Ligand-exchange chromatography of nucleases. 1986

V P Varlamov, and S A Lopatin, and G E Bannikova, and I A Andrushina, and S V Rogozhin

The synthesis of chelating sorbents for ligand-exchange chromatography of enzymes is described. An inorganic support "Silochrom" and organic "Spheron", TSK-Gel HW 55 and cellulose were used as initial supports. The chelating sorbents contained iminodiacetic acid and iminodimethylphosphonic acid as stationary ligands. In order to obtain monofunctional sorbents, iminodiacetic acid was added in the form of its dimethyl ester. The concentration of stationary ligands on the sorbents varied from 10 to 100 mumol per ml sorbent. A chelating sorbent (in nickel form) was shown to be effective in the purification of exonuclease A5 from actinomyces. Electrophoretically homogeneous exonuclease A5 was obtained with a 25% yield. A chelating sorbent with iminodiacetic groups (in copper form) was applied to the isolation of endonuclease from Serratia marcescens directly from the culture medium. The capacity of the chelating sorbents for the endonuclease was studied as a function of the stationary ligand concentration. After one stage of purification, more than 70% pure enzyme was obtained with a yield exceeding 80%.

UI MeSH Term Description Entries
D008024 Ligands A molecule that binds to another molecule, used especially to refer to a small molecule that binds specifically to a larger molecule, e.g., an antigen binding to an antibody, a hormone or neurotransmitter binding to a receptor, or a substrate or allosteric effector binding to an enzyme. Ligands are also molecules that donate or accept a pair of electrons to form a coordinate covalent bond with the central metal atom of a coordination complex. (From Dorland, 27th ed) Ligand
D010727 Phosphoric Diester Hydrolases A class of enzymes that catalyze the hydrolysis of one of the two ester bonds in a phosphodiester compound. EC 3.1.4. Phosphodiesterase,Phosphodiesterases,Hydrolases, Phosphoric Diester
D002845 Chromatography Techniques used to separate mixtures of substances based on differences in the relative affinities of the substances for mobile and stationary phases. A mobile phase (fluid or gas) passes through a column containing a stationary phase of porous solid or liquid coated on a solid support. Usage is both analytical for small amounts and preparative for bulk amounts. Chromatographies
D004720 Endonucleases Enzymes that catalyze the hydrolysis of the internal bonds and thereby the formation of polynucleotides or oligonucleotides from ribo- or deoxyribonucleotide chains. EC 3.1.-. Endonuclease
D000190 Actinomyces A genus of gram-positive, rod-shaped bacteria whose organisms are nonmotile. Filaments that may be present in certain species are either straight or wavy and may have swollen or clubbed heads.
D012706 Serratia marcescens A species of gram-negative, facultatively anaerobic, rod-shaped bacteria found in soil, water, food, and clinical specimens. It is a prominent opportunistic pathogen for hospitalized patients.
D043264 Phosphodiesterase I A phosphoric diester hydrolase that removes 5'-nucleotides from the 3'-hydroxy termini of 3'-hydroxy-terminated OLIGONUCLEOTIDES. It has low activity towards POLYNUCLEOTIDES and the presence of 3'-phosphate terminus on the substrate may inhibit hydrolysis. 5'-Exonuclease,5'-Nucleotidephosphodiesterase,Alkaline Phosphodiesterase,Alkaline Phosphodiesterase I,Oligonucleate 5'-Nucleotidohydrolase,Orthophosphoric Diester Phosphohydrolase,5' Exonuclease,5' Nucleotidephosphodiesterase,Diester Phosphohydrolase, Orthophosphoric,Oligonucleate 5' Nucleotidohydrolase,Phosphodiesterase I, Alkaline,Phosphodiesterase, Alkaline,Phosphohydrolase, Orthophosphoric Diester

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