SUPPORTING INFORMATION
Enzyme activation by Alternating Magnetic Field: importance
of the bioconjugation methodology
Ilaria Armeniaa, María Valeria Grazú Bonaviab,c*, Laura de Matteisd, Pavlo Ivanchenkoe, Gianmario Martrae, Rosalba Gornatia,f, Jesus M de la Fuenteb,c, and Giovanni Bernardinia,f*
a Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell’Insubria, Via J.H. Dunant 3, 21100 Varese, Italy
b CIBER BBN, C Mariano Esquillor S-N, Zaragoza 50018, Spain
c Aragón Materials Science Institute (ICMA), Universidad de Zaragoza, CSIC, Campus Río Ebro, Edificio I+D, Mariano Esquillor Gómez, 50018 Zaragoza, Spain
d University of Zaragoza (UNIZAR), C Mariano Esquillor S-N, Zaragoza 50018, Spain
e Dipartimento di Chimica & Centro Interdipartimentale "Nanostructured Interfaces and Surfaces" – NIS, Università degli Studi di Torino, Via P. Giuria 7, 10125 Torino, Italy
f The Protein Factory, Politecnico di Milano, ICRM CNR Milano, and Università degli Studi dell'Insubria, Via Mancinelli 7, 20131 Milano, Italy
Stability of NP-APTES (S1)
The conjugation strategies herein proposed require specific reaction conditions such as pH and ionic strength, thus it was necessary to investigate the stability of NP-APTES. Zeta-potential measurements were done in order to characterize this aspect.
Measurements were performed using 10 µg/mL of NP-APTES at 25 °C using 90 Plus Particle Size Analyzer in a range of 3 to 12 pH (Figure S1, panel A) and in the range to 0.01-100 mM NaCl (Figure S1 panel B).
Figure S1. Stability of NP-APTES in different pH medium (A) and NaCl concentration (B). Results a media of three independent measurements.
Results indicated a good stability of the particles in the 3-8 pH range, with a zeta-potential ranging between 30 to 40 mV, and in the range of 0.01 to 10 mM of NaCl.
1
DTT reaction (S2)
The second strategy used to conjugate LASPO to the NP-APTES uses the thiol group from cysteine residue present on its surface.
Before the conjugation, the NP-APTES were transformed to thiol-reactive groups with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (Sulfo-SMCC). To evaluate the correct functionalization of the NP-APTES, the residual amino groups present on the NP surface were quantified using Orange II assay: the number of amino-group per mg of NPs decreased from 15 to 11 nmol. Results indicate a reduction of the amino groups of the 27 % suggesting the presence of thiol-reactive groups.
The conjugation reaction was carried on for 30 minutes suspending 1 mg of NP-APTES in 5 mM sodium pyrophosphate buffer pH 5, adding 4 µM od Sulfo-SMCC in a final volume of 1 mL. After several washing, 1 mg of reduced protein was added to the thiol-activated NPs and the reaction was maintained under mechanical stirring for 30 minutes at room temperature.
To demonstrate that the binding of the enzyme was through the cysteine present on the enzyme molecule and not through unspecific binding, the NP-LASPO2 was incubated with DTT, in order to reduce the disulfide bond. Table S2 underline that the 100 % of the molecules are bound to the NPs through the cysteine, indeed, no activity is detected in the NP-LASPO after the DTT addition.
Table S2: activity of NP-LASPO2 before and after DTT addition.
Relative Activity
(%) Uenzyme/mgNPs (%)
NP-LASPO 2 87 0,074
POST-DTT 0,1 0,0003
Surface charges distribution (S3)
In the conjugation strategies, the possibility of ionic interaction of the enzyme with the NP surface must be considered. The side of the protein with the higher charge residues is indeed the one that will faster react with the NP-APTES via a rapid ionic adsorption. A slower site-directed covalent binding reaction occurs via the BS3 activated amino-groups. Given that, we studied the enzyme charge distribution.
m
Figure S3. 3D structure of LASPO showing the positive and negative charge surface distribution. The enzyme structure was taken from the Protein Data Bank (PDB) entry 2E5v and visualized using PyMol v2.1.Negative and positive charges are displayed in red and blue, respectively. (a) graphic representation of interaction plane of the enzyme. (b) Number of charged residues per plane interaction.
Results underline that the region of the active site (front side) is the one with the highest negative charged residues (Figure S3 panel B), while the region with the lowest negative charge is on the bottom part of the LASPO molecule. Interestingly, the region with the active site is also the one with the highest positive charges.
Further analysis were carried on to confirm the possibility of ionic interaction. We performed an analysis of the surface potential of the enzyme using the Adaptive Poisson-Boltzmann Solver (APBS). This software is designed to analyze the solvation properties of small molecules as well as macro-molecules, such as proteins.
The surface potential for was calculated for the different pH used in the conjugation conditions. Figure S4 shows the surface potential (a) and the electrostatic potential isocontours (b), positive and negative charged surface are indicated in blue and red, respectively.
3
pH 5
pH 7
pH 8.2
pH 8.5
b)
a)
Figure S4. Analysis of the surface potential of the enzyme. The PDB entry 2E5V was selected for this 3D representation. APBS was used to calculate the surface potential and the analysis results were visualized using Pymol v2.1. (a) ±10 kT/e electrostatic potential of LASPO in PyMOL plotted on the solvent-accessible surface. (b) ±0.6 kT/e electrostatic potential isocontours of LASPO in PyMOL. The enzyme molecules are oriented to visualized the surface potential in the nearby of the residue used for the conjugation.
PEG conjugation results (S4).
The conjugation of LASPO was carried on through the direct covalent binding of the enzme via the primary amine group of the NP-APTES at its N-terminus. However, at the pH value used (pH 8.2) the enzyme has net negative charge (pI 6.75) while BS3-activated NPs have a net positive one, as demonstrating by ζ potential measurements (+27,95 mV). Thus, we cannot ensure the direct covalent binding. Indeed, a slight ionic adsorption was observed when NP-APTES without activation, i.e. 10 µg of the enzyme is bound to 1 mg of NPs.
To avoid unspecific interactions, we carried out a partial modification of NP-APTES surface with an aminated-750 Da polyethylene glycol (PEG) after the activation with BS3. Results suggest an higher number of molecules per mg of NPs bound. Thus, we this strategy didn’t obtained a specific binding of the enzyme.
Table S4. NP-LASPO5 reaction parameters.
µgenzyme/mgNPs Relative Activity (%) Active enzyme molecules/NP
NP-LASPO5 40 70 21
CD-UV spectroscopy for studying the structure of enzymes conjugated to NPs (S5).
Figure S5. Absorbance (panel A) and related circular dichroism spectra (panel B), in the 185-350 nm of aqueous solutions/suspensions (in D2O) of a) AMY, b) APTES, c) AMY1, d) AMY2 and e)
NP-AMY3. In all cases the AMY concentration was of ca. 0.07 mg·mL-1. Both types of spectra are the result of
measurements carried out in the transmission mode.
The absorbance spectra of NP-APTES, NP-AMY1, NP-AMY2 and NP-AMY3 suspended in D2O (curves b-e, in the orders), show a quite flat profile, due to the scattering of impinging light by suspended NPs, with traces of broad components likely due to light absorption by NPs.
IR spectroscopy for assessment of H2O/D2O (S6).
Figure S6. IR-ATR spectra of α-amylase solutions (in water – blue line, in D2O – red line) and of pure D2O
(black dotted line).
The comparison between the two spectra shown in the figure (with the assignment of the various signals) witnesses for the complete substitution of H2O with D2O by the dissolution-filtration by centrifugation procedure adopted. Moreover, the disappearance of set of bands in the 1500-900 cm-1 range indicates that sucrose molecules were removed during the process, also.
Infrared spectra (IR) (S7).
Figure S7. IR-ATR spectra of samples suspended in D2O of: NP-APTES (grey lines), NP-AMY1 (blue line),
NP-AMY2 (green line) and NP-AMY3 (purple line). Insets are zoomed view of the range where amide I and amide II (in D2O) bands fall.
