SummaryPutative metallothionein (MT) isoforms are readily separated by capillary zone electrophoresis (CZE) with a mixture of tris(hydroxymethyl)aminomethane (Tris) and borate buffers in uncoated polyimide-cladded fused-silica capillary tubing. The influence of buffer concentration and composition, pH, running voltage and temperature on the electroosmotic mobility and on the electrophoretic mobilities of model samples (rabbit liver and horse kidney MT) have been investigated. The use of organic solvents as buffer modifiers and their effect on the separation of putative metallothionein isoforms is also described. It is shown that organic solvent modification significantly enhances the resolutionwith organic solvents as modifiers it is possible to obtain a good separation of putative isoforms of rabbit liver MT in one run whereas two separate runs were needed at different pHs or at different temperatures when the separation was performed with Tris-borate buffer without organic-solvent modification. The effects of the organic solvents as modifiers were found to be solventspecific although the trends were in the same direction (i.e. decreasing electroosmotic mobility with increasing percentage of organic solvent). Differences in effect on individual putative MT isoforms were observed, indicative of different chemical and physical characteristics of the isofroms. A difference in polymorphism of horse kidney MT and rabbit liver MT is clearly apparent.
Several purification methods were tested and the optimal procedure for obtaining L-lysine a-oxidase from fungi Trichoderma sp. and its isoform (minor L-lysine a-oxidase) was worked out. The enzyme and its isoform were obtained in a homogeneous state, the. most important physicochemical properties were studied, and a number of differences between them were found. The most marked differences between L-lysine a-oxidase and its isoform were observed in the molecular weight (120 and 100 kD, respectively), in the isoelectric point (pI 4.4 and 5.6, respectively), and in the specific activity (90-95 and 17-20 U/mg) in experiments where L-lysine where used as substrate.
Key Words: L-lysine a-oxidase; L-amino acid oxidase; isoform; purification of enzyme isoforms; pysicochemieal propertiesData on the purification of the antitumor enzyme L-lysine a-oxidase (LO) from fungi Trichoderma sp. [2,5,7] as well as on some catalytic and biological properties of this enzyme [3][4][5][6] were presented in previous papers. When the composition of culture fluid of Trichoderma sp. was examined, it was found that together with the already known LO it contains another protein which can also catalyze the oxidative deamination of L-lysine and some other Lamino acids and which was dubbed minor L-lysine c~-oxidase (m-LO). Preliminary data on this new enzyme were published earlier [5].Besides working out the optimal procedure for isolating and purifying m-LO, the aim of the present study was to obtain experimental evidence of m-LO existence and to study its main physicochemical properties.
MATERIALS AND METHODSFor the separation of proteins the following chromatographic carriers were used; butyl-silochrom C-
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