Biophysical Properties Of Recombinant Human Endostatin

Endostatin, a 20 kD C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis and tumor growth without acquired drug resistance in animal models. Most of the earlier work on endostatin was focused on therapeutical applications and clinical trials and little has been done to reveal its physical properties and the folding process.In present dissertation, 1H-NMR, Trp fluorescence, and circular dichroism were used to monitor the acid titration behavior of endostatin to test whether or not it forms a folding intermediate. Endostatin is a very acid-resistant protein and only acid can not unfold it completely. Endostatin at pH 2.0 contains little tertiary structure but retains substantial secondary structure with strong ANS binding, agreeing with a folding intermediate. TFE and sodium sulfate strongly stabilize endostatin at pH 2.0, suggesting that endostatin at such acid condition is in a folding intermediate.Stability measurement of endostatin against urea or GdmCl reveals that endostatin at pH 5.0 and 7.4 have similar stability, however, it is only marginal stable at pH 3.5 and can be easily disturbed by low concentration of acid, TFE or Na2SO4. At pH 2.0 or 1.6 the raw data can not be normalized because the base line of the native state of endostatin in the unfolding curve is absent. Stability measurement also reveals that high temperature makes endostatin unstable and the unfolding of endostatin induced by heat is reversible. 5% TFE can destroy the native structure of endostatin and the increase of α-helix induced by TFE is linearly with the concentration of TFE. Salts have the two edges of effect on the acid titration of endostatin: destabilizing endostatin at relative high pH and stabilizing endostatin at lower pH. At neutral pH, sodium sulfate makes endostatin more stable either against denaturants or heat while sodium chloride has no effect on the stability of endostatin, suggesting that the stabilizing of sodium sulfate on the endostatin is specific, independent of the kind of salt. The binding of rate of endostatin and heparin is very slow at normal conditions and a little unfolding of endostatin will make the binding faster. The binding of endostatin and heparin will lead the native structure of endostatin to some extent relax caused by the big molecule of heparin. Such binding of endostatin to heparin can be destroyed by high concentration of urea.