Carnosine (beta-Ala-L-His) has high antioxidant activity, and it is widely used in biology, chemical engineering, medicine and other fields. Its analogue syntheised in non-aqueous solvent and catalyzed by enzymes is high-effective but low-price, so it has great prospect. Here, we synthesized a carnosine analogue imidazole 4(5)-alanylamide-5(4)-carboxylic acid with imidazole-4,5-dicarboxylic acid and L-Alanine as substrates, alpha-chymotrypsin as catalyst in tetrahydrofuran (THF) solvent. Based on the orthogonal experiments, the optimized synthetic conditions are 4,5-dicarboxylic acid: L-alanine = 1:3 (m/m), alpha-chymotrypsin: substrates (4,5-dicarboxyl acid and L-alanine) = 1:200 (m/m), pH 8 phosphate buffer:THF = 1.6:10 (V/V), reaction temperature 35 degrees C, time 1.5 h. We separated the product with silica gel G60 thin-layer chromatography (TLC), and a new spot appeared at Rf (ratio to front) = 0.81; then the new spot was purified and characterized with UV spectra, high performance liquid chromatogram (HPLC) and 13C NMR (13C nuclear magnetic resonance). The UV spectra shows a new absorption peak at 310 nm, and the peak in 253 nm is largely strengthened; HPLC reserve times are all 4.5 min at 253 nm, 310 nm, 330 nm; 13C NMR shows 8 carbons. Combing with the catalytic mechanism of alpha-chymotrypsin, structure of the analogue is confirmed, i.e., imidazole 4(5)-alanylamide-5(4)-carboxylic acid.
Carnosine ; analogs & derivatives ; biosynthesis ; chemistry ; Catalysis ; Chromatography, High Pressure Liquid ; Chymotrypsin ; metabolism ; Furans ; chemistry ; Solvents

The aim is to determine the primary structure of a new hirudin and reteplase fusion protein (HV12p-rPA) by LC-ESI-MS/MS spectrometry. The molecular weight of the hirudin and reteplase fusion protein (HV12p-rPA) was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The HV12p-rPA was digested with trypsin and chymotrypsin separately and the peptides in the digest mixtures were identified by LC-ESI-MS/MS. The molecular weight of HV12p-rPA was 41,472 Da, which was in accordance with the theoretical value. The peptide fragments of HV12p-rPA digested with trypsin were identified by LC-ESI-MS/MS spectrometry and the results indicated that the fusion protein contained r-PA. Then, the peptides of HV12p-rPA digested with chymotrypsin were identified by the same method. The results indicated that the fusion protein contained HV12p and the linker-containing peptide, DEGGGSY. MASDF and LDWIRDNMRP were identified as the N-terminal and C-terminal containing peptides in the chymotryptic digest mixture of the fusion protein. All of the Xcorr values exceeded 1.5, some of which were above 3.0, showing that the results were correct and credible and a sequence coverage of 85% was achieved. HPLC/MS analysis coupled with uncompleted digestion indicated that all these peptides were arranged with the correct order as expected. Thus, sequence of the fusion protein was confirmed and it was consistent with our design in upstream construction.
Amino Acid Sequence ; Chromatography, Liquid ; methods ; Chymotrypsin ; chemistry ; Fibrinolytic Agents ; analysis ; chemistry ; Hirudins ; analysis ; chemistry ; Molecular Sequence Data ; Molecular Weight ; Peptide Fragments ; Recombinant Fusion Proteins ; analysis ; chemistry ; Recombinant Proteins ; analysis ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; methods ; Tandem Mass Spectrometry ; methods ; Tissue Plasminogen Activator ; analysis ; chemistry ; Trypsin ; chemistry

A soluble factor which augments the expression of major histocompatibility complex class I (MHC I) antigens on a number of murine tumor cell lines, has been isolated from the culture supernatants of mixed lymphocyte reaction of spleen cells derived from C57B1/6, Balb/c and Swiss mice. The factor, termed MHC-augmenting factor (MHC-AF) has been partially purified by Sephadex G-100 column chromatography and reverse phase HPLC. MHC-AF activity is associated with an 18 kDa molecule. MHC-AF activity was resistant to pH 2.0 treatment and partially purified MHC-AF preparations did not have any activity in L929 cell/vesicular stomatitis virus (VSV) interferon bioassay system. Antibodies to IFN-gamma did not block the activity of MHC-AF. These results indicate that a MHC-AF distinct from IFN-gamma, is produced by mouse spleen cells undergoing a mixed lymphocyte reaction.
Animal ; Antibodies/pharmacology ; Chymotrypsin/metabolism ; Chymotrypsin/chemistry ; Comparative Study ; Concanavalin A/pharmacology ; Heat ; Histocompatibility Antigens Class I/metabolism* ; Histocompatibility Antigens Class I/drug effects ; Interferon Type II/pharmacology ; Interferon Type II/metabolism ; Interferon Type II/immunology ; Lymphocytes/physiology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Proteins/pharmacology* ; Proteins/metabolism ; Proteins/isolation & purification* ; Spleen/cytology ; Trypsin/metabolism ; Trypsin/chemistry ; Tumor Cells, Cultured/immunology ; Tumor Cells, Cultured/drug effects

Mouse spleen cells activated in a mixed lymphocyte reaction release a soluble factor, which induces a significant proliferative response in fresh mouse spleen cells. This proliferation inducing factor (PIF) was found to be heat stable (90 degrees C for 45 min) and also resistant to trypsin or chymotrypsin treatment. By using a sizing HPLC column, the molecular weight of PIF appears to be 25 kDa. Mouse spleen cells treated with anti-thy-1 + complement lost Con-A induced proliferative responses but responded well to PIF. B cell depleted spleen cells obtained by negative selection panning, did not respond to PIF. These results indicate that B cells proliferated in response to PIF. Polymixin-B, which blocks the B cell proliferative response to LPS, did not inhibit PIF induced proliferation.
Animal ; B-Lymphocytes/physiology* ; B-Lymphocytes/drug effects ; Bone Marrow/metabolism ; Cell Division/physiology ; Chromatography, High Pressure Liquid ; Chymotrypsin/pharmacology ; Dose-Response Relationship, Drug ; Growth Substances/pharmacology* ; Growth Substances/chemistry ; Heat ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Molecular Weight ; Polymyxin B/pharmacology ; Protein Denaturation ; Spleen/metabolism* ; Thymus Gland/metabolism ; Trypsin/pharmacology