Analytical problems in determining peptide nitrogen in the humic fractions of three Italian soils

Abstract

Peptides were released from organic matter fractions of three Italian soils (humin, humic and fulvic acids), when the samples were hydrolyzed in Ba(ON) 2-saturated solution at 105°C for 2 hr. The peptides obtained were separated using electrophoresis and paper chromatography. The presence of polypeptides in the soil organic matter was indicated by: (1) their hydrolysis by pronase; (2) the amino acids released by 6 N HCl hydrolysis; (3) The comparison of i.r. spectra of humic fractions before and after hydrolysis with 6 N HCl. Attempts at isolating the native proteinaceous compounds using electrophoresis in polyacrylamide gel failed; additionally, our attempts to hydrolyze proteinaceous components enzymatically in unfractionated soil organic matter, as well as in its fractions, before and after methylation, with pepsin, papain and pronase, were unsuccessful. Pronase demonstrated a weak proteolytic activity only at very low substrate-enzyme ratios (20 : 1) in humic and fulvic fractions and in whole phyrophosphate extract. Deproteinated substrates treated with pronase also released free amino acids, suggesting autodigestion. In humin, humic and fulvic fractions we found a total amino acid content of 40–45%, 12–24% or 1–85, respectively. Amino acid recovery from single fractions was about 70–80% of the total content in the unfractionated soil.