Optimization of Immobilized Aldose Reductase Isolated from Bovine Liver Sığır Karaciğerinden İzole Edilen İmmobilize Aldoz Redüktazın Optimizasyonu
Total Page：16
File Type：pdf, Size：1020Kb
Turk J Pharm Sci 2019;16(2):206-210 DOI: 10.4274/tjps.galenos.2018.81894 ORIGINAL ARTICLE Optimization of Immobilized Aldose Reductase Isolated from Bovine Liver Sığır Karaciğerinden İzole Edilen İmmobilize Aldoz Redüktazın Optimizasyonu Marya Vakıl NASLIYAN, Sidar BEREKETOĞLU, Özlem YILDIRIM* Ankara University, Faculty of Science, Department of Biology, Ankara, Turkey ABSTRACT Objectives: Isolation of enzymes and experiments on them require great effort and cost and are time-consuming. Therefore, it is important to extend the usability of the enzymes by immobilizing them. In this study our purpose was to immobilize the enzyme aldose reductase (AR) and to optimize the experimental conditions of the immobilized AR and compare them to those of free AR. Materials and Methods: AR was isolated from bovine liver and the enzyme immobilized in photographic gelatin by cross-linking with glutaraldehyde. Then the optimum conditions for free and immobilized AR in terms of pH, temperature, and storage were characterized by determining the enzyme activity. Results: Following immobilization, the optimum pH and temperature levels for free AR, which were pH 7.0 and 60°C, slightly altered to pH 7.5 and 50°C. The enzyme activity of the immobilized AR was maintained at about 65% after reusing 15 times. Moreover, immobilized AR maintained 95% of its original activity after 20 days of storage at 4°C, while the retained activity of the free AR was 85% of the original. Conclusion: Our experiments indicated that the conditions that affect enzyme activity might alter following immobilization. Once the optimum experimental conditions are fixed, the immobilized AR can be stored and reused with efficiency higher than that of free AR. Moreover, this study provides an insight into the advantages of using immobilized AR in enzyme assays rather than free AR. Key words: Aldose reductase, isolation, immobilization ÖZ Amaç: Enzim izolasyonu ve enzimler üzerinde yapılan deneyler, büyük çaba, maliyet ve zaman gerektirir. Bu yüzden, enzimlerin kullanılabilirliğinin immobilizasyon ile uzatılması önemlidir. Bu çalışmadaki amacımız, aldoz redüktaz (AR) enzimini immobilize etmek ve serbest AR’ninkiler ile karşılaştırarak immobilize AR’nin deney şartlarını optimize etmektir. Gereç ve Yöntemler: AR, sığır karaciğer ve böbreğinden izole edildi ve gluteraldehid ile fotografik jelatine çapraz bağlanarak immobilize edildi. Daha sonra, enzim aktivitesi belirlenerek serbest ve immobilize AR’nin optimum pH, sıcaklık ve depolama koşulları tespit edildi. Bulgular: Serbest AR için pH 7.0 ve 60°C olan optimum pH ve sıcaklık seviyeleri, immobilizasyondan sonra pH 7.5 ve 50°C olarak belirlendi. İmmobilize AR’nin enzim aktivitesinin, 15 kez kullanımdan sonra %65 oranında korunduğu tespit edildi. Bununla birlikte, +4°C’de 20 gün boyunca saklanan serbest AR’nin aktivitesi %85 oranında korunurken immobilize AR’nin aktivitesinin %95 oranında korunduğu bulunmuştur. Sonuç: Deneylerimiz, immobilizasyonu takiben enzim aktivitesini etkileyen koşulların değişebileceğini göstermektedir. Ayrıca, immobilize AR, serbest AR’ye göre daha yüksek aktiviteyle korunup tekrar tekrar kullanılabilmektedir. Anahtar kelimeler: Aldoz redüktaz, izolasyon, immobilizasyon *Correspondence: E-mail: [email protected], Phone: +90 532 471 60 46 ORCID-ID: orcid.org/0000-0002-7529-9786 Received: 28.02.2018, Accepted: 29.03.2018 ©Turk J Pharm Sci, Published by Galenos Publishing House. 206 NASLIYAN et al. Immobilized Aldose Reductase 207 INTRODUCTION followed by rotation at 10,000 rpm at +4°C for 25 min. To gain Aldose reductase (AR) (EC 1.1.1.21) is expressed by the AKR1B1 50% and 75% saturations, the same method was carried out by gene in humans. It is located on the 7q35 chromosome, consists adding 5.8 g and 15.9 g of ammonium sulfate to the 100 mL of of 315 amino acids, and its molecular weight is 36 kD. AR is a supernatant solution, respectively. The pellets were dissolved cytosolic monomeric protein.1 It specifically catalyzes the polyol with 50 mM sodium chloride and stored in a freezer at -80°C. pathway, which includes the conversion of glucose-bound Determining the protein amount nicotinamide adenine dinucleotide phosphate (NADPH) cofactor Following isolation, the amount of protein was detected by to sorbitol.2,3 Moreover, it reduces the activity of aldehydes Bradford assay.9 The bovine serum albumin (BSA) standards such as glutathione complexes.4 Indeed, AR is crucial in the were used at the concentrations of 0.4, 0.6, 0.8, 1.0, 1.2, and detoxification of lipid aldehydes produced by oxidative stress.5 1.4 mg/mL. BIO-RAD reagent was added to the BSA standards Under optimum conditions, enzymes are able to display and the sample. Then each solution was measured using a their activities outside of their natural environment. Using spectrophotometer at 595 nm wavelength. Finally, protein this feature, they can be employed on a large scale in health amount was calculated using the standard graph generated science, such as in the diagnosis and treatment of diseases with optical densities of the standards. and drug design. However, their structure tends to alter during the experimental process. Therefore, studies aim to improve Assay of AR enzyme activity the conformational stability of enzymes.6 Immobilization is a AR activity was detected against DL-glyceraldehyde, by method reducing disruption of the enzyme structure. Enzyme monitoring the NADPH oxidation to NADP+ at 340 nm.10 The immobilization avoids the majority of the instability and loss of reaction mixture consisted of AR enzyme (4.54 mg/mL), -5 enzyme activity. This provides a longer time and flexibility to NADPH (9×10 M), Li2SO4 (320 mM-400 mM), DL-glyceraldehyde work on enzymes. Immobilization is not only useful to maintain (6×10-4 M), and KP buffer (50 mM, pH 6.2). NADP+ oxidation the stability of biomolecules but also it reduces the cost of was followed in 0.25 mL of reaction mixture using a multimode enzyme studies. microplate reader at 340 nm for 4 min. Initial and final rates In the present study for the first time AR was isolated from of enzymatic reactions were measured and recorded as nmol/ bovine liver, immobilized, and characterized with respect to min/mg protein. several kinetic properties. In addition, AR derived from bovine Immobilization of AR enzyme kidney was used as a side control and simultaneously applied The immobilization gel consisted of 0.025 g of photographic to identical experiments. The AR enzymes from both sources gelatin and 0.125 M glutaraldehyde in 0.067 M phosphate were immobilized in gelatin by glutaraldehyde and washed to tampon solution (pH 7.4). The photographic gelatin was remove the free enzyme. We compared the behavior of the prepared at 50°C and then cooled to 30-35°C. Different units of free and the immobilized AR under different conditions of pH, AR enzyme were added to the gelatin and then vortexed. Cross- temperature, reusing, and storage. The difference in optimum linking glutaraldehyde was included at the concentration of conditions of the free and the immobilized enzyme may be 5×10-5 M. Then 100 μL of the mixture was placed onto cellulose due to alterations in the structure caused by several factors triacetate. Enzyme and gelatin complexes were dried for 24 h such as carrier material, immobilization method, and activation at room temperature. method.7 Indeed it was previously indicated that immobilization significantly changes the conformation of enzymes and thus The film strips carrying the immobilized AR were assayed their activity.8 for enzyme activity. The reaction mixture consisted of 2.7 mL of potassium phosphate, 0.1 mL of NADPH, and 0.1 mL MATERIALS AND METHODS DL-glyceraldehyde. Absorbance of this reaction mixture was measured at 340 nm before and after 20 min incubation with Chemical materials the film strips to detect enzyme activity. In this research, ethylenediaminetetraacetic acid (EDTA), NADPH, and lithium sulfate (Li2SO4) were purchased from RESULTS AND DISCUSSION Gerbu, Germany. Ammonium sulfate was supplied by Merck. AR isolated from bovine liver was immobilized by cross- Phenylmethylsulfonyl fluoride (PMSF), DL-glyceraldehyde, and linking with glutaraldehyde in photographic gelatin. To detect all other materials used were analytically graded and obtained the amount of AR protein we used the Bradford assay and from Sigma Aldrich, Germany. spectrophotometric measurements. The results indicated that Isolation of AR enzyme from bovine liver the protein amount was 18.39±0.09 mg/mL. In the standard -4 The bovine liver was provided by a slaughterhouse in Kazan, group, the activity values for only AR were 4.172×10 U/L in Ankara, Turkey. Small pieces of liver samples were washed liver. These values are accepted as 100% enzyme activity. with 1.0 mM EDTA. Then they were measured and homogenized Following immobilization, the free enzyme was removed by with threefold 1.0 mM EDTA and 50 μM PMSF and spun at +4°C washing the film strips three times with phosphate buffer and 10,000 rpm for 30 min. To acquire 40% saturation, 22.6 g (0.067 M, pH 6.2) at 25°C for 5 min and spectrophotometry was of ammonium sulfate was added to each 100 mL of supernatant used to detect the amount of free enzyme. Using the washed 208 NASLIYAN et al. Immobilized Aldose Reductase film strips, the effect of enzyme concentration on immobilized We observed that the optimum pH for AR shifted to 7.5 from 7.0 enzyme activity was tested to detect the immobilized enzyme upon immobilization. This alteration should be because of the leakage. Our results showed that AR leakage from the film strips H+ and the OH- ions that are released into the microenvironment increased with enzyme concentration while the enzyme activity during the reaction. Indeed, this result about the optimum was reduced after each wash. On the other hand, three washes pH alteration was expected because of the context of the were sufficient to remove the leakage completely (Table 1).