Lectin purification from carp roe (Cyprinus carpio L.), investigation of its carbohydrate specificity and application in histochemistry

Vol. 57 No. 3, 2016

ROMANIAN JOURNAL of MORPHOLOGY and EMBRYOLOGY

Rostyslav Antonyuk, Alexander Lutsyk, Volodymyr Antonyuk

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 cca. 42 mg/kg roe. Electrophoresis in 15% polyacrylamide gel in the presence of beta-mercaptoethanol showed one band with molecular mass cca. 15 kDa, whereas in the absence of beta-mercaptoethanol, CCRA exposed band with molecular mass cca. 60 kDa. The resulting lectin was thermostable, withstanding heating to 75 degrees 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 (Ca2+ and Mg2+) 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.

Corresponding author: Alexander Lutsyk, Professor, MD, PhD; e-mail: lutsyk@meduniv.lviv.ua, lutsykalexander@gmail.com

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