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Y.C. Lee 《Analytical biochemistry》1984,136(2):536-541
A method for analysis of plasma adenosine which combines the principles of radioisotope dilution and enzymatic catalysis is presented. Plasma from venous heparinized blood containing the adenosine deaminase inhibitor 2′-deoxycoformycin is mixed with a small amount of [3H]adenosine and extracted with perchloric acid. Using highly purified enzyme and [γ-32P]GTP as the phosphate donor, the neutralized extract then serves as substrate for adenosine kinase, and the AMP product is purified by high-performance liquid chromatography. Adenosine concentrations in plasma are linearly proportional to ratios in the enzymatically synthesized AMP and are calculated from a standard curve. The advantages of the method are: ease of sample preparation; sensitivity of 20 nm in as little as 0.3 ml plasma; 20 samples per day can be analyzed by a single operator. Care must be used when obtaining plasma since cellular contamination will affect results. Using this assay, human plasma adenosine levels are 0.121 ± 0.054 μm for males and 0.101 ± 0.067 μm for females. 相似文献
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Carel J. van Oss 《Preparative biochemistry & biotechnology》2013,43(3):361-362
Two MMP-7-ase isoenzymes were purified 100-fold from rat muscle extract to apparent homogeneity, with an overall yield of 10%, using homogenization, ultracentrifugation, high-performance aqueous size-exclusion and high-performance anion exchange chromatography methods. When using a TSK G-2000SW column, the separation resulted in a 6-fold purification and 30% recovery of isoenzymes B and C. This concentrated enzyme extract was then passed through a TSK-DEAE-2SW column, using salt gradient at pH 7.5, with an additional 25-fold purification and 90% recovery of the isoenzymes. Two symmetrical enzyme peaks, representing isoenzymes B and C, were detected when performing purity tests of the active enzymes on the anion exchanger and reversed-phase HFLC columns. The procedures involved are extraction, ultracentri-fugation, chromatographies and enzyme assays and require less than five hours. 相似文献
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