FLOW INJECTION ZYMOGRAPHY FOR THE ON-LINE DETECTION OF ENZYMEACTIVITY

new method - Flow Injection Zymography - has been developed for the detection of enzymeactivity during purification procedures usingflow injection analysis (FIA). Four oxidases and a lipase were used as model enzymes. The method involves frequent sampling ofeluents via a flow injection system and enables swift localization of the active fractions. Furthermore, the dilution technique of zone sampling enables estimation of the purification effect

INVESTIGATIONS ON GLUCOSE BIOSENSORS USING LANGMUIR-BLODGETT TECHNIQUESFOR THE IMMOBILIZATION OF DERIVATIZED GLUCOSE OXIDASE

Anovel method for the detection of glucose using modified glucose oxidase on Langmuir-Blodgett films is presented. A new polymer Langmuir-Blodgett film was used to immobilize modified glucose oxidase together with an oxygen sensitive fluorescent indicator. Five different glucose oxidase preparations were investigated: native, degtycosylated, N-palmitoy!-modified, periodate oxidized and y-carbodiimide treated. The oxygen consumption in the prescence of glucose was measured via dynamic quenchingof the fluorescence of the oxygen sensitive indicator

Inhibitor affinity chromatography: Profiling the specific reactivity of the proteome with immobilized molecules

An inhibitor affinity chromatography (IAC) method has been developed for the analysis of inhibitor-protein interactions as a complementary approach to two-dimensional electrophoresis for functional proteomics studies. The procedure was developed utilizing a cyclin-dependent kinase 2 (Cdk2) inhibitor coupled to a polymeric resin and validated using a number of proteins interacting with the inhibitor with different specificities. Cdk2 and the other kinases bound and eluted from the resin in accordance with the relative in vitro potency of the inhibitor for each enzyme. Molecular interactions with the Cdk2 inhibitor were compared for HCT116 cancer cells versus rat pancreatic acinar cells. Proteins interacting with the ligand on the IAC matrix were identified by mass spectrometry. Isothermal calorimetry was used to confirm and quantitatively evaluate the binding affinity of some of the interacting proteins. Heat-shock protein (Hsp) 70 and Hsp27 were the strongest interactors with the inhibitor, displaying binding affinities comparable to those of Cdk2. These results support the use of IAC as a general method for the rapid identification and qualitative evaluation of the in vivo targets and potential side effects of a given drug