These fractions of Abs were marked in the following text as sle-IgGmix and ms-IgGmix. Ab proteolytic AZ-3146 biological activity activity assay The reaction mixture for analysis of OP-hydrolyzing activity of IgGs containing 20 mM Tris-HCl, 3 mM NaCl, 0.051.0 mM one of two specific or various nonspecific OPs, and 0.0010.02 mg/ml of IgGs, was incubated for 124 h at 30uC. Specific X-OP21 and X-OP25 corresponding to two of four known IgG-dependent specific cleavage sites of hMBP were used. In the case of X-OP21 only 18 N-terminal residues correspond to the antigenic determinant; this OP contains three additional C-terminal amino acid residues. The same products of the hydrolysis separated by RPhC were revealed in several peaks by MALDI spectrometry; the main peaks are marked in bold, while additional peaks are shown in parentheses. C1 reflects the presence of signal corresponding to the analyzed product in the spectrum of total reaction mixture. The same several products of the hydrolysis corresponding to each peak after RPhC were revealed not only by MALDI spectrometry, but also by TLC . All mentioned OPs contained fluorescent residue 6-O-fluorescein ethyl ester on its N-terminus. In addition, three short fluorogenic peptidyl-4methylcoumaryl-7-amides were used as nonspecific controls: Boc-Val-Leu-Lys-MCA, Pro-Phe-Arg-MCA, Boc-Ile-Glu-Gly-Arg-MCA. The cleavage products of different OPs were separated by TLC on Kieselgel F60 plates using acetic acid n-butanol H2O system. The plates were dried and photographed. To quantify the intensities of the fluorescent spots after TLC, X-OP21, X-OP25, and other OPs incubated without 
IgGs was used as controls. Photographs of the plates were imaged by scanning and quantified using GelPro v3.1 software. In some experiments the cleavage products of specific X-OP21 and X-OP25 were first separated by reverse-phase chromatography on Nucleosil C-18 column using 0.05% trifluoroacetic acid and gradient of acetonitrile concentration. The relative amount of various cleavage products was calculated by the fluorescence. Excitation was performed at 320 nm and fluorescence emission detected at 490 nm. The fractions corresponding to different peaks were collected, evaporated to minimal volume and products of the hydrolysis were analyzed by TLC and by MALDI spectrometry. acetonitrile and trifluoroacetic acid was used as the matrix. To 1 ml of the reaction mixture containing hydrolyzed OPs before or after their separation by RPhC or TLC, 1 ml of 0.2% trifluoroacetic acid and 2 ml of the matrix were added, and 1 ml of the final mixture was spotted on the MALDI plate, air-dried, and used for the analysis. Calibration of the MALDI spectra was performed using the protein and OP standards I and II in the external and internal calibration mode. Determination of the kinetic parameters The reaction mixtures contained the standard components and different concentrations of OPs. All measurements were taken under the conditions of the pseudo-first order of the reaction within the linear regions of the time courses. The cleavage products were analyzed by TLC. The activity of IgGmix was determined as a decrease in the percentage of initial X-OPs converted to shorter X-OPs, corrected for the distribution of the fluorescence label between these spots in the control and taking into account the concentration of each OP in every reaction mixture. The KM and Vmax values were calculated from the dependencies of V versus by leastsquares non-linear fitting using Microca