Effects of lanthanide and calcium ions on the polymerisation of collagen

Abstract

The polymerisation of individual collagen molecules into ordered polymeric fibrils is a spontaneous, extracellular process, which helps determine the properties of connective tissues. Our previous work [1, 2] has suggested that lanthanide ions (Ln 3+) might interact with collagen in interesting ways. During experiments to measure the binding of Ln 3+ to collagen, it was discovered that these cations greatly accelerated the rate of collagen polymerisation. Ca 2+ also accelerated polymerisation, but concentrations about 100 times greater than those of Ln 3+ were necessary. For study of polymerisation, pepsin-solubilized calf-skin collagen (1.5 mg/ml) was incubated in 30 m M Tris-HCL, 0.2 M NaCl, pH 7.0 at 37°C. The polymerisation reaction was followed as the increase in absorbance of the solution at a wavelength of 500 nm. As reported by others [ e.g. 3, 4], the resulting curve was sigmoidal; a nucleation phase of 16 min, during which the A 500 remained low, was followed by a growth phase during which the A 500 increased at a maximum rate of 0.05 units/min. A maximum absorbance (A max) of 1.1 units was finally reached. The nucleation phase is thought to represent the length-wise attachment of collagen molecules and the growth phase, their lateral accretion [5]. Sm 3+ reduced the length of the nucleation phase, accelerated the growth phase (Table I) and lowered the A max. These effects were maximal at 100 μ M Sm 3+. Other Ln 3+ also reduced the nucleation phase, but none were as effective as Sm 3+. Er 3+ and La 3+ also accelerated the growth phase, but Lu 3+ was inhibitory. All four Ln 3+ lowered the A max. Ca 2+ reduced the length of the nucleation phase and accelerated the growth phase (Table I), but unlike the Ln 3+, increased the A max. Electron microscopic examination of the fibrils demonstrated that those formed in the presence of increasing concentrations of Sm 3+ were progressively thinner (Table II). Concentration of Sm 3+(μ M Average fibril thickness (nm) 0 320±62 25 210±49 50 130±31 100 78±17 Arrhenius plots of the rates polymerisation at different temperatures gave an activation energy (E a) of 45.8±5.6 kcal/mol for the nucleation phase and 57.2±5.8 kcal/mol for the growth phase. Ca 2+ (5 m M) and Sm 3+ (25 μ M reduced the E a of the growth phase to 34.8±2.3 kcal/mol and 18.0±1.1 kcal/mol respectively. However, the E a for the nucleation phase was little changed by either 5 m M Ca 2+ (E a = 38.2±4.3 kcal/mol) or 25 μ M Sm 3+ (E a = 38.0±2.3 kcal/mol). The ability of physiological concentrations of Ca 2+ to influence the rate of polymerisation of collagen suggests a modulating role for calcium in vivo. The ability of low concentrations of Ln 3+ to mimic these effects suggests that they may prove useful probes of the interaction between Ca 2+ and collagen.