Complexes of the polyamines spermine, spermidine and putrescine with α‐lactalbumins

Abstract

The effects of polyamines on the spectral properties and thermal stability of different α‐lactalbumins were measured. Addition of millimolar concentrations of spermine to the Ca2+‐free (apo) form of bovine or goat α‐lactalbumin resulted in spectral shifts, in both the far‐ and near‐ultraviolet ranges, similar to those induced by Ca2+ binding. Fluorescence emission spectra of tryptophan residues underwent a pronounced blue shift, concomitant with a decrease in quantum yield. Also, in the presence of spermine, thermal stability was increased. In contrast, in the case of human and to some extent also equine α‐lactalbumin, the shifts of the CD and fluorescence spectra and the thermal denaturation curves were in the opposite direction. Direct competition of spermine with Mn2+ could be observed for binding to the Ca2+ site of bovine α‐lactalbumin. As the main binding mode of spermine showed a 1:1 stoichiometry [K1= (2±0.5)×104M−1], we have assumed that binding occurs primarily at this site with one of the distal ammonium groups. In order to accommodate the remaining positively charged ammonium groups of spermine, a binding model was constructed by computer modeling, based on the atomic coordinates of both interacting species. This model makes use of the cluster of negatively charged glutamate residues present in the N‐terminal sequence of bovine α‐lactalbumin, sterically close to the Ca2+ site. The spermine molecule could be nicely fitted in a space delineated by the Ca2+ site, on the one hand, and Glu1, Glu7 and Glu11, on the other. Our model, which has been extended to the shorter polyamines spermidine and putrescine, is able to fully account for the observed stabilization of bovine (and goat) α‐lactalbumins against thermal denaturation, and for the observed CD and fluorescence spectral shifts. It also explains why human (and equine) α‐lactalbumin behave differently. Copyright © 1993, Wiley Blackwell. All rights reserved