Structural comparison of recombinant human macrophage colony stimulating factor beta and a partially reduced derivative using hydrogen deuterium exchange and electrospray ionization mass spectrometry

Hydrogen deuterium exchange, monitored by electrospray ionization mass spec trometry, has been employed to characterize structural features of a deriva tive of recombinant human macrophage colony stimulating factor beta (rhm-CS F beta) in which two of the nine disulfide bridges (Cys157/Cys159-Cys'157/C ys'159) were selectively reduced and alkylated. Removal of these two disulf ide bridges did not affect the biological activity of the protein. Similari ties between CD and fluorescence spectra for rhm-CSF beta and its derivativ e indicate that removing the disulfide bonds did not strongly alter the ove rall three-dimensional structure of rhm-CSF beta. However, differences betw een deuterium exchange data of the intact proteins indicate that more NHs u nderwent fast deuterium exchange in the derivative than in rhm-CSF beta. Re gions located near the disulfide bond removal site were shown to exhibit fa ster deuterium exchange behavior in the derivative than in rhm-CSF beta.