Analysis of Hydroxyproline in Collagen Hydrolysates.

Hydroxyproline (Hyp) is an imino acid posttranslationally formed by sequence-specific hydroxylases in the repeating collagen Gly-Xaa-Yaa triad present in all collagen types of all species. In both Xaa- and Yaa-positions, Pro is the most common residue, often oxidized to 4-Hyp in the Yaa- and rarely to 3-Hyp in the Xaa-positions. Here we describe the qualitative and quantitative analysis of 3- and 4-Hyp-isomers by separating the free imino acids either with hydrophilic interaction chromatography (HILIC) or after derivatization with reversed-phase chromatography (RPC). In both cases the compounds were detected by electrospray-ionization mass spectrometry.

Analysis of hydroxyproline in collagen hydrolysates.

Hydroxyproline (Hyp) is an imino acid post-translationally formed by sequence-specific hydroxylases in the repeating collagen Gly-Xaa-Yaa triad present in all collagen types of all species. In both Xaa- and Yaa-positions, Pro is the most common residue, often oxidized to 4-Hyp in the Yaa- and rarely to 3-Hyp in the Xaa-positions. Here, we describe the qualitative and quantitative analysis of 3- and 4-Hyp-isomers by separating the free imino acids either with hydrophilic interaction chromatography (HILIC) or after derivatization with reversed-phase chromatography (RPC). In both cases, the compounds were detected by electrospray ionization mass spectrometry (ESI-MS).

Mineralization of the metre-long biosilica structures of glass sponges is templated on hydroxylated collagen.

The minerals involved in the formation of metazoan skeletons principally comprise glassy silica, calcium phosphate or carbonate. Because of their ancient heritage, glass sponges (Hexactinellida) may shed light on fundamental questions such as molecular evolution, the unique chemistry and formation of the first skeletal silica-based structures, and the origin of multicellular animals. We have studied anchoring spicules from the metre-long stalk of the glass rope sponge (Hyalonema sieboldi; Porifera, Class Hexactinellida), which are remarkable for their size, durability, flexibility and optical properties. Using slow-alkali etching of biosilica, we isolated the organic fraction, which was revealed to be dominated by a hydroxylated fibrillar collagen that contains an unusual [Gly-3Hyp-4Hyp] motif. We speculate that this motif is predisposed for silica precipitation, and provides a novel template for biosilicification in nature.

Carboxymethylation of the fibrillar collagen with respect to formation of hydroxyapatite.

Control over crystal growth by acidic matrix macromolecules is an important process in the formation of many mineralized tissues. Highly acidic macromolecules are postulated intermediates in tissue mineralization, because they sequester many calcium ions and occur in high concentrations at mineralizing foci in distantly related organisms. A prerequisite for biomineralization is the ability of cations like calcium to bind to proteins and to result in concert with appropriate anions like phosphates or carbonates in composite materials with bone-like properties. For this mineralization process the proteins have to be modified with respect to acidification. In this study we modified the protein collagen by carboxymethylation using glucuronic acid. Our experiments showed unambigously, that N(epsilon)-carboxymethyllysine is the major product of the in vitro nonenzymatic glycation reaction between glucuronic acid and collagen. We hypothesized that the function of biomimetically carboxymethylated collagen is to increase the local concentration of corresponding ions so that a critical nucleus of ions can be formed, leading to the formation of the mineral. Thus, the self-organization of HAP nanocrystals on and within collagen fibrils was intensified by carboxymethylation.

Modification of collagen in vitro with respect to formation of Nepsilon-carboxymethyllysine.

Developing new biopolymer-based materials with bio-identical properties is a significant challenge in modern science. One interesting route to this goal involves the biomineralization of collagen, a pre-structured and widely available protein, into a material with interesting properties. A prerequisite for biomineralization is the ability of cations (e.g., calcium) to bind to the protein and to result in concert with appropriate anions (e.g., phosphate) in composite material with e.g., bone-like properties. In order to increase the number of binding sites it is necessary to modify the protein prior to mineralization. For this glucuronic acid (GA) was used due to its carbonyl and carboxyl groups to derivatize proteinogenic amino groups transferring them into negatively charged carboxyl groups. Our experiments showed for the first time, that Nepsilon-carboxymethyllysine is the major product of in vitro non-enzymatic glycosylation of collagen by glucuronic acid. For an unequivocal determination of the reaction products, the lysine residues of collagen and of the model peptide were carboxymethylated through a reductive alkylation with glyoxalic acid and compared to the glucuronic acid derivatives. Beside their identical mass spectra the common structure elements could be confirmed with FTIR. Thus, in the context of matrix engineering, by producing Nepsilon-carboxymethyllysine, glucuronic acid offers a convenient way of introducing additional stable acidic groups into protein matrices.

Analysis of hydroxyproline isomers and hydroxylysine by reversed-phase HPLC and mass spectrometry.

Collagens, the most abundant mammalian proteins, contain a high content of hydroxylated amino acids, such as, 3- and 4-cis-/trans-hydroxyproline (Hyp) and 5-hydroxylysine (Hyl). Whereas the global content of 4-Hyp was studied by amino acid analysis, no technique to determine all five hydroxyamino acids simultaneously in collagens has been reported. Here, we report the separation of all five hydroxyamino acids as well as two Hyp epimers from all other proteinogenic amino acids after derivatization with N(2)-(5-fluoro-2,4-dinitrophenyl)-l-valine amide (l-FDVA) by RPC-UV-ESI-MS. The general applicability of this method is shown for three Hyp-containing peptides as well as collagen type I.