A Study of His-Tagged Alkaline Phosphatase Immobilization on a Nanoporous Nickel– Titanium Dioxide Film
✍ Scribed by Juan Kun Zhang; Anthony E.G. Cass
- Publisher
- Elsevier Science
- Year
- 2001
- Tongue
- English
- Weight
- 71 KB
- Volume
- 292
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
For the monitoring procedure described here beef heart mitochondria instead of rat liver mitochondria can be used (Fig. 1b). The mitochondria from beef heart offer the advantage that once prepared, they can be stored in a freezer for months (8). Once they are thawed and resuspended in the sucrose medium, the responses are the same as those with freshly prepared rat liver mitochondria. The only difference is that after some minutes the linearity of the response, ⌬A against time, is no longer conserved (Fig. 1b); however, if the initial slope is measured, the results and the sensitivity are the same. This is a significant advantage since beef heart mitochondria can be stored once prepared, and beef heart tissue is readily available.
Interference could be due to the presence of chemical compounds (i.e., organometallic compounds) which induce the opening of a transition pore with consequent swelling even under nonenergized conditions (17). In this case, however, by operating in the presence of Cyclosporine, the inhibitor of the permeant pore, the interferences are excluded. Other interferences could be due to the presence of divalent ions such as Ca 2ϩ or Mg 2ϩ since these compounds alter the membrane structure. The problem is avoided by working in the presence of EDTA in the medium. Organic compounds such as alcohols give interferences only at high concentrations, above millimolar.
Conclusions. A procedure which allows the selective monitoring of detergents in solution has been described. The method utilizes the sensitivity of rat liver mitochondria (or beef heart mitochondria, freshly prepared or thawed) toward detergent compounds since these compounds give rise to mitochondrial swelling. Swelling is measured by an absorbance change and the rate of this change is proportional to the detergent concentration. Since a biosensor for the monitoring of all surfactants (cationic, anionic, and neutral) has not yet been proposed, and since the procedure is not greatly influenced by the presence of interfering compounds, this method is proposed as a prescreening test for direct monitoring of all kinds of surfactants in environmental samples.