ENZYMATIC CHARACTERIZATION OF HUMIC SUBSTANCES

A method is described for the specific hydrolysis of amino acids from humic substances by using the proteolytic enzyme ''Pronase E''. The amino acids are analyzed by a fluorescence detector after precolumn derivatization and HPLC-separation. The pattern and the concentration of amino acids after the enzymatic hydrolysis are set up for humic substances of different origin. Due to the results the enzymatic hydrolysis is suitable for the characterization of humic substances with a nitrogen content higher than 3%. As shown in a kinetic study of the hydrolysis of a fulvic acid the influence of humic substances on the enzyme activity increases in case of lower nitrogen content. Due to the specificity of the proteolytic enzyme the operationally defined method is limited as shown by a protein of a known amino acid content. The amino acids leucine, valine, tyrosine and histidine are released to the highest extent. The determined total extent for the hydrolysis of Insulin B is 26.34% and for Casein 38.37%. By comparison of the enzymatic hydrolysis with the classic acid hydrolysis the advantages of the biochemical method are the simple experimental handling and the mild reaction conditions. Amino acids which are very unstable like glutamine and asparagine can only be analyzed by the enzymatic hydrolysis.