Bordalo, Adriano A.

Mesquita, Raquel

Rangel, António O. S. S.

Santos, Inês C.

English

Repositório Institucional da Universidade Católica Portuguesa

     3PYCheM | Universidade do Porto | 23 Use of solid phase extraction in a sequential injection system for the determination of alkaline phosphatase activity in plant roots  Inês C. Santos1,*, Raquel B. R. Mesquita1,2, Adriano A. Bordalo2, António O. S. S. Rangel1  1CBQF/Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal 2Laboratory of Hydrobiology, Institute of Biomedical Sciences Abel Salazar (ICBAS) and Institute of Marine Research (CIIMAR), Universidade do Porto, Porto, Portugal *icsantos@porto.ucp.pt   Phosphorus (P) is an essential nutrient to all organisms and in soils P is the most limiting nutrient that controls living processes in plants. Dissolved inorganic phosphate is readily available for assimilation by organisms such as bacteria and plants. For that reason, when dissolved inorganic phosphate is depleted compared to other nutrients both in water and soil, phytoplankton, plants and bacteria have the ability to obtain phosphorus from dissolved organic compounds due to the production of an enzyme named alkaline phosphatase. This enzyme hydrolyzes phosphate monoesters releasing inorganic phosphate and organic matter. Its production is regulated by inorganic phosphate concentrations and internal P levels so the values of alkaline phosphatase are an excellent indicator of P status. In this work, a sequential injection methodology was developed for the determination of alkaline phosphatase activity in root plants. The enzymatic activity was assessed using p-nitrophenyl phosphate as substrate and measuring the absorbance of the colored product, p-nitrophenol. The alkaline phosphatase is an extra/intra cellular metalloenzyme, as it requires metal ions in the active site to carry out the catalytic activity. Exploring this feature of the enzyme and aiming for the low levels found, a step for the in-line pre-concentration was included. As alkaline phosphatase needs Zn2+ ions in the active site, a resin, Nitrilotriacetic Acid (NTA) Superflow, was charged with Zn2+ to retain the enzyme. The proposed methodology allowed the determination of alkaline phosphatase activity in plant roots within a range between 0.044  0.441 unit cm-3 enzyme activity and 19  280 mol dm-3 pNP. A determination rate of 17 h-1 and detection limits of 0.025 unit cm-3 enzyme activity and 1.9 mol dm-3 p     Figure 1. Sequential injection manifold for the spectrophotometric determination of alkaline phosphatase activity: P, peristaltic pump; SV, eight-port selection valve; HC, 4.25 m holding coil; S, sample or standard; Col., column with NTA Superflow resin charged with Zn2+ ions; RAP, pNPP or pNP; Eb, eluting buffer; Db, diethanolamine buffer; RC, 0.95 m reaction coil; , spectrophotometer (405 nm); W, waste.   Acknowledgements: I. C. Santos thanks to Fundação para a Ciência e a Tecnologia (FCT, Portugal) and Fundo Social Europeu (FSE) the grant SFRH/BD/76012/2011. R. B. R. Mesquita thanks to Fundação para a Ciência e a Tecnologia (FCT, Portugal) and Fundo Social Europeu (FSE) the grant SFRH/BPD/41859/2007. This work was supported by National Funds from FCT through project PEst-OE/EQB/LA0016/2011. 123 74 658RAPSPSVWRCWDbEbCol.H2OHCOC12

Use of Solid Phase Extraction in a Sequential Injection System for the Determination of Alkaline Phosphatase Activity in Plant Roots

https://repositorio.ucp.pt/bitstream/10400.14/10049/1/ON20.pdf

Use of Solid Phase Extraction in a Sequential Injection System for the Determination of Alkaline Phosphatase Activity in Plant Roots

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Repositório Institucional da Universidade Católica Portuguesa