Effect of Microwave Heating on the Crystallization of Glutathione

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Effect of Microwave Heating on the Crystallization of Glutathione Tripeptide on Silver Nanoparticle Films Edward N Constance, Aysha Zaakan, Fatmah Alsharari, Brittney Gordon, Fareeha Syed, Kevin Mauge-Lewis, Enock Bonyi, Zainab Boone-Kukoyi, and Kadir Aslan J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b11952 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 27, 2017

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The Journal of Physical Chemistry

Effect of Microwave Heating on the Crystallization of Glutathione Tripeptide on Silver Nanoparticle Films Edward N. Constance, Aysha Zaakan, Fatmah Alsharari, Brittney Gordon, Fareeha Syed, Kevin Mauge-Lewis, Enock Bonyi, Zainab Boone-Kukoyi and Kadir Aslan* Morgan State University, Department of Chemistry, 1700 East Cold Spring Lane, Baltimore, MD 21251, USA. * Corresponding Author: [email protected] Abstract Effect of microwave heating on the crystallization of glutathione (GSH) tripeptide of using the Metal-Assisted and Microwave-Accelerated Evaporative Crystallization (MA-MAEC) technique is reported. GSH crystals were grown from supersaturated solutions of GSH (300-500 mg/mL) on the iCrystal plates with silver nanoparticle films (SNFs) and without SNFs in three different microwave systems operating at 2.45 GHz: conventional (multi-mode, fixed power at 900 W), industrial (mono-mode, variable power up to 1200 W), and the iCrystal system (mono-mode, variable power up to 100 W). The efficacy of the MA-MAEC technique, in terms of improvement in the crystallization time, crystal size and quality of GSH, was compared between the three microwave systems and the crystallization at room temperature (no microwave heating, a control experiment). Optical microscopy was used to visualize and quantify the growth of GSH crystals during and after microwave heating. Powder X-ray diffraction (XRD) and Fourier Transform Infra-Red (FTIR) spectroscopy data showed that GSH crystals had identical crystal structure to those grown at room temperature and microwave heating did not alter the chemical structure of GSH molecules during microwave heating, respectively. Using the MA-MAEC technique, the iCrystal system yielded high quality GSH crystals in a rapid manner. 1 ACS Paragon Plus Environment


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INTRODUCTION Crystallization of peptides affords for the structural analysis of important biological molecules and development of biological composite materials.1-2 Furthermore, crystallization of small peptides facilitates new pharmaceutical advances in drug design and delivery for subsequent treatment in medicine.3 One promising candidate for the study of medicine and drug development is glutathione (GSH). GSH, a low-molecularweight tripeptide molecule (γ-glutamyl-cysteinyl-glycine), is one of the most abundant thiol compounds present in animal cells and is the primary antioxidant involved in maintaining redox status in cellular microenvironments.4 GSH also serves as a co-factor for many cytoplasmic enzymes and plays an important role in post-translational modification. GSH metabolism has been linked to HIV treatment, cancer, chronic stress, cardiovascular disease, and neurological disorders, such as, Alzheimer’s disease and Parkinson’s disease.5-8 Thus, the role of GSH supplementation as an effective treatment has been growing rapidly. Several crystallization methods that offer systematic control over the crystallization of GSH have been described in literature. The most prominent of these methods deals with the recovery of GSH from natural sources in high yield and in uncontaminated form.9 However, these methods do not consider the rapidity of GSH crystallization and control over crystal size. Previously, our research group demonstrated the crystallization of biomolecules (i.e., amino acids and proteins) using the MA-MAEC technique.10-11 The MA-MAEC technique offers the advantage of rapid crystallization and improved crystal size and quality for biomolecules of interest.10 The crux of the MA-MAEC technique is the

2 ACS Paragon Plus Environment

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generation of microwave-induced thermal gradients and the subsequent rapid mass transfer of biomolecules directed towards the metal nanostructures on planar surfaces that act as nucleation sites and microwave-transparent medium. Metal nanostructures of silver, gold, copper, nickel, indium tin oxide are deposited on to poly(methyl methacrylate) (PMMA) platforms combined with silicon isolators (i.e., the iCrystal plates).12-16 The unique design of the iCrystal plates ensures that microwave radiation is distributed homogeneously across the wells formed by the silicon isolator.17-18 In studies based on the MA-MAEC technique by our research group to date, two different microwave systems operating at 2.45 GHz was employed: a multi-mode conventional microwave system (fixed power, 700 - 1000 W)10-11 and the iCrystal system (monomode, variable power up to 100 W).19 Conventional microwave system is a commercially available microwave oven that was initially used to demonstrate the proofof-principle demonstration and applications of the MA-MAEC technique for the crystallization of biological compounds.11-16 The iCrystal microwave system is an instrument developed by our research group and was also employed for the crystallization of a variety of biomolecules.19 Our research group seeks to improve the laboratory scale and rapid crystallization of biomolecules and pharmaceutical compounds using the MA-MAEC technique. Although the conventional and the industrial microwave systems were shown to facilitate rapid crystallization of biomolecules to date, a detailed comparative investigation of their capabilities for crystallization of simple amino acids and peptides was never carried out. Subsequently, there is still a need for the selection of a suitable microwave system that can be used with the MA-MAEC technique for the rapid and on-



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demand crystallization of molecules of interest. In addition, there are several commercially available mono-mode industrial and research grade microwave systems that offer variable power up to several kW, which were never used for crystallization of biomolecules. This paper has two main aims: first, we demonstrate the effectiveness of the MAMAEC technique for the rapid crystallization of a tripeptide (GSH) in all three different microwave systems, and second, we establish which of the three microwave systems is most effective for the rapid crystallization of GSH using the MA-MAEC technique. With respect to the latter, we compared several experimental factors, such as, crystallization speeds, crystal size, rate of crystal growth and crystal quality. Images of GSH crystals taken via optical microscopy are used to compare the aspects of crystal growth during microwave heating in all three microwave systems. Additionally, X-ray diffraction and FTIR analysis are used to confirm the preservation of GSH crystal structure. Moreover, computer simulations were carried out to predict and verify GSH crystal morphology based on crystallographic geometrical considerations. Our findings indicate that the use of the MA-MAEC technique improves crystallization rate of GSH in all three microwave systems and that the iCrystal system is the best choice for efficient crystallization in terms of crystal quality and size. EXPERIMENTAL METHODS Materials. Circular PMMA disks (5 cm in diameter) were purchased from McMaster-Carr (IL, USA), the L-glutathione, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS) and sodium acetate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrochloric acid, sodium


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hydroxide and ethanol was purchased from Pharmco-Aaper (Brookfield, CT, USA). The silicon isolator with 21 wells was designed by The Aslan Research Group and manufactured by Grace BioLabs (Bend, OR, USA). Each well had a capacity of 30 microliters of solution. A silver target (57 mm in diameter) was obtained from Electron Microscopy Sciences (Hatfield, PA, USA). Methods. (1) Selection solvent for GSH crystallization studies. Solubility of GSH in several solvents was investigated in the temperature range was between room temperature and 50°C to obtain supersaturated solutions of GSH. The solvents used were DMF, DMSO, ethanol, water, PBS (pH= 7.4), sodium acetate (pH= 4.6), and sodium phosphate dibasic (pH=9.1). Using a micropipette, a milliliter of each of the above solvents was pipetted into a 20-mL glass vial. The pre-measured (

Effect of Microwave Heating on the Crystallization of Glutathione

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