Determination of a-amylase activity using Fourier transform infrared spectroscopy

Abstract

A new method for the determination of alpha-amylase activity in aqueous solutions and human serum with FTIR-spectroscopy is proposed. The chemical reaction catalyzed by the enzyme under study can be followed directly when applying FTIR-spectroscopic detection also in the case, where no colored or electrochemical active species are generated or consumed during the course of the reaction of alpha-amylase with simple starch. Therefore the determination of the alpha-amylase activity could successfully be performed by recording two FTIR-spectra, one immediately after mixing the sample and a substrate (starch-) solution and the other after a 20 min reaction time. From these two FTIR-spectra a difference spectrum was calculated hereby eliminating an unspecific absorption of the matrix. The intensities of the resulting difference spectra corresponded to the extent of the reaction which took place during the investigated time interval and hence could be related to the activity of the enzyme in the sample. The developed method is linear from 80 to 1400 U/l (r.s.d.=5% for 700 U/l) in aqueous solutions and was also successfully applied to the determination of alpha-amylase activity in human serum where a linear working range from 100 to 800 U/l (r.s.d.=11% for 150 U/l) was achieved.

Following Enzyme Activity with Infrared Spectroscopy

“…When a molecular structure is modified in an enzymatic reaction, the infrared spectrum is altered and changes in infrared absorption can be followed to monitor the progress of the reaction. Measurements of enzyme activity with infrared spectroscopy are relatively straightforward and several studies have been published comprising urea hydrolysis by urease [ 1 , 2 ], cefoxitin hydrolysis by β-lactamase [ 3 ], deacylation of cinnamoyl-chymotrypsin [ 4 ], ATP hydrolysis by the Ca 2+ -ATPase [ 5 – 7 ], dephosphorylation of fructose 1,6-bisphosphate by fructose-1,6 bisphosphatase [ 8 ] and of 4-nitrophenylphosphate by alkaline phosphatase [ 9 ], oxidation of d -glucose by glucose oxidase [ 10 ], hydrolysis of sucrose by β-fructofuranosidase [ 11 – 13 ], of maltose by amyloglucosidase [ 13 ] and of starch by amylogucosidase [ 14 , 15 ] and α-amylase [ 15 , 16 ], hydrolysis of amides [ 17 , 18 ] and synthesis of hydroxamic acid derivatives [ 18 ] by amidase, hydrolysis of several organophosphorus compounds by diisopropyl fluorophosphatase [ 19 ], the reaction of α-ketoglutarate and Ala to Glu and pyruvate by glutamic-pyruvic transaminase [ 20 ], consumption of oxalate and production of formate and CO 2 by oxalate decarboxylase [ 21 ], and acetone-butanol fermentation [ 22 ].…”

Analysis of human tear fluid by Fourier transform infrared spectroscopy

“…There have been many reports of the analysis of biomolecular components of human body fluids by Fourier transform infrared spectroscopy (FTIR) technique, particularly in the past 10 years. In these studies, for example, human oral mucosa and saliva,24–28 skin,29–31 and serum32–35 were investigated by using FTIR. However, few studies of the analysis of tear fluid by means of FTIR technique exist.…”

Determination of Amyloglucosidase Activity Using Flow Injection Analysis With Fourier Transform Infrared Spectrometric detection

“…The influence of different starch concentrations on the performance of the FIA-FTIR system was investigated. A high starch concentration increases the linear range of the method because substrate saturation is guaranteed, 11 but the problem of cell clogging is also increased. A concentration of 55 g l 21 was selected, being the highest concentration which did not cause practical problems.…”

“…Application of FTIR spectrometry to molecular specific detection in flow injection analysis has already been demonstrated for enzyme substrate determinations using immobilized enzymes. 9,10 Furthermore, the use of FTIR spectrometry for the determination of aamylase activity in human sera 11 and the assay of bile saltstimulated lipase activity in reversed micelles 12 using batch methods has been reported. Combination of an FTIR spectrometer with an FIA manifold is of advantage as a high degree of automation is achieved, opening up the possibility of using this coupled technique for process analysis and process control.…”