Miniaturization of asymmetrical flow field-flow fractionation and application to studies on lipoprotein aggregation and fusion

Miniaturization of asymmetrical flow field-flow fractionation and application to studies on lipoprotein aggregation and fusion
Received 10 January 2006. Available online 12 May 2006.
Gebrenegus Yohannesa, Mia Sneckb, Sami J.O. Varjoa, Matti Jussilaa, Susanne K. Wiedmera, Petri T. Kovanenb, Katariina ??rnib and Marja-Liisa Riekkolaa
Analytical Biochemistry
Volume 354, Issue 2 , 15 July 2006
ScienceDirect
Copyright ? 2006 Elsevier Inc. All rights reserved.
aLaboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki, FIN-00014 Helsinki, Finland
bWihuriResearch Institute, FIN-00140 Helsinki, Finland
Abstract
Asymmetrical flow field-flow fractionation (AsFlFFF), a technique that provides direct measurement of particle size and diffusion coefficient, is converted into miniaturized scale. In comparison with conventional AsFlFFF, the separation of proteins in miniaturized AsFlFFF is achieved within shorter time periods, with smaller sample amounts, and with lower mobile phase consumption. Minimization of the overloading and optimization of the separation efficiency are prerequisites to good results. Miniaturized AsFlFFF is applied to the measurement of particle sizes of high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL). The average hydrodynamic diameters at pH 7.4 in 8.5 mM phosphate buffer containing 1 mM EDTA and 150 mM NaCl are 8.6 ? 0.5, 11.2 ? 0.2, 22.1 ? 0.7, and 48.9 ? 7.5 nm for subgroups HDL3, HDL2, LDL, and VLDL, respectively. In addition, the effect of different factors on the aggregation and fusion of LDL particles is studied. LDL particle sizes are unaffected by the addition of up to 300 mM NaCl and by an increase of the carrier solution pH from 3.2 to 7.4, but treatment of LDL with a-chymotrypsin, sphingomyelinase, or copper sulfate leads to the formation of aggregated and fused LDL particles.
Keywords: Miniaturized asymmetrical flow-field flow fractionation; Particle size; Lipoproteins; Aggregation and fusion
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