Biophysical Studies of Apolipoprotein A-IM and a Partitioning study

University dissertation from Malin Suurkuusk, Pharmacia&Upjohn, SP 19-5, SE-112 87 Stockholm, Sweden

Abstract: The major part of this thesis deals with the similarities and differences in some biophysical properties of apolipoprotein A-I (apo A-I) and the molecular variant apolipoprotein A-IMilano (apo A-IM), and the influence on lipid binding. A model compound partitioning study has also been performed. Methods that have been utilised are CD spectroscopy, calorimetry both isothermal and temperature scanning, and native gel electrophoresis. Apo A-I is the major protein component in high density lipoproteins (HDL), which are found to be inversely correlated with the occurrence of atherosclerosis. The variant protein, apo A-IM, forms a disulfide linked homodimer that seems to possess an improved anti-atherogenic effect. Apo A-IM was found to have a different thermal and guanidine hydrochloride (guHCl)-induced denaturation behavior in its dimeric form, as compared to the monomer form and to apo A-I. The thermal denaturation process of apo A-IM proceeds via a transition into an intermediate state that is distinct from the unfolded state. This intermediate state has been assigned to be a molten globular state. This state is stabilized by an increase in ionic strength and exists at 37 °C. Unfolding via a stable intermediate state was also observed during guHCl unfolding. Apo A-I and monomeric apo A-IM also seem to melt via an intermediate state, but with the difference that these thermal transitions are not well separated, and occur at higher temperatures. Conclusions to be drawn are that the introduction of a disulfide bridge in apo A-IM favours the molten globular state over the native state at near physiological temperatures. The existence of a molten globular state of apo A-IM may influence the functional properties of the protein compared to apo A-I. Upon lipid binding these proteins form a variety of different sized lipid-protein complexes. At 25 °C, dimeric apo A-IM and apo A-I give similar heterogeneous mixtures of HDL-like complexes (rHDL) when incubated with dimyristoylphosphatidylcholine (DMPC). However at 40 °C, apo A-IM was shown to form smaller, more homogeneous complexes than apo A-I. This observation suggests that the HDL particle formation depends not only on the thermal behaviour of the lipid but also of the protein. In addition to the protein studies, the partitioning of 1-hexanol into DMPC vesicles was studied and was found to be more favourable than partitioning into bulk hydrocarbon. This suggests that the use of a membrane model might be more appropriate to evaluate the partitioning properties of a drug. However, there is a need for defining a standard membrane model where vesicle size and lamellarity in addition to the type of lipid, lipid phase and equilibration time is carefully controlled, since these factors affect the partitioning behaviour.

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