Detection and characterization of an acid-induced folding intermediate of endostatin

Abstract

Endostatin, an important angiogenesis inhibitor, is very acid resistant. We are particularly interested in knowing that whether or not endostatin can form a folding intermediate during acid titration. 1H-NMR, CD spectrum, and ANS binding assay show that endostatin at pH 2.0 contains little tertiary structure, but retains substantial secondary structure with strong ANS binding, and Na 2SO 4 or TFE is found to strongly stabilize endostatin at pH 2.0. All these observations are consistent with the formation of a folding intermediate at pH 2.0. Kinetics studies show that sulfate anions significantly slow down the process for endostatin to reach its equilibrium state at pH 2.0. A regular increase in the amount of α-helix content of the intermediate of endostatin at pH 2.0 is found when the concentration of TFE is increased in the range of 0–40%, suggesting that endostatin has an intrinsic α-helical propensity.