Detection of Egg White Lysozyme Oligomers Based on Fluorescence Lifetime of ThT†

doi:10.7503/cjcu20180397

Abstract

Abstract:

Thioflavin T(ThT) fluorescence lifetime was used to detect aggregates states by time-resolved fluorescence. The protein aggregation was made by the egg white lysozyme in vitro. The characteristics of the oligomer and fibril were determined by means of transmission electron microscopy, ThT steady-state fluorescence and the growth kinetic curve of aggregation. The time-correlated single-photon counting technique was used to measure the fluorescence lifetime of ThT incubation with the aggregations. It calculated the fluorescence lifetime by fitting to the double exponential equation. The results of circular dichroism speculated that the structure of different aggregates was different and affected the fluorescence lifetime of ThT. It demonstrated that ThT fluorescence lifetime can distinguish protein monomers, oligomers and fibrils, and monitor the formation of protein oligomers. The results provide a basis for the monitoring of oligomer substances in the proess of sub-sequent pathological protein aggregation.

Key words: Oligomer, Thioflavin T, Time dependent single photon counting, Fluorescence lifetime

CLC Number:

O629.73

TrendMD:

LI Cheng,SONG Jixue,LIU Tingting,LI Yan,LIU Bingnan,WANG Liang,XIAO Shan,LI Lin,GENG Xuhui,WANG Jihui. Detection of Egg White Lysozyme Oligomers Based on Fluorescence Lifetime of ThT^†[J]. Chem. J. Chinese Universities, 2019, 40(1): 90.

Figures/Tables 6

Fig.1 TEM images of egg lysozyme aggregations at different incubation time(A) The egg lysozyme without any treatment; (B—I) the aggregation samples for egg lysozyme at different time points(1, 2, 4, 6, 9, 12, 16 and 20 d), respectively.

Fig.2 Chemical structure of thioflavin-T Arrows showing rotational movement around the central carbon bond.

Fig.3 Steady state fluorescence emission(A) and the kinetic curve of aggregation(B) for the hen lysozyme samples in the experimental condition

Fig.4 Fluorescence emission decay curves of ThT incubation with the samples of the egg lysozyme Incubation time/d: a. 0; b. 2; c. 16.

Table 1 Kinetic analysis of emission decay for ThT in the presence of egg lysozyme aggregation samples for different incubation time*

Sample	t/d	a₁	τ₁/ns	a₂	τ₂/ns	<τ>/ns
Monomer	0	6553.79	0.22	2060.40	0.76	0.50
Oligomer	1	1405.85	0.41	8060.29	1.13	1.09
	2	5130.40	0.99	5134.20	1.13	1.06
Fibril	4	7893.80	0.96	1699.00	2.62	1.57
	8	7656.82	0.92	2111.02	2.42	1.55
	12	7864.63	1.02	1735.95	2.73	1.66
	16	8323.95	1.00	1289.17	2.88	1.58

Fig.5 CD spectra of the egg lysozyme aggregationIncubation time/d: a. 0; b. 2; c. 16.

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