Enolase Activity Assay

The enzymatic activity of enolase was determined as previously described.¹⁵ Briefly, the substrate 2-PGA was added at 1 mM concentration to test buffer. The conversion of the 2-PGA to phosphoenolpyruvate (PEP) was followed with 8 μg of enolase. The absorbance of the PEP product was monitored at 240 nm at 25 °C for 1 min with a spectrophotometer, JASCO UV–vis V-530. One unit of enolase activity is defined as the amount of 1 μmole of PEP formed in 1 min by 1 mg of the enzyme under the reaction conditions. The molar absorption coefficient was taken as 1520 /(M^-1 cm^-1) to calculate the amount of product formed from 2-PGA. The specific activity was expressed as international units per milligram of enzyme protein.