pH effects on collagen fibrillogenesis in vitro: Electrostatic interactions and phosphate binding

Collagen self-assembly in vitro was conducted in the pH range from 6.0 to 10.5 at 30 °C in order to investigate the electrostatic interactions that occur during fibril formation. A sigmoidal curve was observed in the growth rate of fibrils. Collagen fibril morphologies imaged by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) present bundling of fibrils with a small amount of non-fibrillar collagen. At a low pH of 6.6, collagen molecules form small fibrils with a diameter of 85 nm. In the pH range from 6.9 to 8.0, they form fibrils with a diameter of approximately 200 nm, even though the rate of fibrillogenesis accelerates with increasing pH in this range. Zeta potential measurements of soluble collagen indicate that the net surface charge of collagen molecules is not only affected by the pH of medium but also by the presence of added salts. The acceleration of fibrillogenesis rate with increasing pH from 6.6 to 9.2 is consistent with a reduction of surface net charge since the isoelectric point of soluble collagen is approached. The native D-periodicity of 62 nm was found except at pH 7.1 where collagen molecules form short banding of 50-60 nm in the early stage of fibrillogenesis which might be caused by an unusual alignment of collagen molecules in fibrils.