Protein Denaturation Facilitates Stronger Protein–Crystal Interactions with Calcium Oxalate Monohydrate and Dihydrate than Those in Native States

Kidney stones contain organic components, including proteins, in addition to inorganic components that constitute crystals. In vitro calcium oxalate crystal growth experiments also confirmed that the proteins were incorporated into the crystals. However, the structural states of proteins in stones and crystals remain unclear. In this study, we focused on protein denaturation and compared the effects of native and denatured proteins on the growth of calcium oxalate crystals. Five model proteins with different secondary structures, molecular weights, and isoelectric properties─bovine serum albumin (BSA), soybean trypsin inhibitor (STI), bovine β-lactoglobulin (BLG), chicken egg lysozyme (HEL), and bovine pancreas α-chymotrypsinogen (BPC)─were selected. These proteins were irreversibly denatured under high-temperature and high-pressure conditions. Denatured BSA, STI, and BLG showed significant changes in crystal shape compared to native proteins, indicating that they inhibited crystal growth. In contrast, denatured HEL and BPC stabilized the metastable phases, although the effect of denaturation on the crystal shape was small. Microscopic observations using fluorescently labeled proteins revealed that the denatured proteins exhibited stronger fluorescence from the surface and within the crystals, indicating that protein denaturation enhanced their binding to calcium oxalate crystals. Thus, the structural state of the proteins contained within the stones must be considered.