Removal versus fragmentation of amyloid-forming precursors via membrane filtration.
The presence of even minute amounts of protein aggregates in solution can significantly alter the kinetics of amyloid formation. Removal of such pre-existing aggregates is critical for reproducible analysis of amyloid formation. Here we examine the effects of membrane filtration on insulin fibrillization. We find that filtration of insulin with large pore membranes (≥ 100 nm) generally slows fibril formation relative to unfiltered solutions by removing pre-aggregated protein. Unexpectedly, filtration with small pore membranes (< 100 nm) showed no beneficial effect and, in some cases, accelerated insulin fibril formation. This effect may be due to fragmentation of pre-existing aggregates during filtration through small pore membranes, which can increase the number of amyloid-forming precursors. These findings reveal the complexity of removing protein aggregates via filtration and suggest optimal filtration protocols for conducting fibril formation analysis of insulin and similar amyloidogenic proteins.
Department of Mechanical, Aerospace, and Nuclear Engineering, Rensselaer Polytechnic Institute, JEC 2049, 110 8th St., Troy, New York 12180-3590, USA.
David
D
Posada
Peter M
PM
Tessier
Amir H
AH
Hirsa
D000682:Amyloid / Q000302:isolation & purification* / Q000378:metabolism*; D005374:Filtration / Q000379:methods*; D007328:Insulin / Q000378:metabolism*; D011489:Protein Denaturation; D055503:Protein Multimerization
D000682:Amyloid; D007328:Insulin