Disulfide bridges and configuration of human serum albumin. Relationships between viscosity, optical rotatory dispersion and configuration

Abstract

The effect of oxidative and reductive cleavage of the disulfide bridges of serum albumin on the viscosity, specific rotation, and rotatory dispersion was investigated. Human serum albumin was oxidized with performic acid a t low temperature. Our data show that oxidized human serum albumin (OHA) prepared in this manner represents an unfolded polyelectrolyte. It could be shown that no peptide bonds were hydrolyzed during the oxidation, and that approximately 90% of all -S-S-bonds were oxidized to -SO,H. From viscosity data (in 4M guanidine thiocyanate) it can be concluded that the SS bonds are important for partial folding, and that some of the -SS-bridges are linking distant parts of the polypeptide chain. This was ascertained also by cleaving the SS bridges by reduction and carboxymethylation. The reduced and carboxymethylated albumin exhibited similar optical rotation and viscosity behavior as the OHA. It was also established that the levorotation does not depend solely on the folding but also on the presence or absence of the SS bonds, i.e. cystine content. Finally, i t was concluded that in the instance of human serum albumin the dispersion constant 1 , is a safer criterion for the estimation of configurational changes than the specific rotation. ZUSAMMENFASSUNG:Die Disulfidbriicken in den Makromolekiilen des Serumalbumins wurden durch oxidative oder reduktive Spaltung gesprengt, und die Viskositat, spezifische Rotation und Rotationsdispersion der Losungen untersucht. Die oxydative Spaltung wurde mit Perameisensaure bei niedriger Temperatur vorgenommen. Durch Aminosaureanalysen und Bestimmung der End-Aminosauren wurde gezeigt, daR 90 yo der Disulfidbriicken -S-Szu -SO,H oxydiert worden waren, und daR die Hauptkette dadurch nicht hydrolytisch abgebaut wurde. Die Losungen des oxydierten Albumins waren hochviskos, die Viskositat erniedrigte sich stark durch Salzzusatz. Vergleichende Viskositatsmessungen in 4 m Guanidinrodanid zeigten, daB die oxydierten Makromolekule ein groaeres Volumen beanspruchen als die Makromolekiile des nicht oxydierten Albumins in demselben Losungsmittel;