Isolation, Purification and Characterization of Acid Phosphatase from Germinating Vigna radiata Seeds.

Abstract

The acid phosphatase (EC 3.1.3.2 ) has been purified from germinating seeds of vigna radiata through ammonium sulphate precipitation, DEAE-Cellulose chromatography and concanavalin A-Sepharose 4B chromatography. The specific activity of 1291 nkat.mg⁻¹of protein was obtained with recovery of nearly 1%. About 222 times purification was achieved. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) resolved two bands of acid phosphatase corresponding to molecular weight of 29 kilo Dalton (kDa) and 18 kDa. The molecular weights of native enzyme determined by gel filtration on calibrated Sephadex G-100 column were found to be 29 kDa and 18 kDa The apparent Km value of 29 kDa isoenzyme with p-nitrophenyl phosphate (pNPP) as substrate was 0.3 mM and Vmax was 1336 nmol.sec⁻¹.mg⁻¹ of protein. The optimal pH for this enzyme was 5.5 and pH stability was 4-7. It had optimum temperature of 50oC and temperature stability was 0- 50oC. The enzyme hydrolysed various phosphorylated compounds non-specifically. It was competitively inhibited by phosphate, vanadate while fluoride showed non- competitive inhibition and molybdate exhibited an inhibition of mixed type. It was found insensitive to tartrate and concluded that this enzyme was recognized as tartrate resistant acid phosphatase.