Assessing the relative stabilities of engineered hemoglobins using electrospray mass spectrometry

An ion trap mass spectrometer equipped with an electrospray source was used to examine the relative thermodynamic stabilities of various hemoglobins w ith respect to both tetramer dissociation and hemin dissociation. The resul ts demonstrated that the stability of hemoglobin molecules can be different iated by the amount of applied collision-induced dissociation (CID) energy necessary to break up the intact tetramer into its constituent globins. The stability of the intact tetramer was affected by single mutations in the b eta-globins. The stabilities of the constituent hologlobins were assessed v ia trap CID of selected ions. The results demonstrated the importance of th e contributions of the hologlobin components to the stability of the intact tetramer. Genetic fusion of two alpha-globins, through the introduction of a single glycine residue between the C-terminus of one alpha-chain and the N-terminus of the second, significantly increased the stability of the hem oglobin pseudo-tetramer. Chemical crosslinking of the beta-globins in addit ion to genetic fusion of alpha-globins further stabilized the hemoglobin mo lecule. A dihemoglobin molecule produced by the genetic fusion of two di-al pha-globins with a flexible linker demonstrated a decreased stability relat ive to the corresponding monohemoglobin. (C) 1999 Academic Press.