Rom J Morphol Embryol 2016, 57(3):985–994 ISSN (print) 1220–0522 ISSN (online) 2066–8279 ORIGINAL PAPER Lectin purification from carp roe (Cyprinus carpio L.), investigation of its carbohydrate specificity and application in histochemistry ROSTYSLAV ANTONYUK 1,2) , ALEXANDER LUTSYK 2) , VOLODYMYR ANTONYUK 3) 1) Department of Histology and Embryology, Danylo Halytsky Lviv National Medical University, Ukraine 2) Dubno Central Regional Hospital, Rivne Region, Ukraine 3) Department of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine Abstract A method of lectin purification from carp roe (CCRA) was elaborated, which includes affinity chromatography on cross-linked ovomucoid and copolymers of polyvinyl alcohol and blood group-specific substances. That allowed obtaining lectin with electrophoretic purity and yielding of ≈ 42 mg/kg roe. Electrophoresis in 15% polyacrylamide gel in the presence of β-mercaptoethanol showed one band with molecular mass ≈ 15 kDa, whereas in the absence of β-mercaptoethanol, CCRA exposed band with molecular mass ≈ 60 kDa. The resulting lectin was thermostable, withstanding heating to 75 0 C for 15 minutes, without noticeable loss of hemagglutinating activity. Gel column chromatography on Toyopearl HW-55 determined the lectin molecular weight of 120±3 kDa. For the lectin activity, divalent metal ions (Ca 2+ and Mg 2+ ) were not necessary. CCRA showed the best agglutination titer with pigeon erythrocytes, weaker – with rabbit and dog erythrocytes, and significantly weaker – with human and rat erythrocytes. CCRA lectin was specific to N-acetyl-D-galactosamine and D-galactose group carbohydrates. The best lectin activity inhibition possessed alkaline phosphatase of calf intestine and fetuin. CCRA exposed highest affinity to complex oligosaccharide similar to the receptor of Phaseolus vulgaris erythroagglutinin (PHA-E). A comparative study on the histochemical specificity of CCRA and PHA-E using specimens of normal tissues, and that of colon neoplasia, showed similar, yet not identical binding properties. CCRA lectin rather differentially labeled adenoma and adenocarcinoma of colon, which suggests its prospective applicability in diagnostic histopathology. Keywords: carp roe lectin (CCRA), purification, properties, application in histochemistry.  Introduction Lectins are proteins/glycoproteins that selectively bind carbohydrates (including carbohydrate receptors of living tissues) without causing their chemical transformations. Due to their carbohydrate selectivity, lectins are widely used for detection of specific carbohydrate determinants on the cell surface or in the cellular compartments. For this purpose, lectins with well-defined carbohydrate specificity and preferably with high selectivity to carbo- hydrate determinants are required. Currently, only a limited number of lectins with such properties are renowned. Therefore, the search and investigation of new lectins with rare carbohydrate specificity remains an important issue [1–6]. Plants are considered the main sources for lectin purification, since they often contain lectins in significant amounts and can be cultivated under regular conditions, therefore methods of plant lectins purification are well documented [7, 8]. However, recently more attention is focused on fungal and animal lectins. Earlier we reported purification protocol for a lectin from fruiting bodies of the fungus Mycena pura (Fr.) Kumm. (MPFA), which possess high affinity interaction with alkaline phosphatase of calf intestine [9]. The relative inhibitory intensity of MPFA-alkaline phosphatase inter- action was highest among 20 tested lectins. However, this fungus is of a small size, its fruiting bodies can be found in sufficient quantities during very short seasonal period and only in particular places, therefore its avai- lability is limited. During our studies on lectins from different sources, we founded out that the lectin from carp roe (Cyprinus carpio L., CCRA) also interacts with high affinity with alkaline phosphatase. This lectin purification protocol was described in several publications [10, 11]; moreover, a lectin from roe of a very close species – gibel carp (Carassius auratus gibelio) also was purified [12]. In these papers, lectin purification was maintained by affinity chromatography on Sepharose 4B followed by elution of the adsorbed lectin by 0.4 M N-acetyl-D-glucosamine and its additional purification on Superdex G-75 [10], or ion-exchange chromatography on phosphocellulose, DEAE-52 and subsequent hydroxylapatite column chro- matography [12]. As a result, from 500 g of common carp roe it was obtained 10 mg of galactose-type lectin with molecular mass of 26.68 kDa, which showed carbo- hydrate specificity to N-acetyl-D-glucosamine and, to a lesser extent, to L-rhamnose and L-fucose. For lectin activity, divalent metal ions (Ca 2+ and Mg 2+ ) were not necessary [10]. According to data of other authors [11], rabbit erythrocyte agglutination by recombinant common carp R J M E Romanian Journal of Morphology & Embryology http://www.rjme.ro/