Spermine Oxidase-Substrate Electrostatic Interactions: The Modulation of Enzyme Function by Neighboring Colloidal ɣ-Fe2O3

Abstract

Protein-nanoparticle hybridization can ideally lead to novel biological entities characterized by emerging properties that can sensibly differ from those of the parent components. Herein, the effect of ionic strength on the biological functions of recombinant His-tagged spermine oxidase (i.e., SMOX) was studied for the first time. Moreover, SMOX was integrated into colloidal surface active maghemite nanoparticles (SAMNs) via direct self-assembly, leading to a biologically active nano-enzyme (i.e., SAMN@SMOX). The hybrid was subjected to an in-depth chemical-physical characterization, highlighting the fact that the protein structure was perfectly preserved. The catalytic activity of the nanostructured hybrid (SAMN@SMOX) was assessed by extracting the kinetics parameters using spermine as a substrate and compared to the soluble enzyme as a function of ionic strength. The results revealed that the catalytic function was dominated by electrostatic interactions and that they were drastically modified upon hybridization with colloidal ɣ-Fe₂O₃. The fact that the affinity of SMOX toward spermine was significantly higher for the nanohybrid at low salinity is noteworthy. The present study supports the vision of using protein-nanoparticle conjugation as a means to modulate biological functions.

Keywords: electrostatic interactions; enzyme activity; enzyme nano-immobilization; ionic strength; nanoenzyme; spermine oxidase.

Figure 1

Chemical–physical and morphological characterization of SAMN@SMOX. Comparison of the UV-Vis spectra of naked SAMNs (black line) and SAMN@SMOX: (a) linearized Langmuir isotherm of the SMOX binding onto SAMNs; (b) linear Langmuir model; (c) TEM analyses of the SAMN@SMOX; (d) DLS measurements with the statistical fitting according to the LogNorm function, orange bars for bare SAMNs and blue bars for SAMN@SMOX.

Figure 2

(a) FT-IR spectra of SMOX, naked SAMNs and SAMN@SMOX complex. (b,c) Deconvolution of amide-I band of SMOX and SAMN@SMOX complex, respectively. Experimental amide-I band (black line), Gaussian fitting curve (black dots), β-sheet (red line), random coil (green line), α-helix (blue line), β-turn (light blue line) and β-antiparallel (purple line). (d) Secondary structure contents of native SMOX and of the SAMN@SMOX hybrid according to the deconvolution of the FTIR amide-I band.

Figure 3

(a) Circular dichroism spectra of SMOX (red, 0.075 g L⁻¹), naked SAMNs (black, 0.5 g L⁻¹) and SAMN@SMOX (blue, 0.5 g L⁻¹) in 10 mM HEPPS at pH 8.0. (b) Secondary structure contents of native SMOX and of the SAMN@SMOX hybrid according to circular dichroism.

Figure 4

Kinetic study of the native SMOX and of SAMN@SMOX. Michaelis–Menten curves of native SMOX (a) and of SAMN@SMOX (b); (c) kinetic parameters of SMOX and of SAMN@SMOX.

Figure 5

Spermine chemical structure. Evidencing the four amino groups and the corresponding pKa values used for calculating the electrical charge of the polyamine [36].

Figure 6

(a) Pictorial representation of SMOX–SAMN interaction with the computation model of the protein (represented as alfa, beta and random-coil structures) bridged to the nanoparticle (orange sphere) surface by exemplified His-tag moieties (pink-colored tails). (b) The N-terminus side pointing the solvent. Inset: the exposed catalytic cleft. The surface color scale was obtained using PyMOL, selecting the negative, positive and uncharged amino acids. Blue: ncharged amino acids (Aspartic acid and Glutamic acid), Red: positively charged amino acids (Lysine, Histidine and Arginine) Gray: uncharged amino acids.