The loci of the three amino acid residues that contribute their prosthetic groups to form the stable, nonreducible, trifunctional intermolecular cross-link histidinohydroxylysinonorleucine in skin collagen fibrils were identified. Two apparently homogeneous three-chained histidinohydroxylysinonorleucine cross-linked peptides were chromatographically isolated. They were obtained from a tryptic digest of denatured unreduced 6 M guanidine hydrochloride insoluble bovine skin collagen. Amino acid and sequence analyses demonstrated that the prosthetic groups of alpha 1(I)-chain Hyl-87, alpha 1(I)-chain Lys-16c, and alpha 2(I)-chain His-92 formed the cross-link. The latter results served to define the locus of the stable, nonreducible trifunctional moiety. Identical types of analyses were performed on the three-chained peptides isolated after bacterial collagenase digestion of the cross-linked tryptic peptides. This confirmed the initial identification and location of the three peptides linked by the cross-link. In addition, data reported here provide for a correction of the micromolecular structure for the alpha 2(I) chain. Stereochemical considerations concerning this trifunctional cross-link's specific locus indicate that the steric relationships between the alpha chains of skin and skeletal tissue collagens are fundamentally different and the intermolecular relationships in skin fibrils are specific for skin. The same molecular relationships also indicate that histidinohydroxylysinonorleucine links three molecules of collagen. The stereochemistry of cross-linking for skin collagen is in accordance with and explains the X-ray findings of a 65-nm periodicity found for this tissue [Stinson, R. H., & Sweeny, P. R. (1980) Biochim. Biophys. Acta 621, 158; Brodsky, B., Eikenberry, E. F., & Cassidy, K. (1980) Biochim. Biophys. Acta 621, 162].

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We have independently isolated and characterized cDNA and genomic clones for the human coagulation factor IX. Sequence analysis in both cases indicates that threonine is encoded by the triplet ACT as the third residue of the activation peptide. This is in agreement with some earlier reports but in disagreement with others that show the alanine triplet GCT at this position. The discrepancy can thus be accounted for by natural variation of a single nucleotide in the normal population. Amino acid sequence analyses of activated factor IX from plasma samples of four individuals yielded two cases of alanine and two cases of threonine at the third position of the activation peptide. In factor IX from pooled plasma and in factor IX from a heterozygous individual, however, both alanine and threonine were found. Taken together, the findings show that a prevalent nondeleterious dimorphism exists in the activation peptide of human coagulation factor IX.Factor IX is the plasma protein that is missing or defective in individuals afflicted with the X-chromosome-linked bleeding disorder hemophilia B. Its role in the blood coagulation cascade is to activate factor X through interactions with calcium, membrane phospholipids, and factor VIII. Factor IX circulates as an inactive zymogen until proteolytic release of its "activation peptide" allows it to assume the conformation of an active serine protease.At the molecular level, it is known that hemophilia B may result from a variety of genetic changes (1, 2). Partial and/or complete deletions of the factor IX gene have been shown to be responsible for the disease in some cases (3,4). Several hemophilia B variants have also been described that show normal levels of the factor IX protein by immunological methods but have reduced or negligible activity in clotting assays. These variants have been designated CRM+ (crossreacting material positive). One of the CRM+ variants, factor IX Chapel Hill, results from an amino acid substitution at one of the proteolytic activation sites, blocking cleavage and subsequent activation (5). A change affecting the other cleavage site is likely to be involved in the variant factor IXDeventer (6).The molecular defect in another CRM+ variant, factor IXAlabama (7), is presently under study in our laboratories. The dimorphism described in this report, however, appears to be the result of a nondeleterious mutation which has been fixed in the normal population.As early as 1978, a partial amino acid sequence was reported for the amino-terminal region of the activation peptide of human factor IX (8). This analysis, apparently done on material from pooled plasma, showed an aminoterminal sequence Ala-Glu-Thr-Val-Phe-for the activation peptide, in agreement with a previously determined sequence for the corresponding region from bovine factor IX (9). No mention was made of alanine at the third position. Several years later, however, the same laboratory did report a cDNA sequence for human factor IX which indicated the presence of an alanine codon at the th...

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The reaction products formed during the enzymatic inactivation of heparin cofactor II (HCII) by a proteinase isolated from Echis carinatus were analyzed by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis and by reverse-phase high-performance liquid chromatography. By NaDodSO4-polyacrylamide gel electrophoresis, limited proteolysis of HCII was observed, which resulted in a decrease in the apparent molecular weight of the protein from approximately 68 000 to approximately 53 000. By reverse-phase high-performance liquid chromatography, at least 20 peptides were observed. Primary structure analysis of these peptides indicated that significant proteolysis had occurred in the NH2-terminal region of the protein. HCII inactivation, however, coincided with the appearance of a peptide from the COOH-terminal region of the protein. The peptide differed from the previously identified reactive site peptide [Griffith, M. J., Noyes, C. M., & Church, F. C. (1985) J. Biol. Chem. 260, 2218-2225] by only one residue: a leucyl residue at the NH2-terminal of the peptide. We conclude that leucine, as opposed to the expected arginine, is at the reactive site of HCII.