Electrochemical and spectroscopic study on the interaction between isoprenaline and DNA using multivariate curve resolution-alternating least squares

https://doi.org/10.1016/j.ijbiomac.2011.06.022Get rights and content

Abstract

Interaction of isoprenaline (ISO) with calf-thymus DNA was studied by spectroscopic and electrochemical methods. The behavior of ISO was investigated at a glassy carbon electrode (GCE) by cyclic voltammetry (CV) and differential pulse stripping voltammetry (DPSV); ISO was oxidized and an irreversible oxidation peak was observed. The binding constant K and the stoichiometric coefficient m of ISO with DNA were evaluated. Also, with the addition of DNA, hyperchromicity of the UV–vis absorption spectra of ISO was noted, while the fluorescence intensity decreased significantly. Multivariate curve resolution-alternating least squares (MCR-ALS) chemometrics method was applied to resolve the combined spectroscopic data matrix, which was obtained by the UV–vis and fluorescence methods. Pure spectra of ISO, DNA and ISO–DNA complex, and their concentration profiles were then successfully obtained. The results indicated that the ISO molecule intercalated into the base-pairs of DNA, and the complex of ISO–DNA was formed.

Introduction

Deoxyribonucleic acid (DNA) carries most of the genetic information and facilitates the biological synthesis of proteins and enzymes through replication and transcription of this information [1], [2]. DNA studies often focus on their interaction with molecules, such as small organic compounds and metal complexes, and these often assist in the understanding of the mechanism of such molecules with the biopolymer as well as providing some guidance for the design of new drugs [3]. Interactions between drugs and DNA are important, fundamental issues in life processes, and are crucial for gene therapy, because of the correlations involved in the mechanisms of drug and gene delivery systems [4].

β-Adrenergic receptors are widely distributed in the human body, and their roles are affected by a number of important physiological and biochemical processes. Isoprenaline (ISO, 4-[1-hydroxy-2-[(1-methyl-ethyl) -amino]ethyl]-1,2-benzenediol) is a catecholamine, which has been used for the treatment of bronchial asthma, allergies and ventricular bradycardia [5]. As an adrenergic acceptor, it contributed to the research of other bronchodilators such as, for example, terbutaline, fenoterol and salbutamol [5]. Compared with epinephrine and norepinephrine, the cardiovascular effects of ISO are known to interact with both α- and β-adrenergic receptors, which can relax almost any kind of the smooth musculature that contains adrenergic nerves; however, this effect is particularly pronounced in the bronchus as well as in the gastrointestinal tract. Nevertheless, overdosing on ISO may cause heart failure, and predispose patients to cardiac dysrhythmias [6].

β-Adrenergic receptors are linked through a guanine nucleotide regulatory protein to adenylate cyclase on the inner part of the plasma membrane of target cells. A typical example of the activation of the membrane adenylate cyclase system in order to increase intracellular 3′,5′-cyclic adenosine monophosphate (cAMP) in the liver is observed in the presence of catecholamines. The biological responses to interaction of ISO (a β1- and β2-adrenergic receptor agonist) are generally mediated by an increase of intracellular cAMP, which subsequently initiates the so called biochemical cascade, including glycogenolysis in the liver [7].

There are many methods to study the DNA binding properties, including surface plasmon resonance [8], luminescence [9], capillary electrophoresis [10], NMR [11], circular dichroism [12], UV–vis spectrophotometry [13], fluorescence [14] and electrochemistry [15], [16], [17]. Among these, electrochemical and spectroscopic techniques offer the advantages of high sensitivity and selectivity. However, for complex systems of more than two components, it is usually difficult to distinguish these existing species because their response signals often overlap. This limitation can be overcome with the use of multivariate analysis [18].

Multivariate curve resolution-alternating least squares (MCR-ALS) is a soft-modeling method, which is based on factor analysis, and can be used to analyze spectroscopic data from biomolecular systems at equilibrium in solution [19]. MCR-ALS allows mathematical analysis of more than one data matrix simultaneously, which greatly reduces the number of solutions inherent in the factor analysis model. Extension of the common multivariate spectral studies with the use of the MCR-ALS method has also been carried out on the interaction equilibrium of dyes and drugs with biomolecules [20], [21], [22]. The MCR-ALS method may be applied to all original spectroscopic measurements, obtained by one or several techniques; its output provides the concentration profiles and pure spectra of all the chemically analyzed components: the concentration profiles provide the information on the mechanism of the DNA process, and the features of the recovered pure spectra help to identify the species involved.

The aims of this study were several fold: 1. to use a combination of spectroscopic and electrochemical methods of analysis to investigate the ISO–DNA interaction, and apply data resolution chemometrics methods, such as MCR-ALS so as to extract information from overlapping responses of the reaction participants from spectroscopic data, and 2. given the extracted information from Aim 1 above, to explore the mechanism of ISO–DNA interactions, and in particular, to note any possible damage to the DNA caused by the ISO/DNA interaction.

Section snippets

Reagents

A DNA solution (1.70 × 10−3 mol L−1) was prepared by dissolving 0.050 g DNA (Sigma Co.) in 50 mL of 50 mmol L−1 sodium chloride solution and stored in a refrigerator at 4 °C. The purity of this solution was checked spectrophotometrically (ɛ260 = 6600 L mol−1 cm−1) [23]; 30 μL of the prepared DNA was added to a cuvette containing 3.0 mL phosphate buffer, and the solution gave an absorbance ratio of A260/A280 = 0.1122/0.0613 = 1.83, indicating that the DNA was sufficiently protein free [23]. A stock solution (4.04 × 10

Interaction of ISO with DNA at the GCE

CV was used to study the electrochemical behavior of ISO at a GCE. The first cycle (curve 1, Fig. 1) indicated that the oxidation of ISO (Scheme 1), in the absence of DNA, occurred at 0.321 V, and no corresponding reduction peak was observed; this suggested that the oxidation of ISO at GCE is irreversible.

When DNA was added to the ISO solution (curves 2–14, Fig. 1), ISO reacted with the DNA, and the peak current heights decreased markedly; also, the peak potential shifted from 0.321 V to the more

Conclusions

In this paper, voltammetry, UV–vis spectrometry and spectrofluorimetry were applied to investigate the interaction of isoprenaline (ISO) and DNA with the aid of the chemometrics method—MCR-ALS.

The results of voltammetry suggested that the most probable mechanism of the interaction between ISO and DNA was the intercalation of ISO into the DNA helix. The values of the binding number, m, and the constant, K, were 0.89 (∼1) and 2.1 × 105 M−1, respectively.

The application of the MCR-ALS for the

Acknowledgements

The authors gratefully acknowledge the financial support by the National Natural Science Foundation of China (No. NSFC-21065007), and the State Key Laboratory of Food Science and Technology of Nanchang University (SKLF-MB201007 and SKLF-TS200919).

References (35)

  • M. Yamada et al.

    Int. J. Biol. Macromol.

    (2008)
  • D.O. Wood et al.

    J. Inorg. Biochem.

    (2005)
  • F. Westerlund et al.

    Biophys. Chem.

    (2007)
  • N. Kanayama et al.

    Anal. Chim. Acta

    (2008)
  • B. Pagano et al.

    Biochimie

    (2008)
  • F. Sousa et al.

    Arch. Biochem. Biophys.

    (2007)
  • Y.N. Ni et al.

    Talanta

    (2005)
  • Z.S. Wu et al.

    Anal. Biochem.

    (2006)
  • X. Tian et al.

    J. Electroanal. Chem.

    (2008)
  • A. de Juan et al.

    Biophys. J.

    (1997)
  • R. Gargallo et al.

    Biophys. J.

    (2001)
  • R. Tauler

    Chemom. Intell. Lab. Syst.

    (1995)
  • M. Catalan et al.

    Bioelectrochemistry

    (2010)
  • X. Tian et al.

    Bioelectrochemistry

    (2008)
  • M. Vives et al.

    Anal. Chim. Acta

    (2000)
  • Y. Cao et al.

    Spectrochim. Acta A

    (1998)
  • Q.Y. Chen et al.

    Analyst

    (1999)
  • Cited by (0)

    View full text