PURIFICATION AND CHARACTERIZATION OF SUCCINYL THIOKINASE FROM PIG HEART

In our earlier studies, the concentration of succinate in the body fluid of hard clams was used as an anoxia-stress indicator. The concentration of succinate was monitored by a color kit the key component of which is succinyl thiokinases (STK). Therefore, finding a source of STK is very important for developing the color kit. STK from pig heart was purified over 850-fold to apparent homogeneity. It has a dimeric structure with a relative molecular mass of 69,200, and there are two types of subunits, and , with respective apparent molecular weights of 38,000 and 47,000. Among the isolation steps, the purification factor (14.6) of a CHT ceramic hydroxyapatite column was the highest. STK is heat-labile, and the addition of 2.4 M ammonium sulfate made it more heat-stable than that in 20% glycerol at 60°C. No STK activity was found in the absence of MgCl2. Fifty percent of the activity of STK was inhibited by 8 mM CaCl2 in the presence of 20 mM MgCl2. The stability of STK stored in glycerol was greater than that stored in ammonium sulfate at 4°C. Based on exponential fit data, respective half-lives of STK stored in control, 10% glycerol, 20% glycerol, and 30% glycerol were 1.6, 3.7, 7.5, and 8.2 months.

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