BIOCHEMICAL-CHARACTERIZATION OF AIR-FILLED ALBUMIN MICROSPHERES

Heating and sonication of a solution of human serum albumin (HSA) yiel ds air-filled microspheres that can be used as a contrast agent in ult rasound examinations. The microspheres are stabilized by a thin layer of protein surrounding the air bubbles. As long as the microspheres we re intact, the protein was insoluble in aqueous solutions. After disin tegration of the microspheres, the protein could be solubilized in sev eral solutions. The intermolecular interactions in the microsphere pro tein have been elucidated from its solubility properties. The microsph eres were disintegrated by several detergents which also solubilized t he protein. After pressure disintegration of the microspheres, the pro tein was solubilized immediately in urea and guanidinium chloride, and also in phosphate-buffered saline after incubation overnight. These r esults indicate that the protein was mainly stabilized by non-covalent forces. The solubilization in buffer was inhibited by a high salt con centration, suggesting that hydrophobic interactions were involved in stabilizing the microsphere structure. Analysis of the solubilized pro tein by gel filtration showed that the protein contained substantial a mounts of soluble aggregates of HSA. Reduction of the disulphide bonds dissolved these aggregates into monomeric HSA, showing that intermole cular disulphide bonds were also involved in stabilization of the micr ospheres. The solubilized protein also contained less fatty acids than the soluble HSA used for the production of microspheres. These result s show that the microsphere protein has the same characteristics as he at-denatured HSA.