Molybdenum Speciation in Raw Phloem Sap of Castor Bean

Abstract

Separation and quantification of molybdenum (Mo) in raw phloem sap from castor bean (Ricinus communis L.) was performed by size exclusion chromatography (SEC) and further purification was performed using quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE). For elemental detection, an inductively coupled plasma quadrupole mass spectrometer (ICP-QMS) was applied. Two different SEC columns were utilized: column A, Sephadex G-50 SF (700 mm × 24 mm), and column B, Sephadex G-25 M (28 mm × 9 mm). The protein content of the fractions was determined by the Bradford method. Using column A, two peaks of Mo were detected consisting of a main peak (Mo_A2) in the low molecular weight area (< 1.35 kDa), and a minor peak (Mo_A1, ≥ 30 kDa) at the void volume of the column. Both Mo species were detected at the ultraviolet (UV) active absorption area of raw phloem sap. Two peaks of Mo were also detected using column B, the first peak (Mo_B1) being at the same elution volume as the protein of raw phloem sap, and the second one (Mo_B2) was eluted in the area of 1.5 to 2.4 mL of elution volume. Raw phloem sap digested by proteinase K-enzyme indicates a significant reduction of Mo_B1 peak, which suggests that the peak may contain Mo bound to protein or polypeptides. The raw phloem sap and SEC fraction containing highest Mo concentration (Mo_A2) were furthermore separated by QPNC-PAGE. The result reveals that the Mo-containing fraction from the raw phloem sap was eluted at the same retention volume as the purified sample. This implies that the Mo species were also successfully separated and purified using QPNC-PAGE.

Keywords:

• Castor bean
• Mo speciation
• phloem sap
• QPNC-PAGE
• SEC

The authors are very indebted to Prof. Dr. Klaus Günther and Dr. Björn Thiele for their valuable guidance and encouragement during research activities of principal author at the Institute for Chemistry and Dynamics of the Geosphere: ICG–III Phytosphere, Helmholtz Research Centre Juelich, Germany. authors are also thankful to the institute director (Prof. Dr. Ulrich Schurr) his permission and support during this work. The financial support of Universitas Islam Indonesia is gratefully acknowledged.