Elsevier

Bioelectrochemistry

Volume 79, Issue 1, August 2010, Pages 6-10
Bioelectrochemistry

Interaction of surface-attached haemoglobin with hydrophobic anions monitored by on-line acoustic wave detector

https://doi.org/10.1016/j.bioelechem.2009.09.012Get rights and content

Abstract

The behaviour of proteins on surfaces and at interfaces is an important field with applications in drug development, clinical diagnostics and studies of device biocompatibility. A key factor is the conformation of surface-bound proteins, which can affect chemical signalling and drug binding. A recent study of the interactions of haemoglobin with hydrophobic anions at a liquid–liquid interface has shown that a pH- and orientation-dependent conformational change occurs in the haemoglobin molecule upon interaction with these anions. To corroborate these results, we use an acoustic wave detector to study binding of solution-phase hydrophobic anions to surface-adhered haemoglobin. The orientation of protein is controlled by thiol chemistry, which generates hydrophilic and hydrophobic surfaces. Tetraphenylborate-based anions are introduced to the haemoglobin coated surface via an on-line flow-injection system to monitor the signal in real-time. Changes in the acoustic properties of the surface, measured piezoelectrically, are related to interactions between the protein and the anions. Signal strength is proportional to the degree of interaction between the salts and the haemoglobin, which in turn, is influenced by its conformation.

Introduction

Electrochemical methods have been used to study the behaviour of proteins at the interface between two immiscible liquids [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]. So-called interfaces between two immiscible electrolyte solutions (ITIES) are used to study electron and ion transfer across liquid–liquid interfaces, and have been proposed as biosensors to explore the conformational dynamics of small molecules, peptides, and proteins [2], [3], [12], [13], [14]. In a recent study, the electrochemical behaviour of the serum protein haemoglobin (Hb) was examined at the ITIES [1], [15]. Haemoglobin is an important metalloprotein which is responsible for oxygen transport in blood. As such, it is biologically abundant and interacts with a wide range of other proteins and ligands. Hb is made of four globular subunits, two α units and two β units, each of these connected to an iron-containing heme group. It contains 574 amino acids and has a molecular weight of 64.5 kDa. ITIES electrochemistry studies by Arrigan et al. [1], [15] suggest that aqueous-phase Hb migrates to the liquid–liquid interface, although it remains in the aqueous phase. As well, it likely undergoes a conformational shift such the hydrophobic domains are oriented towards the organic phase. As well, this conformational change was time-dependent, requiring approximately 45 min to occur. Furthermore, Hb is thought to facilitate the transfer of hydrophobic anions across the liquid-liquid interface, an effect that is likely enhanced by a time-dependent conformational shift to a more energetically favourable structure at the ITIES. This reveals that the anionic binding domains and the hydrophobic domains of Hb may be situated close to each other.

Acoustic physics has been used extensively as a method to measure adsorption and conformational changes of surface-bound biochemical species. Specifically, the transverse-shear mode acoustic wave device (TSM) has been applied as a biosensor to measure surface adsorption and interfacial effects at a solid–liquid boundary. This tool operates through the piezoelectric generation of a high-frequency transverse-shear acoustic wave in a quartz wafer, and is capable of detecting surface concentrations on the order of pmol/cm2. It has been widely applied for the detection of nucleic acid binding [16], [17], immunochemistry [18], [19], and protein–small molecule interactions [20]. This sensor has also recently been used to detect living cells on surfaces [21], [22].

Haemoglobin has been studied extensively using the TSM. Shao et al. [23] developed a bovine haemoglobin immunosensor and were able to detect concentrations in the range 0.001 to 0.1 mg/ml. Hook et al. [24] studied adsorption of two structurally similar forms of Hb to a hydrophobic surface with the TSM device. By comparing changes in frequency and dissipation, they inferred that the protein forms two monolayers on the device surface, the first tightly bound and the second less rigidly attached. These authors also found that adsorption kinetics depend on the proximity of the pH to the isoelectric point of haemoglobin. Cavic and Thompson [25] tested Hb adsorption to hydrophilic bare gold and mildly hydrophobic organosiloxane surfaces, and found that the surface energy affects the signal strength and degree of binding. Additionally, it was suggested that the acoustic signal is not caused by adsorption alone, and that the viscoelasticity of the protein plays a role.

In this paper, we use an acoustic wave sensor in a flow-injection setup to investigate the conformational behaviour of Hb on functionalized surfaces when exposed to hydrophobic anions. Different surface treatments are used to orient the Hb molecules, which should influence the availability of the ionic and hydrophobic binding sites. This is shown schematically in Fig. 1. In particular, we record changes in acoustic properties for Hb adsorption to four differently treated surfaces, followed by addition of the electrolytic salts potassium tetrakis(4-chlorophenyl)-borate (TPBCl), sodium tetrakis(4-fluorophenyl)-borate dihydrate (TPBF), and sodium tetraphenylborate (TPB). Changes in the acoustic resonant frequency (Δfs) and the motional resistance (ΔRm) were measured over time. The device response is used to differentiate between the Hb–anion interactions on hydrophilic and hydrophobic surfaces. The surface energy should influence the conformation of the Hb monolayers, which would in turn affect the binding to the anion. These results are used to corroborate the experiments performed on similar systems involving ITIES [1], [15], as described above. The study of these interactions, while not of direct biological importance, could reveal interesting properties of Hb structure, biochemistry and behaviour, in a biomimetic environment.

Section snippets

Reagents

Unless otherwise indicated, all reagents were used as received. Bovine haemoglobin (Hb; MW: 64.5 kDa) was obtained from Sigma-Aldrich Ltd. (Canada). Hb solutions were prepared in 10 mM HCl solutions in deionised water and refrigerated when not in use. The hydrophobic salts were potassium tetrakis(4-chlorophenyl)-borate (TPBCl; MW: 493.12 Da), sodium tetrakis(4-fluorophenyl)-borate dihydrate (TPBF; MW: 450.22 Da), and sodium tetraphenylborate (TPB; MW: 342.23 Da), all purchased from Sigma-Aldrich,

Response due to haemoglobin

Fig. 2 shows a representative plot of changes in fs and Rm for the adsorption of Hb onto a modified surface, followed by addition of TPBCl salt (discussed below). Table 1 shows the frequency and resistance shifts for the Hb adsorption on the four different surfaces, along with contact angles measured for the modified surfaces. These results indicate that surface modification increases the response. However, there is an associated increase in the error, in both Δf and ΔR. There is little

Discussion

There is a marked difference between the results for the cleaned surfaces in Table 2, and the treated surfaces in Table 3. The signal for TPBCl on PhilA is significantly larger than for all the other surfaces. The large signal of Δf =  142 Hz could be due to ionic effects in the liquid or at the interface, and may not necessarily be directly linked to binding of the anion to Hb. Without further study, it is not possible to determine whether this shift is an artefact, or whether it is due to

Conclusions

We have used a transverse-shear mode acoustic device in a flow-injection configuration to demonstrate the adsorption of the blood protein haemoglobin to gold surface cleaned and treated in a variety of ways. Furthermore, we have revealed that the surface energy of the treatment could influence the adsorbed conformation of the immobilised haemoglobin, which in turn affects binding of solution-phase electrolytes.

The resonant frequency decrease for haemoglobin adsorption was consistently larger

Acknowledgments

JSE, SQX, XW and MT are grateful to the Natural Sciences and Engineering Research Council of Canada for the support of this work. GH and DWMA acknowledge the support of the Science Foundation Ireland (grant no. 07/IN.1/B967). This work was initiated while MT was an E.T.S. Walton Visiting Fellow at Tyndall National Institute, sponsored by the Science Foundation Ireland (grant no. 06/W.1/I894).

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    Present address: Tyndall National Institute, Lee Maltings, University College Cork, Cork, Ireland.

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