Abstract
Heparin, a biopolymer possessing high negative charge density, is known to accelerate amyloid fibrillation by various proteins. Using hen egg white lysozyme, we studied the effects of heparin on protein aggregation at low pH, raised temperature, and applied ultrasonic irradiation, conditions under which amyloid fibrillation was promoted. Heparin exhibited complex bimodal concentration-dependent effects, either accelerating or inhibiting fibrillation at pH 2.0 and 60 °C. At concentrations lower than 20 μg/ml, heparin accelerated fibrillation through transient formation of hetero-oligomeric aggregates. Between 0.1 and 10 mg/ml, heparin rapidly induced amorphous heteroaggregation with little to no accompanying fibril formation. Above 10 mg/ml, heparin again induced fibrillation after a long lag time preceded by oligomeric aggregate formation. Compared with studies performed using monovalent and divalent anions, the results suggest two distinct mechanisms of heparin-induced fibrillation. At low heparin concentrations, initial hen egg white lysozyme cluster formation and subsequent fibrillation is promoted by counter ion binding and screening of repulsive charges. At high heparin concentrations, fibrillation is caused by a combination of salting out and macromolecular crowding effects probably independent of protein net charge. Both fibrillation mechanisms compete against amorphous aggregation, producing a complex heparin concentration-dependent phase diagram. Moreover, the results suggest an active role for amorphous oligomeric aggregates in triggering fibrillation, whereby breakdown of supersaturation takes place through heterogeneous nucleation of amyloid on amorphous aggregates.
Highlights
Heparin, a biopolymer possessing high negative charge density, is known to accelerate amyloid fibrillation by various proteins
By analyzing the salt-dependent fibrillation and amorphous aggregation of 2-microglobulin (2m),4 the protein responsible for dialysis-related amyloidosis, we proposed that amyloid fibrils and amorphous aggregates correspond to crystals and glasses of solutes, respectively [8, 9]
Through intermittent measurement of thioflavin T (ThT) fluorescence at 485 nm and 90° light scattering at 445 nm, we simultaneously monitored signals reflecting fibrillation and total aggregate formation [28]
Summary
A biopolymer possessing high negative charge density, is known to accelerate amyloid fibrillation by various proteins. Nonseeded amyloid fibrillation typically occurs slowly, via a homogeneous nucleation pathway defined by a long lag time/high free– energy nucleation barrier [10], amorphous aggregation generally occurs rapidly, absent any such nucleation barrier [8]. This relative “speed out of the gates” can mean that the kinetics of amorphous aggregate formation/dissolution may effectively define the apparent kinetic and thermodynamic behavior of the denatured monomer/amyloid phase transition via its participation as a competitive pathway [13,14,15]
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