Extracellular carbonic anhydrase: Method development and its application to natural seawater

Abstract

We developed an effective fluorometric technique to quantify extracellular carbonic anhydrase (eCA) present in natural seawater samples. The technique includes the separation of eCA from cells to achieve low detection limits through high signal : noise ratios. eCA was efficiently extracted from cell membranes by treatment with 0.1 M phosphate buffer containing 2.5 M NaCl. The free eCA specifically forms a fluorescent complex with dansylamide, and the detection limit of the complex is below 0.1 nM. We applied the technique to samples from different culture solutions and natural seawater collected from the Baltic Sea. We observed eCA concentrations to be in the range of 0.10-0.67 nM in natural seawater. The data indicated that this technique is very sensitive, accurate, and feasible for routine and shipboard measurement of eCA from natural seawater. It is therefore an effective and rapid tool to investigate the carbon acquisition of phytoplankton both in mono culture as well natural communities.Extracellular carbonic anhydrase (eCA) is a zinccontaining enzyme that catalyzes the slow interconversion between HCO 2 3 and CO 2 at the cell surface (Aizawa and Miyachi 1986). In seawater, phytoplankton can be CO 2 -limited because dissolved inorganic carbon occurs mainly as HCO 2 3 . Due to the inefficient conversion toward HCO 2 3 , eCA is important for cellular CO 2 acquisition in phytoplankton.

The carbonic anhydrase activity of sinking and suspended particles in the North Pacific Ocean

“…Our results from the entire North Pacific, which span at least two distinct ecological regimes, are most consistent with those of Mustaffa et al (), who suggest that eCA concentrations in the surface ocean are related to surface ocean ecology. Opal to carbonate ratios increase northward in the Pacific, with the historically highest opal fluxes at around 50° north (Kawahata et al ).…”

“…However, the fact that these data do not collapse onto a single relationship suggests a decoupling between total Chl a fluorescence and total CA activity. This decoupling of CA and Chl a concentration has also been shown by Mustaffa et al () in profiles of the upper 200 m in the Mediterranean Sea. Sta.…”

“…The presence of a large pool of eCA in marine particulate matter is not without precedent. The results of Mustaffa et al () similarly suggest that a significant amount of CA in Mediterranean Sea water is externally bound. Our results are also consistent with the localization of several externally bound isoforms of CA found in marine diatoms (Hopkinson et al ).…”

“…Isoforms of CA have been found intracellulary and extracellularly bound to outer cell walls or membranes, or untethered (Elzenga et al ; Martin and Tortell ; Mustaffa et al ). Transport of CO 2 from seawater into photosynthetic organisms has also been shown to require CA, especially in diatoms (Elzenga et al ; Moroney et al ; Martin and Tortell ; Xu et al ).…”

“…Most CA isoforms require either Zn or Cd in their active site, implicating the cycling of bioactive trace metals such as Zn and Cd in the expression of CA (Christianson and Fierke ; Xu et al ; Twining and Baines ). Despite a few measurements of CA in the surface ocean (Tortell et al ; Mustaffa et al , b ), little is known about the production of marine CA, its fate in material sinking out of the surface ocean, or if it is active in other marine environments such as on particles or in ocean sediments.…”