mouse myeloid differentiation-inducing protein (MGI-2A) and IL-6 are very similar and most likely identical proteins. Recombinant human IL-6 (also called interferon-beta 2 or B-cell differentiation factor), which shows only a 41% similarity to mouse IL-6, has 11 identical amino acid residues out of the 22 in the mouse MGI-2A peptide and also induces differentiation of the same myeloid leukemic cells.

The myeloid blood cell differentiation-inducing protein MGI-2A is interleukin-6

The mouse myeloid blood cell differentiation-inducing protein, macrophage and granulocyte inducer, type 2A (MGI-2A), was purified, and the amino acid sequence of a CNBr cleavage peptide (22 residues) was determined. This amino acid sequence is identical to the sequence found in positions 73 to 94 of mouse interleukin-6 (IL-6). Recombinant mouse IL-6 protein induces differentiation of mouse myeloid leukemic cells that are induced to differentiation by MGI-2, and monoclonal antimouse- MGI-2 antibody, which neutralizes MGI-2, also completely neutralizes this IL-6-induced differentiation. These results show that the major type of mouse myeloid differentiation-inducing protein (MGI-2A) and IL- 6 are very similar and most likely identical proteins. Recombinant human IL-6 (also called interferon-beta 2 or B-cell differentiation factor), which shows only a 41% similarity to mouse IL-6, has 11 identical amino acid residues out of the 22 in the mouse MGI-2A peptide and also induces differentiation of the same myeloid leukemic cells.

Effect of selective chemical modification and CNBr-cleavage at methionine 20 on catalytic activity and substrate binding properties of porcine pancreatic phospholipase A2

Porcine pancreatic phospholipase A 2 contains 2 methionine (Met) residues located at positions 8 and 20, respectively. Reaction of the enzyme with methyliodide and iodoacetic acid resulted in the selective methylation and carboxymethylation, respectively, of Met 20. It was found that porcine pancreatic iso-phospholipase A 2, possessing only Met 8, was not affected by either modification. Reaction of porcine phospholipase A 2 with cyanogen bromide in 0.1 N hydrochloric acid gave rise to cleavage only at Met 20. The enhanced reactivity of Met 20 compared to that of Met 8 is in agreement with the known X-ray structure of phospholipase A 2 which shows that Met 8 is located in the interior of the protein, while Met 20 is at the surface. Both methylation and carboxymethylation of Met 20 do not significantly affect catalytic and substrate binding properties of the enzyme. In contrast, the more rigorous cleavage at Met 20 by CNBr resulted in the loss of catalytic activity, while substrate and Ca 2+ binding was diminished only to a limited extent. Most likely CNBr cleavage at Met 20 perturbs the active site despite the fact that the N-terminal fragment Ala 1-Hse 20 is still bound via the disulfide bridge Cys 11-Cys 77 to the remainder of the protein. The results obtained strongly suggest that the conformation of the sequences Ala 1-Hse 20 and/or Asp 21-Gly 26 are important for the maintenance of the special microenvironment of the active site cleft.

Ca2+/calmodulin-dependent protein kinase II. Identification of a regulatory autophosphorylation site adjacent to the inhibitory and calmodulin-binding domains.

Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) autophosphorylated under limiting conditions (7 microM [gamma-32P]ATP, 500 microM magnesium acetate, 4 degrees C) was analyzed by CNBr cleavage and peptide mapping to determine the site of autophosphorylation that brings about transition of the kinase to the Ca2+-independent form. Reverse phase high performance liquid chromatography (HPLC) (C3) revealed one major CN-Br 32P-peptide (CB1) that eluted at about 6% propanol. This peptide contained [32P]threonine, but almost no [32P]serine, and migrated as a single band (Mr = 3000-3500) in polyacrylamide gels run in the presence of urea and sodium dodecyl sulfate. The properties of CB1 were compared to the properties of a 26-residue synthetic peptide containing the CaM-binding and inhibitory domains as well as a consensus phosphorylation sequence (-Arg-Gln-Glu-Thr-) of rat brain CaM-kinase II (residues 282-307 and 283-308 of the alpha and beta subunits, respectively). CB1 and the synthetic peptide comigrated in urea/sodium dodecyl sulfate gels, co-eluted from reverse phase HPLC (C3 and C18) and from Sephadex G-50, and exhibited Ca2+-dependent calmodulin-binding properties. When the two peptides were subjected to automated Edman sequence analysis, both exhibited a burst of 32P release at cycle 5, which is consistent with the expected amino-terminal sequence of the two peptides, i.e. His-Arg-Gln-Glu-Thr(PO4)-. These findings indicate that autophosphorylation of Thr286 (alpha subunit) and Thr287 (beta subunit) is responsible for transition of CaM-kinase II to the Ca2+-independent form.

Binding of sulfobromophthalein to rat and human ligandins: characterization of a binding-site peptide

Photoaffinity techniques were employed to affect the covalent binding of [ 35S]sulfobromophthalein to proteins of rat and human liver cytosol. In rat liver cytosol at low concentrations, sulfobromophthalein bound to the 22 kDa subunit of ligandin. In human liver cytosol, binding to a 23.5 kDa subunit was observed. At higher concentrations, sulfobromophthalein also bound to 12, 23.5, 37, and 42 kDa peptides. When the peptides resulting from CNBr cleavage of [ 35S]sulfobromophthalein-ligandin complex were resolved by high-performance liquid chromatography, radioactivity was associated with two peptides. The peptide containing 80% of the radioactivity was isolated and characterized. Its molecular weight is 3.4 kDa, it contains the single tryptophan residue of ligandin and has a glutamate (glutamine) as the N-terminal amino acid.

Sequence analysis, expression, and conservation of Escherichia coli uracil DNA glycosylase and its gene (ung).

The complete nucleotide sequence of the Escherichia coli ung gene is described. Transcription initiation and termination sites were determined by S1 nuclease and RNase mapping. The common prokaryotic -35, -10, and the ribosome binding site sequences are represented by TGTTCTGTA, TAAGCTA, and AGGAGAG at their respective locations. A putative hairpin transcription terminator structure is present at the major transcription terminator sites. The open reading frame of the ung gene codes for a protein of 229 amino acids (25,664 daltons). The molecular weight, amino acid composition, and the N-terminal amino acid sequence of the uracil DNA glycosylase purified from E. coli cells match with the open reading frame of the ung gene. The protein sequence analysis shows that the N-terminal methionine is cleaved off in the mature protein. The in vitro transcription coupled translation of the ung gene directs the synthesis of a protein which comigrates with uracil DNA glycosylase. Also, the CNBr cleavage of the protein synthesized in vitro confirms the positions of the methionines deduced from the DNA sequence. The levels of ung gene expression remain constant up to the early stationary phase, but decline in the late stationary phase of the E. coli culture. The E. coli gene showed a strong sequence homology to Shigella, a weak sequence homology to Salmonella and Citrobacter, and a very weak sequence homology to Proteus genes. No sequence homologies were seen for Pseudomonas, Clostridium, Micrococcus, and several eukaryotic genomes.

The amino acid sequence of wheat histone H2B(2)

Two of the four electrophoretic histone H2B variants present in wheat embryos have been isolated. The complete primary structure of the H2B(2) variant has been deduced from sets of overlapping peptides generated by CNBr cleavage, Staphyloccocus aureus V8 protease, endoproteinase Arg-C, the post-proline cleaving enzyme, chymotrypsin and cleavage in dilute acid. A minimum of 17 peptides were required to establish the sequence. This variant has a blocked N terminus and comprises a total of 149 amino acids. The C-terminal two-thirds of the protein are highly homologous to vertebrate H2B. In contrast, the N-terminal third is entirely different and contains an N-terminal extension of 23 residues in which the sequence Ala-Glu-Lys or variants are repeated several times. This region is also highly homologous to the H2B from Tetrahymena pyriformis. It shows in addition similarities to wheat H2A(1), and bovine H1.

Purification, properties and complete amino acid sequence of the ferredoxin from a green alga, Chlamydomonas reinhardtii.

The ferredoxin was purified from the green alga, Chlamydomonas reinhardtii. The protein showed typical absorption and circular dichroism spectra of a [2Fe-2S] ferredoxin. When compared with spinach ferredoxin, the C. reinhardtii protein was less effective in the catalysis of NADP+ photoreduction, but its activity was higher in the light activation of C. reinhardtii malate dehydrogenase (NADP). The complete amino acid sequence was determined by automated Edman degradation of the whole protein and of peptides obtained by trypsin and chymotrypsin digestions and by CNBr cleavage. The protein consists of 94 residues, with Tyr at both NH2 and COOH termini. The positions of the four cysteines binding the two iron atoms are similar to those found in other [2Fe-2S] ferredoxins. The primary structure of C. reinhardtii ferredoxin showed a great homology (about 80%) with ferredoxins from two other green algae.

Effect of oxidation on the properties of apolipoproteins A-I and A-II.

Purified apolipoprotein A-I has been separated by reversed-phase high performance liquid chromatography (HPLC) into multiple peaks and these peaks have been characterized. One peak, apoA-Ib had a relatively longer retention time on HPLC but its retention time could be shortened by treatment by hydrogen peroxide. CNBr cleavage studies indicated that the differences in apoA-Ib and in its oxidation product, apoA-Ia, were due to the different oxidation states of methionine. This phenomenon was also observed in apoA-II, where methionine oxidation produced two more forms of this apolipoprotein in addition to the native form. These isomers were found to have different secondary structures and affinities for lipid. Model peptide analogs of the amphipathic helix with the same sequence but with methionine and methionine sulfoxide at the nonpolar face of the amphipathic helix were synthesized and studied. It was found that the lipid affinities of these synthetic peptide isomers were very different. They also differed in their secondary structures as studied by circular dichroism (CD). We propose that methionine oxidation introduces hydrophilic residues at the nonpolar face of the amphipathic helical domains of these apolipoproteins and, therefore, alters their secondary structure and lipid affinity.

Charybdotoxin and noxiustoxin, two homologous peptide inhibitors of the K +(Ca 2+) channel

We show that noxiustoxin (NTX), like charybdotoxin (CTX) described by others, affects Ca 2+-activated K + channels of skeletal muscle (K +(Ca 2+) channels). Chemical characterization of CTX shows that it is similar to NTX. Although the amino-terminal amino acid of CTX is not readily available, the molecule was partially sequenced after CNBr cleavage. A decapeptide corresponding to the C-terminal region of NTX shows 60% homology to that of CTX, maintaining the cysteine residues at the same positions. While CTX blocks the K +(Ca 2+) channels with a K d of 1–3 nM, for NTX it is approx. 450 nM. Both peptides can interact simultaneously with the same channel. NTX and CTX promise to be good tools for channel isolation.

Preparation of overlapping peptides of bovine retinal S-antigen and their localization by immunoblotting with peptide-specific antibodies

Bovine retinal S-antigen was cleaved by three chemical cleavage procedures including o-iodosobenzoic acid (IBA), mile acid and cyanogen bromide. The resultant peptides were used to study antibody-defined epitopes. Treatment with IBA, which cleaves primarily at tryptophanyl peptide bonds, produced at least 4 major fragments and several minor fragments. The peptides have been identified by their migration on SDS-PAGE and tested for their immunoreactivity to several affinity-purified anti-CNBr-peptide antibodies and to affinity-purified anti-IBA peptide antibodies. The presence of a single tryptophan residue 194 residues from the amino-terminus should result in 2 fragments of approximately 23,000 and 26,000 molecular weight based on the known size of intact S-antigen. The additional fragmentation is due to the presence of acid labile bonds and cleavage at IBA-sensitive tyrosyl residues associated with a side reaction. Western immunoblots using affinity-purified antibodies against the various IBA and CNBr peptides have allowed location of these peptides within the intact molecule. Specifically, IBA23K and IBA21K are overlapping fragments on the carboxy end, mutally exclusive of all other peptides. IBA15K and IBA5.6K overlap, and IBA18K and IBA10K overlap within IBA26K which comprises the N-terminal half of S-Ag. Additionally, IBA10K contains an antibody epitope destroyed by CNBr cleavage of the methionyl residue between CB53 and CB56. Further characterization of these IBA peptides will expedite the location of possible additional uveitogenic epitopes in the amino-terminal half of S-Ag as well as epitopes lost by other peptide generating techniques.

Amino-acid sequence of the cytochrome-b5-like heme-binding domain from Hansenula anomala flavocytochrome b2.

Flavocytochrome b2 (L-lactate dehydrogenase) from baker's yeast is composed of two structural and functional domains. Its first 100 residues constitute the heme-binding core, which is homologous to cytochrome b5 [B. Guiard, O. Groudinsky & F. Lederer (1974) Proc. Natl Acad. Sci. USA 71, 2539-2543]. We report here the amino acid sequence of the heme-binding domain isolated by tryptic proteolysis of Hansenula anomala flavocytochrome b2. The sequence was established by automated degradation of the whole fragment and of peptides obtained by CNBr cleavage at the unique tryptophan and by proteolysis with thermolysin and endoproteinase Lys C. As isolated, the domain consists of 84 residues without any sulfur amino acids. It shows 49 identities with the heme-binding domain from Saccharomyces cerevisiae and 28 with beef microsomal cytochrome b5. Using the recently published three-dimensional structure of S. cerevisiae flavocytochrome b2 [Z-x. Xia, N. Shamala, P. H. Bethge, L. W. Lim, H. D. Bellamy, N. H. Xuong, F. Lederer and F. S. Mathews (1987) Proc. Natl Acad. Sci. USA 84, 2629-2633], it can be seen that there are only positively charged side chains close to the accessible heme edge, the only negative charges in that area being those of the heme propionates. The implications of this result are discussed in the light of Salemme's model for the cytochrome b5/cytochrome c complex [F. R. Salemme (1976) J. Mol. Biol. 102, 563-568].

Identification of erythro-β-hydroxyasparagine in the EGF-like domain of human C1r

Previous studies [(1987) Biochem. J. 241, 711–720] have shown that position 150 of human C1r is occupied by a modified amino acid that, after acid hydrolysis, yields erythro-β-hydroxyaspartic acid. In view of further investigations on the nature of this residue, peptide CN1a T8/T9 TL8 (positions 147–155) was isolated from C 1 r A chain by CNBr cleavage followed by enzymatic cleavages by trypsin and thermolysin. Amino acid analysis, sequential Edman degradation and FAB-MS of this peptide indicate that the residue at position 150 is an erythro-β-hydroxyasparagine resulting from post-translational hydroxylation of asparagine.