An ESR assay for alpha-amylase activity toward succinylated starch, amylose and amylopectin.
The esterification of the three polysaccharides, starch, amylose and amylopectin was carried out in pyridine-DMSO by succinic anhydride. The carboxylic groups in the succinylated polysaccharides were measured by FT-IR spectroscopy. The succinic derivatives were tested as alpha-amylase (1,4-alpha-D-glucan glucano hydrolase, E.C. 3.2.1.1) substrates. A colorimetric assay of the alpha-amylase activity indicated that this enzyme is active on succinic esters of starch and amylose and that the activity shows a linear decrease with the number of succinic units introduced into the polysaccharide. Since the colorimetric test was not suitable for the detection of the alpha-amylase activity when succinylated amylopectin was the substrate, we set-up an assay based on the labeling by a paramagnetic probe of the free carboxylic groups of succinylated polysaccharides. The kinetics of the alpha-amylase reaction were monitored by ESR spectroscopy through the increase of the mobility of the paramagnetic probe. The spin label used was the commercially available 4-amino-tempo. By this method we demonstrated that alpha-amylase is active on succinylated amylopectin. The utility of the assay for monitoring alpha-amylase activity when other methods (i.e. colorimetric tests) fail, is discussed.
Marcazzan
M
M
Department of Biological Chemistry, University of Padova, Italy.
Vianello
F
F
Scarpa
M
M
Rigo
A
A
D000687:Amylopectin / Q000737:chemistry*; D000688:Amylose / Q000737:chemistry*; D002264:Carboxylic Acids / Q000737:chemistry; D011198:Solanum tuberosum / Q000737:chemistry; D013055:Spectrophotometry, Infrared; D013057:Spectrum Analysis / Q000379:methods*; D013387:Succinic Anhydrides / Q000737:chemistry; D013997:Time Factors; D014908:Triticum / Q000737:chemistry; D000516:alpha-Amylases / Q000032:analysis*
D002264:Carboxylic Acids; D013387:Succinic Anhydrides; C031801:succinic anhydride; D000688:Amylose; D000687:Amylopectin; D000516:alpha-Amylases