#105

Protein/Drug Interaction: Ferulic Acid and Bovine Serum Albumin
SPR has been demonstrated to be a powerful optical technique for bioaffinity studies at thesolid/solution interface. In general, SPR measures the change in refractive index originated from thebinding of a solution species with a molecule pre-immobilized onto the SPR sensor chip. The
advantages of SPR include its simplicity, high sensitivity, obviation of sample labeling, and amenability
for real-time analysis. This application note describes the kinetic studies of ferulic acid and bovine
serum albumin (BSA) pre-immobilized onto an alkanethiol self-assembled monolayer (SAM)-modified
gold sensor chip.
Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA), whose structure is shown in FIG. 1a, is aphenolic acid commonly found in foods and plants. It has been confirmed to have antioxidant,
antimicrobial and anti-inflammatory activities, and purported to possess even an anti-cancer effect [1].
BSA is a frequently used model protein for developing new analytical methods, since it is the mostabundant protein in plasma and its structure has been well-characterized (FIG 1b) [2].

(a)(b)

FIG. 1 Chemical structure of ferulic acid (a) and the ribbon structure of bovine serum albumin (b).

A BI-SPR 1000 system, equipped with a dual-channel flow cell was used. To measure the binding
constant (K) between FA and BSA, BSA was first adsorbed onto an aminoethanethiol SAM-covered
gold sensor chip. Borate buffer was delivered by a syringe pump to the dual-channel PEEK
(polyetheretherketone) flow cell equipped with a PDMS (polydimethylsiloxane) gasket. In this setup,
one channel can be employed for sample analysis while the other serves as the reference.
Alternatively, both channels can be used to perform replicate analyses or measurements of different
samples. To introduce a sample into the flowing buffer stream, a six-port, through-the-handle injection
valve can be used. Since the valve, fittings, flow cell, and gasket are all constructed from biocompatible
materials, the system is ideal for measurements of biomolecules.
FIG. 2 is an overlay of three representative SPR sensorgrams showing the interaction between
BSA on the sensor chip and different concentrations of FA in solution. The flow rate used was 80µL/min. A series of FA solutions were analyzed. By plotting the SPR dip shifts (Δθ) as a function of FA
concentrations, one can deduce the maximum dip shift (Δθmax). It was found that the plot of SPR dipshifts vs. FA concentrations followed the Langmuir adsorption isotherm. Thus K for FA binding to BSA
can be estimated using the following equation [3]:

1
1
1
1
=+Δθ Δθ max K [ FA] Δθ maxwhere Δθmax is the maximum SPR dip shift when the immobilized BSA molecules have all been boundto FA, and [FA] is the FA concentration. The average K value for the FA/BSA binding was determined
by SPR to be 200 × 109 M-1. Such a value is consistent with results obtained with spectrofluorometry[4]. Moreover, it is in excellent agreement with affinity capillary electrophoresis (ACE) assays [5]. In theACE assays, a lower pH resulted in a considerable BSA adsorption onto the capillary, limiting the useof ACE to assess the binding affinities at various pH values. SPR overcomes this deficiency since thedetection is achieved through attachment of FA onto surface-confined BSA (i.e., the adsorption of BSA
onto surfaces is not a problem). Apparently, the consistency with data collected by solution-based
techniques indicates that immobilization of BSA onto surfaces does not alter the interaction between
BSA and FA or possibly other phenolic compounds of similar chemical structure.

FIG. 2 Representative SPR
sensorgrams corresponding tobindingofFA
onto
preimmobilizedBSA
molecules.
Three different FA concentrations(0.2, 0.4, 1.0 µM) were injected. A
flow rate of 80 µL/min was used.

Time (s)References[1] Ou, S. Y., Kwok, K. C., J. Sci. Food Agric. 2004, 84, 1261-1269.
[2] Kragh-Hansen, U., Pharmacol. Rev. 1981, 33, 17-53.
[3] Hancock, M. A., Spencer, C. A., Koschinsky, M. L., Biochemistry 2004, 43, 12237-12248.
[4] Rawel, H. M., Meidtner, K., Kroll, J., J. Agric. Food Chem. 2005, 53, 4228-4235[5] Zhang, Y., Xu, M., Du, M., Zhou, F., Electrophoresis, 2007, 28(11),1839-45

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