Krieg P, Lendl B, Vonach R, Kellner R
Institute for Analytical Chemistry, Vienna Technical University, Getreidemarkt 9/151, A-1060, Vienna, Austria.
Anal Bioanal Chem. 1996 Dec;356(8):504-7. doi: 10.1007/s0021663560504.
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.
提出了一种利用傅里叶变换红外光谱法测定水溶液和人血清中α-淀粉酶活性的新方法。在α-淀粉酶与简单淀粉反应过程中，当没有有色或电化学活性物质生成或消耗时，应用傅里叶变换红外光谱检测，也可以直接跟踪所研究酶催化的化学反应。因此，通过记录两个傅里叶变换红外光谱，一个在将样品与底物（淀粉）溶液混合后立即记录，另一个在反应20分钟后记录，就可以成功地测定α-淀粉酶活性。从这两个傅里叶变换红外光谱计算出差分光谱，从而消除了基质的非特异性吸收。所得差分光谱的强度与在所研究的时间间隔内发生的反应程度相对应，因此可以与样品中酶的活性相关联。所开发的方法在水溶液中80至1400 U/l范围内呈线性（700 U/l时相对标准偏差=5%），并且也成功地应用于人血清中α-淀粉酶活性的测定，实现了100至800 U/l的线性工作范围（150 U/l时相对标准偏差=11%）。
