Method for assessing the content of molybdenum enzymes in the internal organs of fish

Molybdenum enzymes (Mo-enzymes) contain a molybdenum cofactor (MoCo) in the active site. These enzymes are potentially interesting for studying the survival mechanism of fish under hypoxic water conditions. This is because Mo-enzymes can synthesize nitric oxide from nitrates and nitrites, which are present in high concentrations under hypoxic water conditions. However, there is currently no method for assessing the Mo-enzymes content in the fish internal organs. Methods capable of determining Mo-enzymes content in the fish are of major importance. For this purpose, a method for quantitative determination of MoCo from plant tissues was modified. We demonstrated the Mo-enzyme content assessment by isolated MoCo from the fish's internal organs and the Neurospora crassa nit-1 extract containing inactive NADPH nitrate reductase. The Mo enzyme content was calculated using a calibration curve in nM of nitrites as a product of restored NADPH reductase activity after complementation with MoCo. Here we present a robust laboratory method which can be used to assess the content of Mo-enzymes in the internal organs of fish. • Mo-enzymes play a crucial role in detoxifying toxic compounds. Therefore, it is important to develop a method to accurately determine the amount of Mo-enzymes present. Notably, the method demonstrated the efficiency and accuracy as detected high content of Mo-enzymes in the liver and intestines (P < 0.0001). The obtained data on the distribution of Mo-enzymes in the internal organs of this species correspond to that of other vertebrates. Here, we present a rapid, sensitive, accurate and accessible method. • The developed method is simple and easy to use. Importantly, the protocol does not require complex manipulations, and the equipment used is available in most laboratories. The article provides step-by-step instructions for reproducing the method.