Interaction of bovine gallbladder mucin and calcium-binding protein: Effects on calcium phosphate precipitation

Background & Aims Gallstones consist of calcium salts and cholesterol crystals, arrayed on a matrix of gallbladder mucin (GBM), and regulatory proteins like calcium-binding protein (CBP). To determine if interactions between CBP and GBM follow a biomineralization scheme, their mutual binding and effects on CaHPO₄ precipitation were studied. Methods Binding of CBP to GBM was assessed by inhibition of the fluorescence of the complex of GBM with bis-1,8-anilinonaphthalene sulfonic acid (bisANS). The effects of the proteins on precipitation of CaHPO₄ were assessed by nephelometry and gravimetry. Precipitates were analyzed for calcium, phosphate, and protein. Results CBP and bis-ANS competitively displaced each other from 30 binding sites on mucin, with a 1:1 stoichiometry and similar affinity. The rate of precipitation of CaHPO₄ was retarded by mucin and CBP. Precipitate mass was unaffected by GBM alone but decreased with the addition of CBP. Complexing CBP with GBM abolished or moderated this latter effect, altered precipitate morphology, and changed the stoichiometric ratios of Ca to PO₄ in the precipitates from 1:1 to 3:2. Mucin and CBP were incorporated into the precipitates. Conclusions These studies suggest that the formation of calcium-containing gallstones is a biomineralization process regulated by both GBM and CBP.