Amyloid Fibrils from Hemoglobin

Abstract

Amyloid fibrils are a class of insoluble protein nanofibers that are formed via the self-assembly of a wide range of peptides and proteins. They are increasingly exploited for a broad range of applications in bionanotechnology, such as biosensing and drug delivery, as nanowires, hydrogels, and thin films. Amyloid fibrils have been prepared from many proteins, but there has been no definitive characterization of amyloid fibrils from hemoglobin to date. Here, nanofiber formation was carried out under denaturing conditions using solutions of apo-hemoglobin extracted from bovine waste blood. A characteristic amyloid fibril morphology was confirmed by transmission electron microscopy (TEM) and atomic force microscopy (AFM), with mean fibril dimensions of approximately 5 nm diameter and up to several microns in length. The thioflavin T assay confirmed the presence of β-sheet structures in apo-hemoglobin fibrils, and X-ray fiber diffraction showed the characteristic amyloid cross-β quaternary structure. Apo-hemoglobin nanofibers demonstrated high stability over a range of temperatures (-20 to 80 °C) and pHs (2-10), and were stable in the presence of organic solvents and trypsin, confirming their potential as nanomaterials with versatile applications. This study conclusively demonstrates the formation of amyloid fibrils from hemoglobin for the first time, and also introduces a cost-effective method for amyloid fibril manufacture using meat industry by-products.

Keywords: amyloid fibrils; hemoglobin; nanofiber; nanofibril.

Figure 1

Formation of amyloid fibrils from hemoglobin. (A) Thioflavin T (ThT) fluorescence intensity overtime of aqueous apo-hemoglobin (AHB) solution extracted from pure hemoglobin after incubating at pH 2.8, 125 mM NaCl, 80 °C for 24 h. Each reading represents an average of triplicate well readings, with the error bars representing the standard error of mean. (B) Far ultraviolet (UV) circular dichroism (CD) spectrum of AHB solution following incubation at pH 2.8, 125 mM NaCl, 80 °C for 0 h (black), 12 h (green), 24 h (orange); (C) transmission electron microscopy (TEM) images of AHB solution following incubation at pH 2.8, 125 mM NaCl, 80 °C for 0 h (a), 6 h (b), 12 h (c), 24 h (d). Scale bars are 200 nm. (D) TEM images of AHB fibrils formed from pure hemoglobin (a) and waste blood hemoglobin (b). All the images were captured at 15,000 magnifications. Scale bars represent 200 nm. (E) Atomic force microscopy (AFM) image of AHB fibrils formed from waste blood hemoglobin. Images were acquired in air using tapping mode at a resonance frequency of 175 kHz and force constant of 1.5 N/m to 15 N/m. Scale bar represents 500 nm.

Figure 2

AFM analysis of hemoglobin amyloid fibrils. (A) A representative AFM micrograph of AHB fibrils formed from waste blood hemoglobin. Images were acquired in air using a tapping mode at a resonance frequency of 175 kHz and force constant of 1.5 N/m to 15 N/m. Scale bar is 50 nm. (B) Representative line traces along individual fibrils. Scale bar is 50 nm. (C) Table detailing the characterization of fibrils in terms of the diameter and peak-to-peak distances; (D) Distribution of peak-to-peak distances of hemoglobin amyloid fibrils (n = 112).

Figure 3

X-ray fiber diffraction pattern of AHB fibrils (A) and schematic diagram of the cross-β quaternary structure of amyloid fibrils (B). X-ray fiber diffraction showed characteristic reflections at 4.71 Å and 10.36 Å, corresponding to the molecular spacing between β-strands and β-sheets, respectively, as shown in (B). The X-ray fiber diffraction images were analyzed using Adxv software (version 1.9.12, Andrew Arvai, The Scripps Research Institute, La Jolla, CA, USA).

Figure 4

Solvent stability of AHB fibrils. (A) ThT fluorescence of AHB fibrils suspended in MeOH, EtOH, MeCH(OH)Me, dimethyl sulfoxide DMSO, and MeCN at 6 and 24 h, following buffer exchange to the appropriate solvent. ThT fluorescence is presented as % control (0 h). Each reading represents an average of triplicate well readings, with the error bars representing the standard error of mean. * Significantly different (two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test) when compared to control, p < 0.05. (B) Representative TEM micrographs of AHB fibrils suspended in (a) fibrillation buffer; MeOH at (b) 6 h, (c) 24 h; EtOH at (d) 6 h, (e) 24 h; MeCH(OH)Me at (f) 6 h, (g) 24 h; DMSO at (h) 6 h, (i) 24 h; MeCN at (j) 6 h, (k) 24 h. The images were captured at 15,000× magnification and the scale bars represent 200 nm.

Figure 5

Temperature stability of AHB fibrils. (A) ThT fluorescence of AHB fibrils resuspended in the fibrillation buffer incubated at −20 °C, 4 °C, 22 °C, 37 °C, and 80 °C. ThT readings were reported post-buffer exchange. ThT fluorescence is presented as % control (0 h). Each reading represents an average of triplicate well readings, with the error bars representing the standard error of mean. * Significantly different (two-way ANOVA followed by Bonferroni post hoc test) when compared to control, p < 0.05. (B) Representative TEM micrographs of AHB fibrils suspended in (a) fibrillation buffer; −20 °C at (b) 6 h, (c) 24 h; 4 °C at (d) 6 h, (e) 24 h; 22 °C at (f) 6 h, (g) 24 h; 37 °C at (h) 6 h, (i) 24 h; 80 °C at (j) 6 h, (k) 24 h. The images were captured at 15,000× magnification and the scale bars represent 200 nm.

Figure 6

pH stability of AHB fibrils. (A) ThT fluorescence of AHB fibrils resuspended at pH 2.0 (30 mM glycine), 4.0 (30 mM sodium acetate), 6.0 (30 mM sodium citrate), 8.0 (30 mM Tris HCl), and 10.0 (60 mM sodium phosphate). ThT readings were reported post-buffer exchange with the original buffer used for fibrillation. ThT fluorescence is presented as % control (0h). Each reading represents an average of triplicate well readings, with the error bars representing the standard error of mean. * Significantly different (two-way ANOVA followed by Bonferroni post hoc test) when compared to control, p < 0.05. (B) Representative TEM micrographs of AHB fibrils suspended in (a) fibrillation buffer; pH 2.0 at (b) 6 h, (c) 24 h; pH 4.0 at (d) 6 h, (e) 24 h; pH 6.0 at (f) 6 h, (g) 24 h; pH 8.0 at (h) 6 h, (i) 24 h; pH 10.0 at (j) 6 h, (k) 24 h. The images were captured at 15,000× magnification and the scale bars represent 200 nm.

Figure 7

Protease resistance of AHB fibrils. (A) ThT fluorescence of AHB fibrils incubated with trypsin in 50 mM Tris HCl, 1 mM CaCl₂ at pH 8.0 and 37 °C. ThT readings were reported post-buffer exchange with the original buffer used for fibrillation. ThT fluorescence is presented as % control (0 h). Each reading represents an average of triplicate well readings, with the error bars representing the standard error of mean. (B) Representative TEM micrographs of AHB fibrils treated with trypsin at (b) 3 h, (c) 6 h, and (d) 24 h time points with respect to (a) the control. The images were captured at 15,000× magnification and the scale bars represent 200 nm.