A COMPARISON OF SOIL ADENINE-NUCLEOTIDE MEASUREMENTS BY HPLC AND ENZYMATIC ANALYSIS

A high pressure liquid chromatography (HPLC) method was developed to measure the soil adenine nucleotides ATP, ADP and AMP. This method was compared with conventional measurement of these nucleotides by enzymatic conversion of ADP and AMP to ATP and final measurement of ATP by the luciferin-luciferase enzyme system. Sufficiently sensitive measurement of these nucleotides with the HPLC system was achieved by chemical derivatization with chloracetaldehyde, which formed highly fluorescent 1-N6-ethenoadenosine nucleotides. The efficiency of this reaction was 100% for ATP and AMP and 93% for ADP. The nucleotides can be quantified in the low picomole range using this derivatization. The concentrations of adenine nucleotides in four arable soils were measured following extraction with two different reagents: an acid H2SO4-based reagent and an alkaline NaHCO3-based reagent. The difference between amounts of ATP, ADP measured by the HPLC and the enzymatic conversion technique was mostly < 10%. Higher differences were observed for AMP in some extracts. The comparison of the two extractants showed that the NaHCO3-based reagent extracted about twice as much nucleotides from two of the four soils. This trend was also found in the extracts of the two other soils, but the differences were less pronounced. It is concluded that, depending upon the soil under investigation, different extractants differ in their capabilities to extract the particular adenine nucleotides from soil.