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Related Experiment Videos

Determination of intracellular nitrate.

J M Romero1, C Lara, M G Guerrero

  • 1Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla y C.S.I.C., Spain.

The Biochemical Journal
|April 15, 1989
PubMed
Summary

This study presents a new method for measuring intracellular nitrate using ion-exchange h.p.l.c. The technique accurately quantifies nitrate in cyanobacteria, aiding in the study of nutrient transport.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Microbiology

Background:

  • Nitrate is a crucial nutrient for many organisms.
  • Accurate measurement of intracellular nitrate is essential for understanding cellular processes.
  • Existing methods may lack sensitivity or be prone to interference.

Purpose of the Study:

  • To develop a sensitive and reliable method for determining intracellular nitrate concentrations.
  • To optimize the method for analysis in microbial systems, specifically cyanobacteria.
  • To address potential interferences in nitrate determination.

Main Methods:

  • Cell lysates prepared in 2 M phosphoric acid (H3PO4) after silicone oil centrifugation.
  • Nitrate analysis performed using ion-exchange high-performance liquid chromatography (h.p.l.c.).
  • Mobile phase optimized with H3PO4 and tetrahydrofuran (THF), adjusted to pH 1.9 with sodium hydroxide (NaOH).
  • Interference from nitrite eliminated using sulfamic acid.

Main Results:

  • A sensitive procedure for intracellular nitrate determination was established.
  • The method successfully quantified nitrate in the unicellular cyanobacterium Anacystis nidulans.
  • Interference from chloride, sulfate, and nitrite was addressed and managed.

Conclusions:

  • The developed ion-exchange h.p.l.c. method is effective for intracellular nitrate measurement.
  • This technique facilitates the study of nitrate uptake and transport mechanisms in microorganisms.
  • The procedure offers a robust tool for cellular nutrient analysis.

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