Nα,Nim-Bis-Boc-D-histidine benzene

A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of Nα-benzoyl-l-arginine ethyl ester to Nα-benzoyl-l-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas.

We examined the dielectric (DE) and nuclear magnetic resonance (NMR) spectroscopic behaviour of N-methyl-2-pyrrolidone (NMP) and N,N-dimethylacetamide (DMAc), which are tertiary amide compounds that bear five-membered cyclic and open structures, respectively. They were examined to investigate the formation of anti-parallel dimers and tetramers in a dipole configuration in the pure liquid state and in benzene solution over a wide concentration range at 25 °C. Because the Kirkwood correlation factors of electric dipoles considerably decreased with increasing concentrations in both the NMP and DMAc systems and because the second DE relaxation modes were clearly observed to exhibit relaxation times that are longer than those of the first modes assigned to the rotational relaxation of monomeric molecules, anti-parallel dimers are formed due to dipole-dipole interactions with increasing concentrations. Assuming a chemical process between the monomers (MONs) and the anti-parallel dimer (DIM), 2MON ⇄ DIM, the equilibrium constants for the anti-parallel dimer formation were determined to be KDEd ~ 1.2 M-1 for NMP and 0.35 M-1 for DMAc, based on the first DE relaxation strength. The fact that the evaluated KDEd values substantially increased with increasing concentrations in the range above ca. 1.0 M strongly suggested the formation of higher order intermolecular associations instead of dimers. Chemical shifts in the 1H-NMR spectra assigned to (N)CH3 and (5)CH2 protons of NMP and the two (Nα)CH3 and (Nβ)CH3 protons of DMAc dissolved in (d)Bz substantially changed with increasing concentrations due to the formation of intermolecular associations. The states occupied by MONs, DIMs and tetramers (TETs) were evaluated from chemical shift data as a function of the concentrations in the NMP and DMAc systems. Then, the equilibrium constants for the dimer and tetramer formation (2DIM ⇄ TET) were determined to be KNMRd ~ 1.2 M-1 and KNMRt ~ 0.2 M-1 for NMP and KNMRd ~ 0.35 M-1 and KNMRt ~ 0.25 M-1 for DMAc. The reason for the differences in the equilibrium constants between NMP and DMAc may be related to the difference in motional freedom between the cyclic and open structures. The formation of anti-parallel DIMs and TETs was also confirmed by semi-empirical quantum chemical calculations for both NMP and DMAc molecules.

Proteases play an important role during mammalian fertilization. Their function is frequently investigated using specific inhibitors. We analyzed four serine protease inhibitors [4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF), soybean trypsin inhibitor from glycine max (STI), Nα-tosyl-L-lysine-chloromethyl ketone hydrochloride (TLCK) and N(p)-tosyl-L-phenylalanine-chloromethyl ketone (TPCK)] for their in vitro effect on fertilization and sperm quality in pigs. Inhibitor concentrations were chosen based on the reduction of fertilization rate during preliminary dose-response experiments with cryopreserved epididymal spermatozoa. The inhibitor effects on in vitro fertilization (IVF) and sperm parameters (membrane and acrosomal integrity, motility and mitochondrial membrane potential - MMP) were evaluated using diluted fresh semen. AEBSF (100 μM), TLCK (100 μM) and TPCK (100 μM) decreased total fertilization and polyspermy rates by at least 50%. STI (5 μM) lowered total fertilization rates but not the level of polyspermy. AEBSF and TPCK reduced fertilization parameters to a similar degree using cryopreserved epididymal spermatozoa (dose-response experiment) or diluted fresh semen. Inhibition by STI was more pronounced using cryopreserved epididymal spermatozoa, whereas TLCK inhibited IVF only with diluted fresh semen. AEBSF and STI had no effect on sperm parameters, and TLCK significantly reduced motility. TPCK diminished MMP and motility and affected membrane and acrosomal integrity in a negative way. In summary, serine protease inhibitors differed in the way they reduce the fertilization rate. These results emphasize the necessity of inhibitor testing before they can be applied in fertilization studies. AEBSF and STI can be used in the future IVF studies without compromising sperm quality.