Determination of Free Ammonium and Asparagine and Glutamine Amide-Nitrogen in Extracts of Plant Tissue

Abstract

Summary. A relatively simple and rapid procedture for the measturement of free ammonium and the amides in plant extracts is describedl. The method was developed by combining a cation-exchange method for blood ammon;a with a differential acidhydrolysis procedure for asparagine and glutamine amide-nitrogen.The recovery of standard samples (100-400 ,ug of ammoniutm-or amide-nitrogen) of free ammonitum, asparagine, and glutamine after being run through the extraction, column, and analytical procedures ranged between 99 and 102 %.The harvest, extraction, and analytical procedtures were tested on shoots from 4 to 6-day-old germinating barley seeds. The high levels of the amides and the low level of free ammoniuim present in the tissue extracts indicated that the extraction and analytical procedures resutlted in little if any hydrolysis of the amides. Methods (3,8,9) often employed to estimate the levels of free ammonium, asparagine, and glutamine in extracts of plant tissule have the disadvantage of requiiring a time-consuiming distillation step in the determination of free ammonium. This distillation step, unless controlledl very careftlly, can restult in the partial hydrolysis of the amide bond of glutamitne and the stubseqtuent recovery of this amidenitrogen with the free-ammonitum fraction.Duiring the past several vears, enzymatic assays for total amide-nitrogen (5), and the amide-nitrogen grotups of asparagine (7) and gluitamine (10)

Determination of free ammonium in plant tissue.

“…For the determination itself use has been made of colorimetric methods (Nessler reagent or indophenol blue) (Yuen & Pollard, 1952), or NH3 has been distilled over from the extract buffered at a relatively low pH value by, for example, a borate buffer (Breteler, 1972). A separation of ammonium from organically bound nitrogen by means of a cation exchange resin was also proposed (Routchenko, 1967;Henderlong & Schmidt, 1966).…”