Catalytically active bovine serum amine oxidase bound to fluorescent and magnetically drivable nanoparticles

Figures & data

Figure 1 Structures of the fluorescent dyes used as probes for nanoparticle surface properties.

Table 1 Optical characteristics of fluorescent dyes used to test maghemite nanoparticle surface properties

Fluorescent probe	Molar extinction coefficient (m^−1cm^−1)	Excitation wavelength (nm)	Emission wavelength (nm)	Quantum yielda
Fluorescein	65,000	494	520	0.79
Fluorescein isothiocyanate	65,000	494	520	0.79
Rhodamine B	106,000	554	580	0.49
Rhodamine B isothiocyanate	106,000	554	580	0.49
Rhodamine 110	90,000	498	520	0.9

Note:

a The quantum yield was determined in ethanol.³³

Figure 2 FTIR spectra of bare maghemite nanoparticles, rhodamine B isothiocyanate and SAMN–RITC complex. Samples were lyophilized, homogenized with KBr powder, and pelleted by an 8.0 ton hydraulic press. (—) SAMN, (---) RITC; (········) SAMN–RITC. Inset: Comparison of FTIR spectra of RITC and SAMN–RITC complex in the region 1365–1000 cm⁻¹. (—), RITC; (········), SAMN–RITC.

Abbreviations: SAMN, surface-active maghemite nanoparticles; RITC, rhodamine B isothiocyanate; KBr, potassium bromide; FTIR, Fourier-transform infrared.

Figure 3 RITC binding to maghemite nanoparticle surface as a function of pH.

Note: Experiments were carried out in 50 mM tetramethylammonium perchlorate in the presence of 10 μM RITC and 100 mg L⁻¹ SAMN, measuring the decrease of solution absorbance at 554 nm.

Abbreviations: SAMN, surface-active maghemite nanoparticles; RITC, rhodamine B isothiocyanate.

Table 2 Binding of RITC derivatives to bare maghemite nanoparticles

RITC-derivative	Number of bound μmol RITC per g nanoparticle
	Experimental conditions
	pH 7.0	pH 10.0
None	2.5	1
–NH–CH2–NH2	2	5
–NH–CH2–CH2–COOH	11	7
–NH–CH2–CH2–OH	17	12

Note: The binding process was carried out in 50 mM tetramethylammonium perchlorate in the presence of 100 mg/L maghemite nanoparticles.

Figure 4 TEM image of maghemite nanoparticles derivatized with RITC and with immobilized BSAO (SAMN–RITC–BSAO).

Abbreviations: BSAO, bovine serum copper-containing amine oxidase; SAMN, surface-active maghemite nanoparticles; RITC, rhodamine B isothiocyanate; TEM, transmission electron microscopy.

Table 3 The ten most abundant 10 common peptides obtained after trypsin digestion of native and SAMN bound BSAO

Peptide number	Score	Bound BSAO	Native BSAO	Bound/free
1. YLYLASK	36	747655	132977
2. ALDPADWTVQK	67	7693036	10761424
3. GGPYLHPVGLELLVDHK	55	11988500	17391553
4. YMDSGFGMGYFATPLIR	101	9442405	4823422
5. ALDPADWTVQKVFFQGR	44	1239335	539496
6. KQLETEEQAAFPLGGASPR	87	13640798	9686214
7. SQVPPGPTPPLQFHPQGPR	63	113110365	15533939
8. IQTVSFAGGPMPQNSPMER	132	44140030	11272321
9. LAYEISLQEAGAVYGGNTPAAMLTR	169	3758314	1981715
10. HHSDFLSHYFGGVAQTVLVFR	41	4856416	1648758
Sum		210616854	73771819	2.85

Figure 5 Amino acid sequence coverage of native and bound BSAO.

Notes: Bold letters indicate amino acid total sequence coverage (42%) obtained after trypsin digestion and analysis by MS/MS mass spectrometry of nanoparticle-bound BSAO. Underscore, bold letters indicate the amino acid sequences that were used for relative quantification of nanoparticle-bound BSAO.

Abbreviations: BSAO, bovine serum copper-containing amine oxidase; MS/MS, tandem mass spectroscopy.

Table 4 Kinetic parameters of native and SAMN-bound BSAO

Enzyme form	Kinetic parameters
	KM (μM)	kc (min^−1)	kc/KM (μM^−1 min^−1)
Native BSAO	7.4	46.8	6.3
SAMN–RITC–BSAO	14.0	14.4	1.0

Abbreviations: BSAO, bovine serum copper-containing amine oxidase; SAMN, surface-active maghemite nanoparticles; RITC, rhodamine B isothiocyanate.

ValeurBMolecular Fluorescence: Principles and ApplicationsWeinheim GermanyWiley-VHC2002

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