Assay for spermidine synthase activity by micellar electrokinetic chromatography with laser-induced fluorescence detection.

An assay for spermidine synthase (SPDS) activity in rat liver has been developed using micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection to enable the discovery of SPDS inhibitors. The assay was established by estimating the amount of spermidine (SPD) produced from the putrescine (PUT) present by SPDS. The SPD in an enzyme reaction mixture of homogenized rat liver could directly react with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent. The NBD derivatives of SPD and PUT could be separated and detected by MEKC-LIF detection within 15 min. The IC(50) value measured for SPDS inhibitor, 4-methylcyclohexylamine, in rat liver by this assay was consistent with published data. Our SPDS assay using MEKC-LIF is simple and allows easy determination of SPDS activity in homogenized samples without troublesome procedures such as preparation of antibody or fluorescence-labeled substrate. The assay should be effective for discovering the SPDS inhibitors using biological samples.

Assay of collagenase activity for native triple-helical collagen using capillary gel electrophoresis with laser-induced fluorescence detection.

Three collagenase assays for two native triple-helical collagens have been developed using capillary gel electrophoresis (CGE) with laser-induced fluorescence (LIF) detection in order to discover the matrix metalloproteinases (MMPs) inhibitors. These collagenase assays include measurement of the activities of interstitial collagenase (MMP-1) and neutrophil collagenase (MMP-8) against type I collagen, and collagenase-3 (MMP-13) against type II collagen, and the enzyme activities could be readily measured by determining the 3/4 fragments produced from the cleavage of the native collagens. The highly desired sensitivity of the assays could be achieved, employing a dynamic fluorescence labeling technique with the running buffer containing 0.05% sodium dodecylsulfate and non-covalent fluorescent dye for protein, NanoOrange. The collagen, its 1/4 and 3/4 fragments of type I or II collagen could be separated and detected within the run time of 20 min by CGE mode using the gel buffer (pH 8.8) containing 4% polyacrylamide. Good linearity of the peak area of the 3/4 fragment was obtained over each assay range of collagenase (15-150 ng/tube for MMP-1, 3-30 ng/tube for MMP-8, and 1.5-30 ng/tube for MMP-13, respectively). The relative standard deviation of the peak areas of the 3/4 fragment produced from type II collagen by MMP-13 cleavage was calculated to be less than 13.4%, indicating that the assay was reproducible. Also, IC50 values of three MMPs inhibitors, which were calculated for estimation by the variation of the peak areas of the 3/4 fragments using 90 ng/tube for MMP-1, 30 ng/tube for MMP-8 or 15 ng/tube for MMP-13, were almost consistent with data from other assays. The CGE-LIF method is expected to be very useful for proteinase assay and its application to the estimation of inhibitors because this method enables an assay of collagenase activity using native substrate to be conducted without experimentally troublesome procedure such as preparation of antibody or fluorescence-labeled substrate.

Enzyme assay for protein kinase using micellar electrokinetic chromatography with laser-induced fluorescence detection.

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection has been developed for a protein kinase assay. This protein kinase assay could readily determine the phosphorylation activity of substrate peptide kemptide using cAMP-dependent protein kinase (PKA) as a model enzyme. Kemptide and phosphorylated kemptide could be reacted with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent for LIF detection by directly adding NBD-F into the PKA enzymatic reaction mixture. These derivatives of substrate and product were separated and detected within the analysis time of 5 min by micellar electrokinetic mode using a mixture of sodium dodecylsulfate and methanol as a running buffer. Good linearity of the peak response of the phosphorylated kemptide was obtained over the range of 1-20 mU/tube of PKA in the assay. The relative standard deviation of the peak areas of the phosphorylated kemptide using 2, 5 and 10 mU/tube of PKA were calculated to <10.4%, indicating that the assay was reproducible. Also, IC(50) values of six PKA inhibitors, the K(i) value and the inhibition pattern of one inhibitor, which were calculated to estimate by the variation of the peak area of the phosphorylated kemptide using 5 mU/tube of PKA, were consistent with the published data. The sensitivity of the assay was higher than that of enzyme-linked immunosorbent assay (ELISA) for PKA phosphorylation activity, as IC(50) values, K(i) value, and the inhibition mechanism of inhibitors could be estimated using one-tenth amounts of PKA, compared with that of ELISA. The MEKC-LIF is expected to be very useful for protein kinase assay and its application to the estimation of inhibitors because this method does not entail experimentally troublesome procedures such as the preparation of antibody or fluorescence-labeled substrate.