结合凝胶电泳、同步辐射X射线荧光和原子光谱法对金属结合蛋白进行扫描和测定。

Metal-binding proteins scanning and determination by combining gel electrophoresis, synchrotron radiation X-ray fluorescence and atomic spectrometry.

作者信息

Verbi F M, Arruda S C C, Rodriguez A P M, Pérez C A, Arruda M A Z

机构信息

Departamento de Química Analítica, Instituto de Química, UNICAMP, PO Box 6154, 13084-971 Campinas, São Paulo, Brazil.

出版信息

J Biochem Biophys Methods. 2005 Feb 28;62(2):97-109. doi: 10.1016/j.jbbm.2004.09.008. Epub 2004 Nov 23.

DOI:10.1016/j.jbbm.2004.09.008

PMID:15680280

Abstract

In the present work, protein bands from in vitro embriogenic callus (Citrus sinensis L. Osbeck) were investigated using micro-synchrotron radiation X-ray fluorescence (muSR-XRF) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation. Metal-binding protein quantification was done after microwave oven decomposition of gel by synchrotron radiation total reflection X-ray fluorescence (SR-TXRF), flame atomic absorption spectrometry (FAAS) and flame atomic emission spectrometry (FAES). According to the analysis of the protein bands, it is possible to observe that both 81 and ca. 14 kDa proteins present different Fe signal intensity at different positions. The analysis of 53 kDa protein, showed even more interesting results. Besides Fe, the muSR-XRF experiments indicate the presence of Ca, Cu, K and Zn. Chemical elements such as Cu, K, Fe and Zn were determined by SR-TXRF, Mg by FAAS and Na by FAES. Ca was determined by SR-TXRF and FAAS only for accuracy check. In the mineralised protein bands of 81 and around 14 kDa band, only Fe was determined (105 and 21.8 microg g(-1)). For those protein bands (86-ca. 14 kDa) were determined, Ca, K, Cu and Zn in a wide concentration range (42.4-283, 2.47-96.8, 0.91-15.9 and 3.39-29.7 microg g(-1), respectively).

摘要

在本研究中，体外胚性愈伤组织（甜橙）的蛋白质条带经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离后，采用微同步辐射X射线荧光光谱（μSR-XRF）进行研究。通过同步辐射全反射X射线荧光光谱（SR-TXRF）、火焰原子吸收光谱法（FAAS）和火焰原子发射光谱法（FAES）对凝胶进行微波炉分解后，进行金属结合蛋白定量分析。根据蛋白质条带分析，可以观察到81 kDa和约14 kDa的蛋白质在不同位置呈现不同的铁信号强度。对53 kDa蛋白质的分析显示了更有趣的结果。除铁外，μSR-XRF实验表明还存在钙、铜、钾和锌。通过SR-TXRF测定铜、钾、铁和锌等化学元素，通过FAAS测定镁，通过FAES测定钠。仅通过SR-TXRF和FAAS测定钙以进行准确性检查。在81 kDa和14 kDa左右条带的矿化蛋白质条带中，仅测定到铁（分别为105和21.8 μg g-1）。对于所测定的那些蛋白质条带（86 kDa-约14 kDa），钙、钾、铜和锌的浓度范围较宽（分别为42.4-283、2.47-96.8、0.91-15.9和3.39-29.7 μg g-1）。