J. Sep. Sci. 2008, 31, 1803 – 1809 T. Lemma et al. 1803 Tibebe Lemma 1 Rupasri Mandal 2 Xing-Fang Li 2 Janusz Pawliszyn 1 1 Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada 2 Environmental Health Science, Department of Public Health Sciences, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta Canada Original Paper Investigation of interaction between human hemoglobin A 0 and platinum anticancer drugs by capillary isoelectric focusing with whole column imaging detection CIEF with whole column imaging detection (WCID) was used to investigate the inter- action of platinum-based anticancer drugs, cis-platinum(II) diamine dichloride (cis- platin) and [SP-4-2-{1R-trans)]-(1,2-cyclohexanediamine-N,N9)[ethanedioata(2 – )-O,O9]pla- tinum (oxaliplatin), with human hemoglobin A 0 (Hb). This technique facilitates the investigation and characterization of the formation of adducts between drugs and proteins. Cisplatin and oxaliplatin were mixed with the target protein at different concentrations (0:1, 1:1, 1:10, 1:50, and 1:100), and the reaction mixtures were incu- bated for 0, 0.5, 1, 12, 24, 48, and 72 h at 378C in a water-bath. The focused Hb – drug adduct profiles were imaged by WCID. At higher drug to protein molar ratios (for both oxaliplatin and cisplatin), the results exhibit significant changes in the peak shapes and heights, which may indicate the destabilization of the protein. However, the conformational change was less evident at lower molar ratios. In addition, a major pI shift was observed for the oxaliplatin reaction mixtures (for 1:10, 1:50, and 1:100 ratios). In comparison with previously reported findings obtained by other analytical methods, conclusions were drawn about the validity of CIEF as a simple and convenient method for the investigation of protein – drug interactions. These results may provide useful information for further understanding the activity and toxicity of these chemotherapeutic drugs and improving their clinical performance. Keywords: Capillary isoelectric focusing / Human hemoglobin A0 / Platinum anticancer drugs / Whole column imaging detection / Received: September 5, 2007; revised: January 17, 2008; accepted: January 18, 2008 DOI 10.1002/jssc.200700418 1 Introduction Platinum-based drugs are among the most active anti- cancer agents and have been widely used in the treat- ment of a variety of human tumors. Over the last 30 years, a large number of platinum analogs have been synthesized to enlarge the spectrum of activity, over- come cellular resistance, and/or reduce the toxicity of both first (i. e., cis-platinum(II) diamine dichloride (cispla- tin)) and second generation (i. e., carboplatin) platinum drugs [1]. Of these platinum-based compounds, [SP-4-2- {1R-trans)]-(1,2-cyclohexanediamine-N,N9)[ethane- dioata(2–)-O,O9]platinum (oxaliplatin), a novel compound containing a trans-1-(R,R)-1,2-diaminocyclohexane (DACH) carrier ligand, has recently been approved for the treatment of metastatic colorectal carcinoma in con- junction with fluoropyrimidines [2]. Oxaliplatin has shown in vitro and in vivo efficacy against many tumor cell lines and tumors including those that are resistant to cisplatin and carboplatin [1, 3–5]. In addition to its pos- itive effects, oxaliplatin also shows several toxic effects. The main cumulative dose-limiting toxicity of oxalipla- tin is progressive peripheral sensory neuropathy [6, 7]. It is also associated with an acute neuropathy mild which reverses in several hours or days [6, 7]. Oxaliplatin can also produce diarrhea, vomiting, and hematological sup- pression [7–9]. However, the mechanisms of action and toxicity are not clear. Pt-containing anticancer drugs are believed to induce apoptosis in cancer cells by covalently binding to DNA [10–12], however, they also react with a number of pro- Correspondence: Professor Januz Pawliszyn, Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1 E-mail: janusz@uwaterloo.ca Fax: +1-519-746-0435 Abbreviations: cisplatin, cis-platinum(II) diamine dichloride; Hb, hemoglobin; oxaliplatin, [SP-4-2-{1R-trans)]-(1,2-cyclohexane- diamine-N,N9)[ethanedioata(2–)-O,O9]platinum; WCID, whole col- umn imaging detection i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com