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A novel basic protein from human kidney which inhibits calcium oxalate crystal growth
1Department of Medical Biochemistry, Dr ALM PG Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, India.
Objectives:
To isolate calcium oxalate-binding proteins from human kidney and characterize the functional properties.
Materials And Methods:
Calcium oxalate crystals were prepared and allowed to interact at two different pH values with Triton-extracted human kidney homogenate. The proteins in the homogenate were isolated and fractionated on a cellulose column, and purified by high-performance liquid chromatography. The protein with the greatest oxalate binding activity at pH 4.5 was analysed for its amino-acid composition and characterized by Scatchard plot analysis, crystal growth, nucleation and aggregation studies.
Results:
Three major protein fractions were eluted when calcium oxalate monohydrate was adsorbed at both pH values (designated as fractions I-III, according to their order of elution). The yield of fraction I and III was increased when adsorbed at an acidic pH. However, only fraction III had maximum oxalate binding activity at pH 4.5. When purified, this protein had maximum oxalate binding activity of approximately 270 pmol/mg protein and a molecular weight of approximately 23 kDa. Amino acid analysis showed that 18% of the total molar proportion was of basic amino acids, e.g. lysine and arginine, while acidic amino acids accounted for only 11%. Both alanine and glycine constituted approximately 41% of the total molar proportion. Modifications to the lysine group abolished oxalate-binding activity of the protein. The protein inhibited crystal growth by 82% at 0.8 micromol/L, while it inhibited the nucleation and aggregation of the crystals by 6% and 28%, respectively, at 49 nmol/L. The inhibition of both nucleation and aggregation was higher at pH 5.7 than at pH 7.4. Significantly, the protein induced the formation of intertwined calcium oxalate dihydrate crystals in a medium known to induce the formation of individual dihydrate crystals.
Conclusion:
The protein described here is the first reported basic inhibitor of calcium oxalate crystal growth with oxalate-binding activity at pH 4.5 that modulates calcium oxalate crystallization. It is suggested that this protein may play a physiologically significant role in inhibiting stone formation in acidic urine.