Isolation and characterization of the cyanogen bromide peptides derived from the human .alpha.2(V) collagen chain

Abstract

The human alpha 2(V) collagen chain when cleaved with cyanogen bromide yields ten peptides which can be recovered in approximately equimolar quantities. Characterization of the purified peptides with regard to molecular weight and amino acid composition establishes the uniqueness of the peptides and reveals that the alpha 2(V) chain recovered following limited pepsin digestion contain 956 amino acid residues. Possible homologies between the alpha 2(V) peptides and peptides derived from other collagen chains were noted. In addition, a high-performance liquid chromatography system is described for the separation of three of the alpha 2(V) chain peptides which were not resolved by using conventional separation techniques.

Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

“…The nine Hyp residues mapped here to the X-positions of Gly-Hyp-Hyp repeats in the COL1 domain of human recombinant pro-␣1(V) chains falls within this range (15,69). However, the 22 X-position Hyp residues mapped here within the bovine placenta ␣1(V) chain lies beyond this range and is instead reminiscent of the range of 10 to 20 3-Hyp residues previously estimated by amino acid analysis to lie within the collagen IV chains of some tissues (75,76).…”

“…chains from human skin (15) or placenta (69) or produced in 293-HEK cells (61) produced estimates of 4-Hyp content of from ϳ106 to ϳ111 per 1000 amino acids. The 106 and 98 Y-position Hyp residues directly localized here by MS n for the COL1 domains of bovine placenta ␣1(V) and human recombinant pro-␣1(V) chains, respectively, are in the same range as these numbers, consistent with identification of the Y-position Hyp residues mapped here as 4-Hyp residues.…”

Cyanogen Bromide Peptides of the Fibrillar Collagens I, III, and V and Their Mass Spectrometric Characterization: Detection of Linear Peptides, Peptide Glycosylation, and Cross-Linking Peptides Involved in Formation of Homo- and Heterotypic Fibrils

“…Hydroxyproline occurs mainly as 4-hydroxyproline and in small amounts (<5%) as 3-hydroxyproline. 5,7,8 Both species cannot be distinguished by the mass spectrometric approach presented here. The hydroxyproline and hxdroxylysine content was taken from previous works: The values of proline, hydroxyproline, lysine, and hydroxylysine for R1(I) were taken from Rauterberg et al, 5 for R1(III) from Rauterberg et al, 6 for R1(V) from Rhodes et al, 7 and for R2(V) from Rhodes et al 8 The corresponding values of the R2(I)-chain are based on c-DNA data: The sequence ranges No.…”

“…3,4 For biochemical analysis, instead of using enzymes, collagens are frequently cleaved with cyanogen bromide (CNBr). [5][6][7][8] This approach has some advantages: Typically, the cleavage reaction is almost complete and the relatively small number of methionine residues in the R-chains has the consequence that on average not more than about a dozen CB peptides per chain are formed. The presence of larger peptides, which will in part contain more than 200 amino acids, facilitates their identification and, hence, the determination of the collagen type.…”

The pro α2(V) collagen gene is evolutionarily related to the major fibrillar-forming collagens

“…Figure 4: Partial distribution of the methionyl residues of the human a 2(V) chain. The CB peptides are numbered according to Rhodes et al, (33). The small peptide previously undetected by protein analysis is numbered with a 0.…”