We also evaluated whether the fibrillation kinetics was related to heparin concentration to understand better the effect of heparin on the lag phase. The addition of increasing concentrations of heparin led to a reduction of the lag and a progressive increase in the magnitude of the Enzalutamide fluorescence plateau value . The dependence of the transition midpoint on the heparin/apomyoglobin molar ratio indicates that the number of W7FW14F apomyoglobin molecules per heparin molecule varies from 40 to 5 on increasing polyanion concentration. In summary, incubation of W7FW14F apomyoglobin with heparin resulted in a substantial acceleration of the aggregation and fibrillation processes, and the magnitude of the acceleration effect was related to GAG concentration. To probe further the mechanism by which heparin induced amyloid formation, we investigated its effect on the partially folded soluble conformation that W7FW14F apomyoglobin adopts near pH 4.0 . Under this condition, the addition of heparin caused aggregation with a kinetics rate faster than that observed at pH 7.0, i.e., 0.09660.015 min21 vs. 0.05660.006 min21. The CD spectra recorded during the first 6 h of the process revealed the presence of two distinct successive conformational populations. The first appeared soon after the addition of heparin , whereas the second appeared much later . It is Palbociclib interesting to note that the CD spectra obtained soon after the addition of heparin at pH 4.0 are similar, if not identical, to those recorded at neutral pH in the presence of heparin, whereas the spectra obtained at longer times are practically identical with that of mature fibrils . FTIR analysis revealed that the amide I�� maximum of both forms is close to 1625 cm21 , which supports the concept that the aggregated protein is essentially b-structured. Moreover, the heparin-induced aggregates formed at pH 4.0 were also able to bind ThT . The intensity of ThT fluorescence measured soon after heparin addition was three-fold higher than that detected at pH 7.0 in the absence of heparin, which indicates that the aggregates have a prevalently cross-b structure, consistent with FTIR analysis. The initial instantaneous appearance of ThT fluorescence was followed by further increases and reached maximum value 5 days after the addition of heparin.