Succinate Dehydrogenase Activity Assay in situ with Blue Tetrazolium Salt in Crabtree-Positive Saccharomyces cerevisiae Strain
Dorota Kregiel*, Joanna Berlowska and Wojciech Ambroziak
Institute of Fermentation Technology and Microbiology, Technical University of Lodz, Wolczanska 171/173, PL-90-924 Lodz, Poland
Article history:
Received July 18, 2007
Accepted May 15, 2008
Key words:
succinate dehydrogenase (SDH), Saccharomyces cerevisiae, enzymatic activity in situ, blue tetrazolium
Summary:
A spectrophotometric method for determining succinate dehydrogenase (SDH) activity assay in azide-sensitive yeast Saccharomyces cerevisiae has been developed. The permeabilization of yeast cells by 0.05 % digitonin permitted to study yeast enzymatic activity in situ. The reduction of blue tetrazolium salt (BT) to blue tetrazolium formazan (BTf) was conducted in the presence of phenazine methosulphate (PMS) as an exogenous electron carrier, and sodium azide (SA) as an inhibitor of cytochrome oxidase (Cyt) pathway. Various factors such as type of substrate, BT concentration, cell number, temperature and time of incubation, and different Cyt pathway blockers were optimized. In earlier studies, dimethyl sulfoxide (DMSO) had been selected as the best solvent for extraction of BTf from yeast cells. The linear correlation between permeabilized yeast cell density and amount of formed formazan was evidenced in the range from 9·107 to 5·108 cells per sample solution. Below the yeast cell concentration of 107 the absorbance values were too low to detect formazans with good precision. This standarized procedure allows the estimation of SDH activity in whole cells, depending on vitality level of yeast populations. Significant increases of succinate dehydrogenase activities were observed in sequential passages as the result of the increase of activity of the strain and adaptation to cultivation conditions.
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