Miniaturization of asymmetrical flow field-flow fractionation and application to studies on lipoprotein aggregation and fusion.
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 8.5mM phosphate buffer containing 1mM 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 alpha-chymotrypsin, sphingomyelinase, or copper sulfate leads to the formation of aggregated and fused LDL particles.
Yohannes
Gebrenegus
G
Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki, FIN-00014 Helsinki, Finland.
Sneck
Mia
M
Varjo
Sami J O
SJ
Jussila
Matti
M
Wiedmer
Susanne K
SK
Kovanen
Petri T
PT
Oörni
Katariina
K
Riekkola
Marja-Liisa
ML
D002918:Chymotrypsin; D019327:Copper Sulfate; D031521:Fractionation, Field Flow / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D008074:Lipoproteins / Q000737:chemistry* / Q000302:isolation & purification*; D008075:Lipoproteins, HDL / Q000737:chemistry / Q000302:isolation & purification; D053439:Lipoproteins, HDL2; D053440:Lipoproteins, HDL3; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000302:isolation & purification; D008079:Lipoproteins, VLDL / Q000737:chemistry / Q000302:isolation & purification; D008904:Miniaturization / Q000295:instrumentation / Q000379:methods; D046912:Multiprotein Complexes; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010316:Particle Size; D013108:Sphingomyelin Phosphodiesterase
D008074:Lipoproteins; D008075:Lipoproteins, HDL; D053439:Lipoproteins, HDL2; D053440:Lipoproteins, HDL3; D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL; D046912:Multiprotein Complexes; D013108:Sphingomyelin Phosphodiesterase; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D019327:Copper Sulfate