J. Phys. Chem. B 2009, 113, 14189–14195
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Structure and Function of Hemoglobin Confined Inside Silica Nanotubes Shobhna Kapoor, Soumit S. Mandal, and Aninda J. Bhattacharyya* Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560012, India ReceiVed: April 9, 2009; ReVised Manuscript ReceiVed: May 22, 2009
Investigations on the structure and function of hemoglobin (Hb) confined inside sol-gel template synthesized silica nanotubes (SNTs) have been discussed here. Immobilization of hemoglobin inside SNTs resulted in the enhancement of direct electron transfer during an electrochemical reaction. Extent of influence of nanoconfinement on protein activity is further probed via ligand binding and thermal stability studies. Electrochemical investigations show reversible binding of n-donor liquid ligands, such as pyridine and its derivatives, and predictive variation in their redox potentials suggests an absence of any adverse effect on the structure and function of Hb confined inside nanometer-sized channels of SNTs. Immobilization also resulted in enhanced thermal stability of Hb. The melting or denaturation temperature of Hb immobilized inside SNTs increase by approximately 4 °C as compared with that of free Hb in solution. 1. Introduction Nanostructured inorganic materials1-3 are promising for diverse biotechnological applications,4-6 such as controlled drug delivery,7 biosensing,8-12 and human implants.13 The rich structural diversity observed in inorganic nanomaterials is rarely observed in other class of materials, namely, organic materials. Versatility in function of inorganic nanomaterials is attributed to several factors, important among them being their higher chemical and structural stability under physiological conditions as compared to the organic counterparts, such as polymers. Additionally, existence of several well-established procedures for synthesis allows easier processing of materials in various spatial dimensions with optimized architectures tailored to perform specific tasks. Despite these advantages, generation of biodegradable inorganic materials in an aqueous environment is a formidable task. Polymers or, in general, organic-based systems14,15 offer greater opportunities on issues of biocompatibility and biodegradability. However, in the case of organic systems, too, the success rates have been rather low due to correlation of several nontrivial issues, such as toxicity associated with the process of biodegradation. Despite being in the nascent stages, inorganic nanostructured systems offer an attractive alternative to organic systems and even might aid in the development of newer systems, such as hybrids consisting of both organic and inorganic components.16 Inorganic porous materials with pore sizes ranging from e2 to >50 nm have received considerable attention as hosts for several biotechnological applications. Porous materials have been successfully employed for the immobilization of a variety of guest biomolecules of various sizes ranging from macromolecules, such as proteins, to smaller molecules, such as vitamins and drugs.17-19 Nanostructured porous materials such as mesoporous oxide (e.g., MCM or SBA silicas)20,21 nanotubes21,22 have already been demonstrated as promising hosts for drug delivery. The pore characteristics of these hosts (such as size, surface area, and surface chemical moieties) determine the degree of drug loading and release kinetics. It has also been shown that confinement of the biological molecules such as * Corresponding author. Fax: +91 80 23601310. E-mail: aninda_jb@ sscu.iisc.ernet.in.
proteins inside the pore space of the inorganic host18,23,24 does not have any adverse affect on the biomolecular structure and function. In fact, for certain situations, confinement resulted in enhancement of molecular activity compared to that in the free state.21,25 Globin proteins26-32 such as hemoglobin, myoglobin, neuroglobin, and cytoglobin bind small ligand molecules such as dioxygen and carbon monoxide reversibly at the central iron site of a heme prosthetic group embedded in an R-helical globin fold. Ligand binding to globin proteins in solution has been used successfully as model systems to probe protein dynamics, structure, and function. It has been shown that the environment around the heme has a strong influence on the binding kinetics of gaseous ligands to Hb. In the present work, we study systematically the influence of confinement on the ligand binding activity of hemoglobin. We discuss the reversible binding of liquid-based ligands of pyridine and its derivatives to hemoglobin confined inside silica nanotubes synthesized via the sol-gel template method.33 The consequence of confinement inside nanometer-sized channels on protein structure is further discussed via thermal stability studies. 2. Experimental: Methods and Materials Preparation of Silica Nanotubes (SNTs). Silica nanotubes were prepared by the procedure described in ref 33. Briefly, commercially available anodisc membranes (Whatman Anodisc) with pore diameter of 200 ( 50 nm were immersed for 2-3 min in a solution of 10 mol % silicon tetrachloride (SiCl4, Sigma) in carbon tetrachloride (CCl4, Merck). Following immersion, the membranes were first quickly washed with CCl4 for removal of remnant reagents from the faces and were again placed in a fresh portion of CCl4 for 30 min to remove unbound SiCl4 from the pores. After this washing procedure, the membranes were soaked in a mixture of CCl4 and methanol (1:1 v/v) for 2 min; then in ethanol for 5 min to displace CCl4; and, finally, dried under argon atmosphere. The dried membranes were immersed in deionized water for 5 min, washed with methanol for 2 min, and then redried under argon stream. The membranes were then dissolved by keeping them immersed overnight in 70% H2SO4 aqueous solution at 70 °C. After removal of acid by centrifugation, water (Millipore) was added
10.1021/jp9032707 CCC: $40.75 2009 American Chemical Society Published on Web 07/28/2009
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to the white sediment, and the process was repeated at least 9-10 times for complete elimination of the acid. Additionally, each time, the decanted liquid was also centrifuged to ensure extraction of all nanomaterials. The sediment from the centrifugation process was dried in an air oven at 55-70 °C. Preparation of SNT and Hb Composite. Hb (human Hb, MW 65 000) in the amount of 2.5 mg was mixed with a 10 mg/mL solution of SNT in PBS (0.1 M, pH 7.0) and incubated at 4 °C for 24 h. After this, the solution was centrifuged for 1 min (5000-6000 rpm) to remove the supernatant. Concentration of Hb in silica nanotubes estimated using UV-vis spectroscopy was 1.4 mg mL-1. Hereafter, SNTs impregnated with Hb has been abbreviated as SNT-Hb. Preparation of Modified Electrode. Prior to modification, a glassy carbon electrode (GCE, diameter: 3 mm) was rinsed with water and ultrasonicated in a water bath. Ten microliters of SNT-Hb and Hb were dropped on the shiny surface of the GCE and dried for 3-4 h in air at room temperature. The modified electrodes are abbreviated as SNT-Hb/GCE and Hb/ GCE and were dipped in the PBS buffer solution (0.1 M, pH 7.0) for various measurements. Electrochemical Measurements. The cyclic voltammetric measurements were performed on CHI608 (CH Instruments). The working, counter, and reference electrodes were SNT-Hb/ GCE or Hb/GCE and a saturated calomel and Pt wire, respectively. The working solution (5 mL of 0.1 M PBS, pH 7.0) was deoxygenated for 30 min prior to the start of the measurement, and a nitrogen atmosphere was maintained throughout the experiment. Fourier Transform Infrared Spectroscopy (FTIR) and Ultraviolet (UV-vis) Spectroscopy. For room temperature FTIR (Perkin-Elmer FTIR Spectrometer Spectrum 1000) measurements, 100 µL of both Hb (2.5 mg/mL) and SNT-Hb composite solutions were dropped on a clean glass plate and dried at room temperature for 3-4 h. After drying, they were scratched and the requisite amount of sample was mixed with pure spectroscopic grade potassium bromide (KBr) and was cast into a pellet of diameter 1.3 cm and thickness ∼0.1 cm. A UVvis absorption spectrum (Perkin-Elmer, Lambda 35 UV spectrometer) was also obtained for studying the native structure of Hb. Electron Microscopy. Transmission electron microscope (TEM) images were observed and recorded on a FEI Tecnai F30 with an acceleration voltage of 200 kV. Two microliters of sample solution was dropped on a Cu grid with a carbonreinforced plastic film. Scanning electron microscopy was performed using FEI SIRION in a voltage range of 200-300 kV. Circular Dichroism. Twenty microliters of Hb and SNTHb solution were mixed with 380 µL of the fresh buffer. A 200 µL portion of this solution was taken in a 1 mm cuvette for recording the CD spectrum (JASCO J-715). A CD spectrum at different temperatures ranging from 25 to 85 °C was recorded at 1 °C per min intervals. 3. Results and Discussion 3.1. Evidence of Impregnation of SNT with Hb from Transmission and Scanning Electron Microscopy. Figure 1a shows the general morphology of silica nanotubes characterized by transmission electron microscope. The nanotubes have a diameter of 250 nm and length of 10 µm approximately. The detailed morphology has been described elsewhere.21 The sol-gel template synthesized SNTs display several heterogeneities, such as nonuniformity in tube diameter, the presence
Kapoor et al. of internal junctions (such as Y-junctions), or an opening only at one end. These structural heterogeneities of SNTs arise essentially from the Anodisc templates employed in their synthesis. We have recently shown21 that these morphological features of SNTs significantly affect in vitro release of ibuprofen from the SNTs. At this juncture, we are unable to ascertain the extent of influence of the structural heterogeneities on confined hemoglobin. However, on the basis of the experimental results discussed in the subsequent sections, we propose that the structural heterogeneities do not have any adverse effect on the native structure and function of the proteins. Confinement of hemoglobin inside SNT leads to an enhancement of electroactivity as well as greater thermal stability. The transmission electron micrographs shown in Figure 1b, d, and e exhibit successful intercalation of hemoglobin inside the SNTs. No special sample treatment was done for transmission electron microscopy. Impregnation of the silica nanotubes with hemoglobin was judged by comparing the contrast changes between several transmission electron micrographs of unloaded and loaded SNTs. This approach was successfully employed in a previous work involving impregnation of silica nanotubes with ibuprofen (C13H18O2) for controlled drug delivery.21 Impregnation of SNTs by Hb is also evident via the scanning electron micrograph shown in Figure 1c. 3.2. Spectroscopic Evidence of Impregnation of SNT with Hb. UltraWiolet-Visible (UV-Wis) Spectroscopy. The shape and position of a Soret absorption band associated with the porphyrin ring provides insight into the environmental influence on the native structural configuration of Hb, especially around the heme site. For free Hb (Figure 2), the Soret band is well-resolved and is observed at 406 nm. For Hb immobilized inside SNT, the Soret band is considerably broadened and is slightly redshifted by 3 to 409 nm. A shift in the Soret band due to confinement inside nanostructured materials has been reported in ref 34. We attribute both the broadening and red shift to the interaction of Hb with SNT as well as to enhanced molecular interaction.35 Neither interfacial nor intermolecular interactions result in any form of structural distortion. Confinement does result in a decrease in the total number of structural configurations as compared to that in solution; that is, the free state. Confinement however, does not eliminate the favorable protein conformations requisite for primary protein activities, such as ligand binding. We propose that confinement may also reduce protein dynamics, leading to retention of the favorable conformations for longer periods of time. Our proposition of the absence of any structural distortion will be further demonstrated in light of the ligand binding and thermal stability studies. Fourier Transform Infrared (FTIR) Spectroscopy. Figure 3 shows the Fourier transform infrared spectroscopy of various samples. FTIR results also confirm the retention of the native protein structure as well as supplement findings of the UV-vis spectroscopy. Hb shows absorption bands at around 1655 cm-1 (amide I) caused by the CdO stretching vibration of peptide linkages in the protein backbone and around 1540 cm-1 (amide II) resulting from a combination of N-H bending and C-N stretching of the porphyrin ring.36-38 These provide detailed information on the secondary structure of the polypeptide. In the case of SNT-Hb, the amide I and amide II bands appear at nearly the same wavenumbers as observed for free Hb in solution. This suggests an unchanged secondary structure of Hb confined inside the SNTs. However, the bands for SNT-Hb are considerably broader compared to that of free Hb. The broadening is attributed to the SNTs’ interaction with the protein39 as well as intermolecular interaction. The complete disappearance
Hemoglobin Confined Inside Silica Nanotubes
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Figure 1. TEM images (a, b, d, e) showing the general morphology of the bare SNT nanotubes. (a) Bundles of SNT nanotubes approximately 10 µm long. Inset: open end of a SNT. (b, d, e) Impregnation of SNT with hemoglobin (Hb). Micrograph e shows a certain fraction of Hb residing outside a SNT. (c) Scanning electron microscope image of SNTs impregnated with hemoglobin (Hb).
Figure 2. Room temperature (at 25 °C) UV-visible spectra showing the Soret band for free Hb and SNT-Hb.
of the band (Si-O-Si symmetric stretch) at 800 cm-1 acts as further evidence for existence of the interaction between Hb and SNT. Circular Dichroism (CD) Spectroscopy. Information on the three-dimensional structures of macromolecules containing
Figure 3. Fourier transform infrared spectra (FTIR) at 25 °C for various samples: SNT; before heating, Hb; before heating, SNT-Hb; after heating, Hb; and after heating, SNT-Hb.
chiral centers can also be obtained from CD spectroscopy. The far UV region (
