Simultaneous determination of enzyme activities by FTIR-spectroscopy in an one-step assay R. Schindler, B. Lendl * Institute of Analytical Chemistry, Vienna University of Technology, Getreidemarkt 9/151, A-1060 Vienna, Austria Received 6 October 1998; received in revised form 5 February 1999; accepted 16 February 1999 Abstract The simultaneous quanti®cation of b-fructofuranosidase (invertase, EC 3.2.1.26) and glucan 1,4-a-glucosidase amyloglu- cosidase, EC 3.2.1.3) activities in an one-step assay is reported. The approach is based on chemometric evaluation of the mid- IR spectra of the reaction mixture, measured with a sequential injection (SI) system with FTIR spectroscopic detection. Two partial least squares (PLS) calibration models were established automatically. The ®rst model was calculated from reaction mixtures after the action of standard mixtures of both enzymes. The other was built up with mixtures of the substrates (maltose, sucrose) and products (fructose, glucose) without any enzymatic reaction. The establishment of the second model was more rapid as no time for an enzymatic reaction is necessary. Both models gave comparable results for the prediction of mixtures containing 0±2.0 U/ml of each enzyme, with mean errors between 0.08 and 0.05 U/ml. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Enzyme activity; Glucan 1,4-a-glucosidase; b-Fructofuranosidase; Invertase; Amyloglucosidase; Fourier transform infrared spectroscopy; Sequential injection analysis; Multivariate calibration 1. Introduction Assays for enzyme activities and kinetic studies of enzymatic reactions are frequently based on a sequence of reactions or often employ specially designed substrates in order to get an easily detectable species as e.g. NAD(P) or some released dye. Two inherent drawbacks of this methodology can be iden- ti®ed, ®rstly it may be dif®cult to relate changes in the measured signal unambiguously to changes occurring in the monitored reaction. Secondly, the simultaneous monitoring of several enzymatic reactions is hindered as the assay conditions for different enzymes are often incompatible or if their reactions ®nally lead to the same product. For the reasons mentioned, an one-step assay with the possibility to monitor several enzymatic reactions simultaneously is of high interest. Mutual in¯uences of the enzymes could be identi®ed more easily and lengthy reaction chains using expensive reagents can be avoided. Separation techniques like HPLC or GC could be used to separate educts or products, but these techniques are time-consuming and only single-point assays are possible. Multidimensional spectroscopic Analytica Chimica Acta 391 (1999) 19±28 *Corresponding author. Tel.: +43-1-58801/4828; fax: +43-1- 5867813; e-mail: blendl@mail.zserv.tuwien.ac.at 0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII:S0003-2670(99)00192-0