Interaction of Neuroglobin Mutant H64V with Azide Anion†

doi:10.7503/cjcu20170316

Abstract

Abstract:

Mutating the histidine 64 to valine, the gene of Neuroglobin(Ngb) mutant H64V was constructed and its protein was expressed and purified. This mutation changed the coordination geometry of neuroglobin from hexa- to penta-coordinated. The interaction of neuroglobin mutant H64V with azide anion() was investigated by spectroscopy. UV-Vis absorbance spectra showed that the binding of mutant H64V to induced the change of the Soret-band absorption peak from 406 nm to 418 nm. Fluorescence spectra indicated that the fluorescence of mutant H64V was quenched by through static quenching mechanism. The binding equilibrium dissociation constant and thermodynamic parameters between mutant H64V and were calculated. In addition, the effects of different anions on their binding were studied, the results showed that the anions reduced the binding ability between mutant H64V and azide anion.

Key words: Neuroglobin, Mutant H64V, Azide anion, Spectroscopy

CLC Number:

TrendMD:

GAO Chaohui, LI Haili, WANG Lei, QIU Zhanglei, MU Jianshuai, LI Lianzhi. Interaction of Neuroglobin Mutant H64V with Azide Anion^†[J]. Chem. J. Chinese Universities, 2018, 39(3): 476.

Figures/Tables 6

Fig.1 UV-Vis absorbance spectra of neuroglobin(Ngb)(A) and its mutant H64V(B) before and after reaction with c(Ngb)=5.0 μmol/L, c(NaN3)=3.0 mmol/L.

Fig.2 UV-Vis absorbance spectra of neuroglobin mutant H64V with different concentrations of at 298 K(A) and 303 K(B)(A) c(Ngb)=2.0 μmol/L; c(NaN3)=0, 25.0, 50.0, 75.0, 100.0, 200.0, 300.0, 400.0, 500.0, 600.0, 700.0, 950.0 and 1200.0 μmol/L; (B) c(Ngb)=3.0 μmol/L; c(NaN3)=0, 75.0, 100.0, 200.0, 300.0, 400.0, 500.0, 600.0, 850.0, 1100.0, 1350.0 and 1600.0 μmol/L. The arrows inclicate the increasing of the concentration of NaN3.

Fig.3 Plot of Y-1 versus x-1 of neuroglobin mutant H64V with at 298 K(A) and 303 K(B)

Fig.4 Fluorescence spectra of neuroglobin mutant H64V with different concentrations of c(Ngb)=2.0 μmol/L, c(NaN3)=0, 16.7, 33.3, 50.0, 66.7, 83.3, 100.0 μmol/L. The arrow indicates the increasing of the concenttation of NaN3.

Fig.5 Stern-Volmer curve for fluorescence quenching of neuroglobin mutant H64V with diffe-rent concentrations of N3-

Table 1 Equilibrium dissociation constant(Kd) of neuroglobin mutant H64V with in the presence of different anions at 800 mmol/L determined by UV-Vis absorbance spectra

Anion	No anion	Cl	S	Cl^-	Br^-	N
10⁴K_d/(mol·L^-1)	1.805	3.286	3.143	2.086	2.524	2.160

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