On the Stability of Clathrate Hydrates Encaging Polar Guest Molecules: Contrast in the Hydrogen Bonds of Methylamine and Methanol Hydrates

Abstract

The stability of clathrate hydrates encaging highly polar guests has been investigated in order to explain the experimental observation that some amines form clathrate hydrates but alcohols act as inhibitor to hydrate formation. We choose methylamine and methanol as guest species and examine the stable structure, at which the total potential energy has a minimum value. At the local minima of those two hydrates, the potential energies of water-water and guest-water, and their hydrogen bonded networks are compared. It is found that methanol does not retain the host lattice structure, while the host-network structure is kept in the presence of methylamine. It is shown that the difference in the magnitude of the partial charge on the hydrogen atom between the hydroxyl and amino groups plays a much more significant role on the stability of both clathrate hydrates than the difference in molecular geometry. This is supported from the result of a methylamine-like model that has the same partial charges on the atoms in the hydrophilic site as methanol.     

Isolation and characterization of a monoclonal nonspecific suppressor factor (MNSF) produced by a T cell hybridoma

By fusing Con A-activated BALB/c mice spleen cells with AKR thymoma BW5147 cells, we prepared a hybridoma producing a monoclonal nonspecific suppressor factor (MNSF). This factor inhibits a generation of LPS-induced immunoglobulin-secreting cells. We used ELISA for the bioassay of MNSF activity. With this method, a stable E17 hybridoma clone was selected, and its product in culture medium was isolated and characterized. MNSF fractionated on Sephadex G-100 in saline buffer shows a form with multiple m.w., but fractionated in 0.4 M pyridine-acetic buffer, it is limited to two species of approximately 24Kd and 16Kd. The MNSF was purified by hydroxyapatite chromatography, with marked effectiveness. MNSF activity was found exclusively in the 0.35 M sodium phosphate elution, and the content was further fractionated on subsequent gel filtration in the high ionic strength buffer described above. The purified factor exhibited two forms, of 24Kd and 16Kd, and showed peaks of pI 5.3 and 5.7, respectively, on isoelectric focusing. The MNSF preparation described here is stable at 56 degrees C and unaffected by 2-mercaptoethanol, but is unstable at pH 2.0 and is sensitive to tryptic proteolysis. We injected the hybridoma cells into the peritoneal cavity of pristane-primed F1 (AKR/J X BALB/c) mice, and a large amount of pure MNSF was obtained from the ascites, the characteristics of which were similar to those in the culture supernatant. Thus, the MNSF obtained from the E17 hybridoma consists of functionally identical but physicochemically different discrete proteins. This simple method of purification can serve as a probe for further characterization of MNSF and its application in in vivo experiments.     

The conformation of the lysyl side chain of substrates at the active center of trypsin

In order to study the conformation of the side chain of lysine substrates bound to the active center of trypsin, two lysine analogs, cis- and trans-2,6-diamino-4-hexenoic acids (4,5-dehydrolysines) were synthesized and kinetic parameters for the hydrolysis of benzoyl methyl esters and phenylthiazolones of these analogs by this enzyme were compared with those of the corresponding lysine derivatives. The derivatives of cis-4,5-dehydrolysine were hydrolyzed much more slowly than those of lysine, owing largely to the small kcat values for the former. On the other hand, the derivatives of the trans-isomer were hydrolyzed at about the same rates as those of lysine and the values of both Km and kcat of the former are also similar to those of the latter. These results indicate that the conformation of the side chain of the lysine derivatives hydrolyzed by trypsin is such that the beta- and epsilon-carbons are in a trans-like conformation, as suggested by X-ray crystallographic studies of inhibitor-trypsin complex.     

Interactions of derivatives of guanidinophenylalanine and guanidinophenylglycine with Streptomyces griseus trypsin

The rates of hydrolysis of the ester, amide and anilide substrates of p-guanidino-L-phenylalanine (GPA) by Streptomyces griseus trypsin (S. griseus trypsin) were compared with those of arginine (Arg) substrates. The specificity constant (kcat/km) for the hydrolysis of GPA substrates by the enzyme was 2-3-times lower than that for arginine substrates. The kcat and Km values for the hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) by S. griseus trypsin are in the same order of magnitude as those of N alpha-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt), although both values for the former when hydrolyzed by bovine trypsin are higher by one order of magnitude than those for the latter. The specificity constant for the hydrolysis of Bz-GPA-OEt by S. griseus trypsin is much higher than that for N alpha-benzoyl-p-guanidino-L-phenylglycine ethyl ester (Bz-GPG-OEt). As with the kinetic behavior of bovine trypsin, low values in Km and kcat were observed for the hydrolysis of amide and anilide substrates of GPA by S. griseus trypsin compared with those of arginine substrates. The rates of hydrolysis of GPA and arginine substrates by S. griseus trypsin are about 2- to 62-times higher than those obtained by bovine trypsin. Substrate activation was observed with S. griseus trypsin in the hydrolysis of Bz-GPA-OEt as well as Bz-Arg-OEt, whereas substrate inhibition was observed in three kinds of N alpha-protected anilide substrates of GPA and arginine. In contrast, no activation by the amide substrate of GPA could be detected with this enzyme.     

Hydropiperoside,a novel coumaryl glycoside from the root of Polygonum hydropiper

From the methanol extract of the root of Polygonum hydropiper, a novel coumaryl glycoside hydropiperoside was isolated together with anthraquinone, ellagic acid 3,3′-di-O-methyl ether, gallic acid, two quercetin glycosides and an unidentified aromatic δ-lactone possessing antifertility activity. The structure of hydropiperoside was established as β-d-(1,3,6-tri-p-coumaryl)-fructofuranosyl-α-d-glucopyranoside by combination of extensive ¹H NMR and ¹³C NMR spectra, and the FD/MS spectrum.     

Peroxyoxalate chemiluminescent assay for oxidase activities based on detecting enzymatically formed hydrogen peroxide

A sensitive peroxyoxalate chemiluminescent (PO-CL) assay for activities of oxidases (uricase, choline oxidase, cholesterol oxidase and xanthine oxidase) which catalyse a formation of hydrogen peroxide was developed using 4,4′-oxalyl-bis[(trifluoromethylsulphonyl)imino]trimethylene-bis(4-methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8-tetramorpholinopyrimido[5,4-d]pyrimidine as a fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 × 10^?7-1 × 10^?4 mol/L. Relative standard deviations for oxidase assays were 5.1–12.7% (n = 10). Detection limits were 1 × 10^?3 U/mL for uricase, 5 × 10^?4 U/mL for choline oxidase, 5 × 10^?3 U/mL for cholesterol oxidase and 5 × 10^?4 U/mL xanthine oxidase (sample to blank ratio, 3).     

High-performance liquid chromatographic determination of short-chain aliphatic aldehydes using 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole as a fluorescence reagent

High-performance liquid chromatographic determination of four short-chain aliphatic aldehydes using fluorescence detection was carried out with 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H). DBD-H derivatives with three aliphatic aldehydes — formaldehyde, acetaldehyde and propionaldehyde — were synthesized and their fluorescence properties were examined. Relative fluorescence intensities of these compounds in acetonitrile were ca. ten-fold larger than those in aqueous acetonitrile. DBD-hydrazones could be separated by reversed-phase chromatography using aqueous acetonitrile as eluent and detection at 560 nm with excitation at 445 nm. Submicromolar levels of formaldehyde, acetaldehyde, propionaldehyde and butylaldehyde could be determined. The HPLC procedure using propionaldehyde as internal standard was applied to the measurement of acetaldehyde levels in normal human plasma before and 30 min after ingestion of ethanol.     

Sesquiterpenes from Porella species

The Porella liverworts contain abundant sesquiterpenes. ent-Biocyclogermacrene, three ent-aromadendrenes, a unique hydrocarbon, α-pinguisene and two drimane type sesquiterpenes were obtained together with the intensly pungent component, tadeonal, from P. vernicosa and P. gracillima. P. macroloba contained the same sesquiterpenes except for the absence of ent-bicyclogermacrene and the ent-aromadendrenes. The fragrant odor of P. perrottetiana was composed of α-pinene and camphor.