High-Throughput Analysis of Enzyme Activities
- Iowa State Univ., Ames, IA (United States)
High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surface of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different detection modes. The detection limits are 22, 15, and 3.6 μM ADP for using fluorometer, ICCD and PMT respectively. The Michealis constant K m(ATP) for protein kinases ranges from 5 to 100 μM. For inhibitor screening, in order to get the most accurate result, ATP concentration should be closed to Km. In this case, further lower the detection limit of ADP is needed before the direct detection of ADP can be actually used in kinase inhibitor screening.
- Research Organization:
- Ames Lab., Ames, IA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC)
- DOE Contract Number:
- AC02-07CH11358
- OSTI ID:
- 933034
- Report Number(s):
- IS-T 2294; TRN: US200814%%285
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
AUTOMATION
CAPILLARIES
CATALYSTS
CHARGE-COUPLED DEVICES
DETECTION
ELECTROPHORESIS
EMISSION SPECTRA
ENZYME ACTIVITY
ENZYMES
FLUORESCENCE
IMMOBILIZED ENZYMES
KINETICS
OPTICAL PROPERTIES
PHOSPHOTRANSFERASES
PROTEINS
REACTION KINETICS
ROBOTS
SENSITIVITY
SUBSTRATES