muramidase and formic-acid

The success of wound healing depends upon the proper growth of vascular system in time in the damaged tissues. Poor blood supply to wounded tissues or tissue engineered grafts leads to the failure of wound healing or rejection of grafts. In present paper, we report the synthesis of novel organosoluble and pro-angiogenic chitosan derivative (CSD) by the reaction of chitosan with 1,3-dimethylbarbituric acid and triethylorthoformate (TEOF). The synthesized material was characterized by FTIR and

Topics: Adenocarcinoma; Apoptosis; Barbiturates; Biocompatible Materials; Cell Line, Tumor; Chitosan; Chorioallantoic Membrane; Drug Delivery Systems; Formates; Humans; Hydrogels; Magnetic Resonance Spectroscopy; Muramidase; Neovascularization, Pathologic; Neovascularization, Physiologic; Polyesters; Solubility; Solvents; Spectroscopy, Fourier Transform Infrared; Stomach Neoplasms; Tissue Engineering; Tissue Scaffolds; Viscosity; Wound Healing

The effect of acids on the structure of lysozyme (Lyz) during electrospray ionization (ESI) was studied by comparing the solution and gas-phase structures of Lyz. Investigation using circular dichroism spectroscopy and small-angle X-ray scattering demonstrated that the folded conformation of Lyz was maintained in pH 2.2 solutions containing different acids. On the other hand, analysis of the charge state distributions and ion mobility (IM) distributions, combined with molecular dynamics simulations, demonstrated that the gas phase structures of Lyz depend on the pKa of the acid used to acidify the protein solution. Formic acid and acetic acid, which are weak acids (pKa > 3.5), induce unfolding of Lyz during ESI, presumably because the undissociated weak acids provide protons to maintain the acidic groups within Lyz protonated and prevent the formation of salt bridges. However, HCl suppressed the formation of the unfolded conformers because the acid is already dissociated in solution, and chloride anions within the ESI droplet can interact with Lyz to reduce the intramolecular electrostatic repulsion. These trends in the IM distributions are observed for all charge states, demonstrating the significance of the acid effect on the structure of Lyz during ESI.

Topics: Acetic Acid; Animals; Chick Embryo; Circular Dichroism; Egg Proteins; Formates; Hydrochloric Acid; Molecular Dynamics Simulation; Muramidase; Protein Conformation; Protein Unfolding; Spectrometry, Mass, Electrospray Ionization

Femtosecond (fs) laser vaporization is used to transfer cytochrome c, myoglobin, lysozyme, and ubiquitin from the condensed phase into an electrospray (ES) plume consisting of a mixture of a supercharging reagent, m-nitrobenzyl alcohol (m-NBA), and trifluoroacetic acid (TFA), acetic acid (AA), or formic acid (FA). Interaction of acid-sensitive proteins like cytochrome c and myoglobin with the highly charged ES droplets resulted in a shift to higher charge states in comparison with acid-stable proteins like lysozyme and ubiquitin. Laser electrospray mass spectrometry (LEMS) measurements showed an increase in both the average charge states (Zavg) and the charge state with maximum intensity (Zmode) for acid-sensitive proteins compared with conventional electrospray ionization mass spectrometry (ESI-MS) under equivalent solvent conditions. A marked increase in ion abundance of higher charge states was observed for LEMS in comparison with conventional electrospray for cytochrome c (ranging from 19+ to 21+ versus 13+ to 16+) and myoglobin (ranging from 19+ to 26+ versus 18+ to 21+) using an ES solution containing m-NBA and TFA. LEMS measurements as a function of electrospray flow rate yielded increasing charge states with decreasing flow rates for cytochrome c and myoglobin.

Topics: Acetic Acid; Animals; Benzyl Alcohols; Cattle; Chickens; Cytochromes c; Formates; Horses; Indicators and Reagents; Lasers, Solid-State; Muramidase; Myoglobin; Protein Denaturation; Protein Stability; Solvents; Spectrometry, Mass, Electrospray Ionization; Trifluoroacetic Acid; Ubiquitin; Volatilization

Quality data collection for macromolecular cryocrystallography requires suppressing the formation of crystalline or microcrystalline ice that may result from flash-freezing crystals. Described here is the use of lithium formate, lithium chloride and other highly soluble salts for forming ice-ring-free aqueous glasses upon cooling from ambient temperature to 100 K. These cryosalts are a new class of cryoprotectants that are shown to be effective with a variety of commonly used crystallization solutions and with proteins crystallized under different conditions. The influence of cryosalts on crystal mosaicity and diffraction resolution is comparable with or superior to traditional organic cryoprotectants.

Topics: Cryoprotective Agents; Crystallography, X-Ray; Formates; Freezing; Ice; Lithium Chloride; Macromolecular Substances; Muramidase; Ribonucleases; Salts

The protonation state and activity of enzymes in low-water media are affected by the aqueous pH before drying ("pH memory"). However, both protonation and activity will change if buffer ions can be removed as volatile or organic-extractable weak acids or bases. With NH4OOCH buffers, in which both ions can be removed, pH memory disappears completely for subtilisin-catalyzed transesterification in hexane. Only weak pH memory is found with buffers having one volatile component, NH4-phosphate and NaOOCH. The changes in ionization state result from proton exchanges like Protein-COO-NH4+ --> Protein-COOH + NH3 (g) and Protein-NH3+HCOO- --> Protein-NH2 + HOOCH (g). An equivalent, complementary picture is that net charges on the protein and buffer ions must remain equal and opposite. With NaOOCH buffers, loss of some HCOO- ions gives a more negative net charge on the protein, balanced by the excess Na+. With NH4-phosphate buffers, loss of NH3 gives protein with a more positive net charge. The resulting catalytic activities were high and low, respectively, similar to those after drying from Na-phosphate buffers of optimal (8.5) and acid pH. All of the above effects have been demonstrated for both covalently immobilized subtilisin and the lyophilized free enzyme. Subtilisin lyophilized from NH4OOCH buffers gave pH approximately 4 after redissolution in water, probably because removal of HCOO- counterions remains incomplete. The resulting catalytic activity was low. The effects are discussed in relation to the possible locations, in low-dielectric media, of the positive charge that balances the net negative catalytic triad in active subtilisin.

Topics: Animals; Buffers; Catalysis; Enzymes, Immobilized; Formates; Freeze Drying; Hydrogen-Ion Concentration; Kinetics; Muramidase; Myoglobin; Subtilisins

The cyanogen bromide (CNBr)/formic acid cleavage reactions of wild-type and trifluoromethionine (TFM)-containing recombinant lambda lysozyme were studied utilizing ESI and MALDI mass spectrometry. Detailed analysis of the mass spectra of reverse-phase HPLC-purified cleavage fragments produced from treatment of the wild-type and labeled proteins with CNBr indicated cleavage solely of methionyl peptide bonds with no observation of cleavage at TFM. N-Acetyl-TFM was also found to be resistant to reaction with CNBr, in contrast to N-acetyl-methionine. The analysis also indicated differential reactivity among the three methionine positions in the wild-type enzyme. Additionally, formylation of intact enzyme as well as peptide fragments were observed and characterized and indicated that serine, threonine, as well as C-terminal homoserine side chains are partially formylated under standard cleavage protocols.

Topics: Amino Acid Sequence; Bacteriophage lambda; Cyanogen Bromide; Formates; Indicators and Reagents; Methionine; Molecular Probes; Molecular Sequence Data; Muramidase; Recombinant Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Capillary zone electrophoresis (CZE) of five model proteins (lysozyme, myoglobin, ribonuclease A, alpha-lactalbumin, and trypsinogen), using ammonium formate as the electrophoretic buffer and triethylamine (TEA) as a buffer additive at pH 2.5, was used for protein separation. The electrophoretic behavior of these proteins was examined with respect to various concentrations (10-40 mM) of TEA and of ammonium formate. Based on the experimental parameters of electrophoretic resolution, current, and peak separation time, an electrolyte (30 mM each of TEA and ammonium formate) was empirically derived as the optimum for scale-up separation. The loading limit for proteins, covering a wide range of injection volumes (60-990 nl) and amount of protein (1-21 pmol of each protein), was investigated on 75 and 100 microns I.D. untreated fused-silica capillaries. Protein adsorption (average < 15%) was experimentally determined using this volatile buffer system.

Topics: Adsorption; Animals; Buffers; Cattle; Chickens; Electrophoresis, Capillary; Ethylamines; Formates; Horses; Hydrogen-Ion Concentration; Indicators and Reagents; Isoelectric Point; Lactalbumin; Molecular Weight; Muramidase; Myoglobin; Proteins; Ribonuclease, Pancreatic; Trypsinogen

Topics: Anti-Infective Agents, Local; Dermatologic Agents; Formates; Muramidase