Capillary electrophoresis of large cyanogen bromide peptides of fibre-forming collagens with special reference to cross-linking
Abstract
Cyanogen bromide (CNBr) peptides derived from collagen types I and III and having a relative molecular mass of more than 13 500 exhibit a linear relationship between molecular mass and migration time. This behavior is similar to the elution of CNBr peptides of collagen types I and III from wide pore (30 nm) C-18 reversed-phase columns.
The electrophoretic procedure is very rugged and the linearity is preserved over the pH range 2.0-2.5, with a background electrolyte concentration of 20-100 mmol/l and an applied voltage of 8-25 kV [per 70 cm (effective length 63 cm)x 75 mu m I.D. capillary]. Modification of the inner capillary surface or addition of an organic modifier ruins the separation, The separation mechanism is apparently multimodal, as no correlation between the number of total amino acid residues forming a peptide, the number of proline and hydroxyproline residues or the number of glycines could be established.
Also, there is no secondary structure involved, as the results with native and denatured peptides were the same. Application of this approach to reveal higher molecular mass peptides (similar to 40 000) in rat tail tendons of two-year-old rats compared with three-month-old rats is presented.