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Combining electrophoresis with detection under ultraviolet light and multiple ultrafiltration for isolation of humic fluorescence fractions
Authors:Trubetskaya Olga E  Shaloiko Lubov A  Demin Dmitrii V  Marchenkov Victor V  Proskuryakov Ivan I  Coelho Christian  Trubetskoj Oleg A
Institution:aBranch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 142290 Pushchino, Moscow region, Russia;bInstitute of Basic Biological Problems, Russian Academy of Sciences, 142290 Pushchino, Moscow region, Russia;cInstitute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow region, Russia;dLaboratoire de Photochimie Moléculaire et Macromoléculaire, UMR CNRS-Université Blaise Pascal 6505, 63177 Aubière Cedex, France
Abstract:Polyacrylamide gel electrophoresis of chernozem soil humic acids (HAs) followed by observation under UV (312 nm) excitation light reveals new low molecular weight (MW) fluorescent fractions. Ultrafiltration of HAs sample in 7 M urea on a membrane of low nominal MW retention (NMWR, 5 kDa) was repetitively used for separation of fluorescent and non-fluorescent species. Thirty ultrafiltrates and the final retentate R were obtained. Fluorescence maxima of separate ultrafiltrates were different and non-monotonously changed in the range of 475–505 nm. Fluorescence maxima of less than 490 nm were detected only in the four first utrafiltrates. For further physical–chemical analyses all utrafiltrates were combined into a fraction called UF < 5 (NMW < 5 kDa). Retentate R demonstrated very weak fluorescence under 270 nm excitation, while fluorescence intensity of UF < 5 was about six times higher than of the bulk HAs. Fraction UF < 5 was further ultrafiltrated on membranes of MNWR 3 kDa and 1 kDa, yielding three subfractions UF3-5, UF1-3 and UF < 1 with NMW 3–5 kDa, 1–3 kDa and <1 kDa, respectively. The validation of the UF procedure was performed by size exclusion chromatography on Sephadex G-25 column. The fluorescence maxima were found to be at 505, 488 and 465 nm for UF3-5, UF1-3 and UF < 1, respectively, with increasing of fluorescence intensity from UF3-5 to UF1-3 to UF < 1 fraction. EPR analysis showed that the amount of free radicals was the largest in retentate R and drastically decreased in fluorescent ultrafiltrates. The results demonstrate that more than one fluorophore is present in chernozem soil HAs complex.
Keywords:Humic acid  Fluorescence  Electrophoresis  Multiple ultrafiltration
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