A simple procedure for isolating microgram quantities of biologically active bikunin from human urine

Abstract

To report a simple, relatively rapid protocol to isolate biologically active bikunin from human urine using ion-exchange-trypsin affinity chromatography. Bikunin is a protease inhibitor which has been shown to play a role in various processes, including inhibition of calcium oxalate crystallization, the regulation of proliferation and modulation of carcinogenesis. The unavailability of the purified protein has hampered studies on bikunin's expanding role in these processes. Female human urine was dialysed (15 kDa threshold) and crudely fractionated with a double-saturated ammonium sulphate precipitation. The first precipitation was with 35% saturated ammonium sulphate, and the supernatant was harvested, and the second with 90% saturated ammonium sulphate, and the precipitate collected. The protein mixture was then passed over Sepharose SP-fast-flow cation exchange and Sepharose Q-fast-flow anion exchange columns connected in series. The final purification was with a trypsin-affinity column which selectively bound bikunin. This procedure could recover 1 microg of bikunin per 2 mL of urine, and the final product was essentially free of contaminating inter-alpha-trypsin inhibitor heavy chains or bikunin-heavy chain conjugates. Product purity was confirmed by two-dimensional polyacrylamide gel electrophoresis combined with silver staining or Western blot. All isolations contained the 17 kDa minimally glycoslyated/sulphated form of bikunin and the 28 kDa form of bikunin. Some preparations also contained 33-48 kDa forms of bikunin. The protein cores of all three proteins were confirmed to be bikunin by mass spectrometry and Western blot. Harvested bikunin retained its trypsin inhibitory activity (L-benzoylarginine-p-nitroanilide assay). Preparations containing the 33-45 kDa form had two to three times more trypsin inhibitory activity than preparations without this band. This novel ion exchange-trypsin affinity chromatography protocol uses only two chromatographic steps. The product consists of three isomers of biologically active bikunin, free of contaminating heavy chains or bikunin-heavy chain conjugates. The ready availability of purified bikunin should facilitate future studies of bikunin's emerging role in urolithiasis, proliferation and carcinogenesis.