Biochemistry

Metals are essential in many biological processes, including oxygenic photosynthesis. Here we described a method to measure the metal pool in whole cells and thylakoids, including the bioactive pool in intact photosynthetic protein complexes in the model oxygenic cyanobacterium Synechocystis PCC6803. In the first part of the protocol, whole cells and thylakoid membranes are carefully prepared, in which the total metal concentrations are measured by inductively coupled plasma triple-quadrupole mass spectrometry (ICP-QQQ-MS). In the second part of the protocol, isolated thylakoids are solubilized to release the integral membrane proteins and the metal binding protein complexes. These intact photosynthetic protein complexes are subjected to size exclusion chromatography (SEC) and metal binding in the size separated complexes is analyzed by hyphenation with ICP-QQQ-MS.

Manganese (Mn) is an essential micronutrient for all photoautotrophic organisms. Two distinct pools of Mn have been identified in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), with 80% of the Mn residing in the periplasm and 20% in cytoplasm and thylakoid lumen (Keren et al., 2002). In this protocol, we describe a method to quantify the periplasmic and intracellular pools of Mn in Synechocystis accurately, using inductively coupled plasma mass spectrometry (ICP-MS).

Organisms have developed many protective systems to reduce the toxicity from heavy metals. The nematode Caenorhabditis elegans has been widely used to determine the protective mechanisms against heavy metals. Responses against heavy metals can be monitored by expression of reporter genes, while sensitivity can be determined by quantifying growth or survival rate following exposure to heavy metals.

Lichens are good biomonitors for air pollution because of their high enrichment capability of atmospheric chemical elements. To monitor atmospheric element deposition using lichens, it is important to obtain information on the multi-element concentrations in lichen thalli. Because of serious air pollution, elemental concentrations in thalli of lichens from North China (especially Inner Mongolia, Hebei, Shanxi and Henan province) are often higher than those from other regions, therefore highlighting the necessity to optimize ICP-AES/MS (Inductively coupled plasma-atomic emission spectroscopy/mass spectrometry) for analyzing lichen element content. Based on the high elemental concentrations in the lichen samples, and the differences in the sensitivity and detection limits between ICP-MS and ICP-AES, we propose a protocol for analyzing 31 elements in lichens using ICP-AES/MS. Twenty-two elements (Cd, Ce, Co, Cr, Cs, Cu, K, La, Mo, Na, Ni, Pb, Rb, Sb, Sc, Sm, Sr, Tb, Th, Tl, V and Zn) can be identified by using microwave digestion- ICP-MS, and 9 elements (Al, Ba, Ca, Fe, Mg, Mn, P, S and Ti) by using ashing-alkali fusion digestion- ICP-AES.