26^th Annual Meeting of the German Association for Stable Isotope Research (GASIR) October, 6 to 8, 2003, Cologne, Germany">A novel electrophoretic technique designed to modify the ratio of magnesium isotopes inside the creatine kinase active sites. A preliminary report

Authors: Dmitri Kouznetsov¹; Stanislav Arkhangelsky¹; Alana Berdieva²; Peter Khasigov²; Marina Orlova³

Source: Isotopes in Environmental Health Studies, Volume 40, Number 3, September 2004 , pp. 221-227(7)

Abstract:

A simple and efficient preparative electrophoretic technique has been proposed to obtain a modified creatine kinase (CK, E.C.2.7.3.2) molecule with an increased content of ²⁵Mg in the active site. A key point of the method is the special design of a 0.9 × 12.0 cm column for ascendent electrophoresis, packed consecutively, from the bottom to the top, with layers of 30 % PAAG (polyacrylamide grade), ²⁵Mg²⁺-containing 7.5 % PAAG, enzyme-binding ADP Sepharose and 2.2 % agarose gels, based on different tris-glycine and tris-HCl separation buffer systems. The isotope substitution process was a result of simultaneous desorption of enzyme from ADP Sepharose and electrically directed extensive flow of ²⁵Mg²⁺ cations through the porous gel matrix. Greater than 8-fold ²⁵Mg enrichment, i.e. a 10.2-86.3 % increase of ²⁵Mg contribution to total enzyme magnesium, has been reached. The modified ²⁵Mg-rich CK samples manifest higher (2.4-fold increase) values of specific catalytic activity when compared with intact (control) ones.

Keywords: Creatine kinase; Isotope effect; Isotope enrichment; Magnesium-25; Spin-selective process

Document Type: Research article

DOI: 10.1080/10256010410001689916

Affiliations: 1: N. N. Semenov Institute of Chemical Physics Russian Academy of Sciences Ul. Kosygina 4 117977 Moscow Russia 2: I. M. Sechenov Moscow Medical Academy Moscow Russia 3: M. V. Lomonosov Moscow State University Moscow Russia

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26th Annual Meeting of the German Association for Stable Isotope Research (GASIR) October, 6 to 8, 2003, Cologne, Germany">A novel electrophoretic technique designed to modify the ratio of magnesium isotopes inside the creatine kinase active sites. A preliminary report

26^th Annual Meeting of the German Association for Stable Isotope Research (GASIR) October, 6 to 8, 2003, Cologne, Germany">A novel electrophoretic technique designed to modify the ratio of magnesium isotopes inside the creatine kinase active sites. A preliminary report