Quantitative determination of the extent of neutralization of carboxylic acid functionality in carbopol 974P NF by diffuse reflectance fourier transform infrared spectrometry using Kubelka-Munk function.

PURPOSE: The purpose of this study was to develop an analytical method for the quantitative determination of the extent of neutralization of the carboxylic acid function in Carbopol 974P NF using Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFT) with Kubelka-Munk function analysis. METHODS: Carbopol 974P NF is a high molecular weight, chemically crosslinked polymer of acrylic acid, that has the C=O stretching band of the unionized carboxylic acid function at 1695 cm(-1). The quantitative determination of the extent of neutralization of the carboxylic acid function in Carbopol 974P NF is based upon the asymmetrical C=O stretching of the carboxylate anion at 1570 cm(-1) measured by DRIFT spectroscopy. RESULTS: To overcome spectral differences arising from sample preparation (powders, granules and tablets) and in an effort to increase the precision of the analytical method, the following approaches were used: (1) an internal standard, (2) first derivative of the spectrum to eliminate the effect of baseline drift and (3) the ratio of the first derivative of the C=O stretch of the carboxylate anion peak (1570 cm(-1)) in the neutralized Carbopol 974P NF to that of the peak of the internal standard (866 cm(-1)). The above data treatment techniques proved to be superior to the usual methods of peak height or peak area. The calibration curve of the ratio of the first derivative (1570 cm(-1)/866 cm(-1)) was a linear function of the mass of sodium carboxylate over the range from 0.0% to 100.0% neutralization of the carboxylic acid function in Carbopol 974P NF (Fig. 1a). No particle size or sample preparation effects were noted within the experimental error. CONCLUSIONS: DRIFT spectroscopy using the Kubelka-Munk function is a powerful tool for the routine determination of the extent of neutralization of the carboxylic acid function in Carbopol 974P NF in complex pharmaceutical formulations.

Transformation of cadmium-binding complexes during cadmium sequestration in fission yeast.

Fission yeast responded to environmental cadmium by producing a family of small Cd-binding peptides, phytochelatins (PCn). A low molecular weight (LMW) complex essentially composed of PC2, and PC3 was produced and then disappeared gradually in 24 hrs after Cd treatment, which served as a transient form for a temporary but quick relief of Cd in the cytosoL It had been reported that the LMW complex was further transported into the vacuole by an ABC-type protein (HMT1), and a higher molecular weight (HMW) complex was formed in the vacuole. Results from gel filtration chromatography and HPLC analysis showed that the transformation of the LMW to the HMW complex was accompanied with a rearrangement of its PCn component. Besides, the molecular conformation of the HMW complex changed from a relaxed form in the early stage to a more condensed conformation during cell aging. And the transformation of the LMW into the HMW complex by the addition of sulfide in the test tube was demonstrated.

Partial purification and characterization of invertase isozymes from rice grains (Oryza sativa).

Four invertase isozymes have been isolated from the milky stage rice grains. According to the pH optima, they are classified as one alkaline (IT7) and three acid invertases. The acid invertases are further divided into two soluble forms (IT4 and IT5) and one cell wall-bound (ITb) form which was solubilized in 1 M NaCl. The pH optima of ITb, IT4, IT5 and IT7 are 4.5, 3.5-4.0, 5.0 and 7.0, and the molecular masses are 42, 60, 64 and 260 kDa, respectively. Both IT4 and IT5 were bound to Con A-Sepharose suggesting that these enzymes are glycoprotein. The Km of ITb, IT4, IT5 and IT7 for sucrose are 4.3, 0.9, 12.1 and 70.1 mM, respectively. IT4 and IT5 have a higher Km for raffinose, and the maximum activities are 64% and 27% of that using sucrose as the substrate. IT7 did not hydrolyze raffinose at all. These invertases also exhibit distinct isoelectric points (pI) and different susceptibility to various inhibitors.

Two purine biosynthetic enzymes that are required for cadmium tolerance in Schizosaccharomyces pombe utilize cysteine sulfinate in vitro.

In plants and in certain fungi, exposure to heavy metals induces the synthesis of metal-binding peptides commonly known as phytochelatins. With cadmium, phytochelatins can sequester the metal into a sulfide-containing complex. From genetic analysis of fission yeast mutants, we previously reported that two genes in purine biosynthesis, encoding adenylosuccinate synthetase and succinoaminoimidazole carboxamide ribonucleotide (SAICAR) synthetase, are required for the biogenesis of the phytochelatin-cadmium-sulfide complex in vivo. We suggested that a sulfur analog of aspartate, cysteine sulfinate, might be utilized by these enzymes and that the cysteine sulfinate-derived products would then become intermediates or carriers in a sulfur transfer pathway leading to the sulfide found within the metal chelate. In this paper, we report that partially purified adenylosuccinate synthetase and SAICAR synthetase are capable of utilizing cysteine sulfinate in vitro to form sulfur analog products. Adenylosuccinate lyase, however, fails to catalyze further conversion of these sulfur derivatives. These observations support the genetic data implicating a link among purine biosynthetic enzymes, sulfur metabolism, and cadmium tolerance.

Purification and characterization of 5'-phosphodiesterase from barley rootlets.

A phosphodiesterase was purified from barley rootlets by polyethylene glycol fractionation, calcium phosphate gel-cellulose adsorption, Sepharose CL-4B gel filtration, DEAE-Sepharose CL-6B ion exchange and preparative polyacrylamide gel electrophoresis (PAGE). The enzyme was purified by 103.6 folds and 11% of the enzyme activity was recovered. The purified enzyme was apparently homogeneous when examined on PAGE. It had a molecular weight of 100 kD, an optimum pH of 9.5, and a Km of 0.28 mM on the hydrolysis of p-nitrophenyl thymidine 5'-phosphate. SDS-PAGE revealed that the enzyme molecule might be composed of two or three subunits. Reducing agents, CuSO4, EDTA and 5'-nucleotides were inhibitory to the enzyme activity. This enzyme was able to hydrolyze RNA and denatured DNA efficiently, whereas native DNA was not a good substrate.

Presence of three rice sucrose synthase genes as revealed by cloning and sequencing of cDNA.

By sequencing cDNA clones, we have concluded that three distinct sucrose genes are expressed in rice (Oryza sativa cv. Tainong 67). When the amino acid sequences deduced from these cDNAs as well as those of known sucrose synthase are compared, the highest divergence is found in the C-termini. The most suitable DNA sequences for use as specific for the mRNA derived from these genes have been suggested.

Sucrose synthase in rice plants : growth-associated changes in tissue specific distributions.

Different parts of the rice (Oryza sativa L.) plant at different growth stages were analyzed for sucrose synthase (SS) by enzyme activity assay and enzyme-linked immunosorbent assay directly on the extracts or on the eluates from a gel filtration column. On a dry matter basis, the amount of soluble protein and SS activity decreased significantly, but the amount of enzyme protein changed little in growing leaves. In the grain, the SS activity was the highest at the early ripening stage and decreased later, but the amount of SS protein increased with the increase in maturity. In the root, a low activity of SS was detectable only in the tillering but not in other stages. Immunoblotting of SS protein extracted from different parts of rice showed two bands. Elution patterns of crude extracts from a gel filtration column showed the presence of several types of SS protein. Among them, two to three types with larger elution volumes had the SS activity but others with smaller elution volumes (considered as the aggregated forms) had no activity. The SS purified from different parts of the plant showed similar but distinctly different electrophoretic mobilities in a native gel. It has been concluded that different isozymes are expressed in different tissues at different growth stages.

Ethanol and water sorption into stratum corneum and model systems.

Sorption of ethanol and water into stratum corneum, delipidized stratum corneum, and triolein as a simple model lipid was investigated. Optima in ethanol sorption and flux are related to dehydration of keratins. There was no optimum for solubility in triolein; a linear cosolvency is observed with ethanol:triolein mixtures. A model is proposed which qualitatively predicts the key features of ethanol-enhanced skin permeation on the basis of these solubility phenomena and a constant diffusion coefficient.

Ethanol: water mutually enhanced transdermal therapeutic system II: skin permeation of ethanol and nitroglycerin.

An optimal concentration range of aqueous ethanol produces 5-10-fold increases in nitroglycerin flux across skin and ethanol skin permeation that are far greater than reported previously. For aqueous ethanol solutions saturated with nitroglycerin with an ethanol volume fraction less than or equal to 0.7, the flux of nitroglycerin across skin is linear with the ethanol flux and is traced to a linear solubility relationship and a constant diffusion coefficient.

Starch Phosphorylase Inhibitor Is beta-Amylase.

The proteinaceous noncompetitive inhibitor of starch phosphorylase isolated from the root of sweet potato (Ipomoea batatas [L.] Lam.) (TC Chang, JC Su 1986 Plant Physiol 80: 534-538) has been identified as a beta-amylase. The starch phosphorylase inhibitor and beta-amylase activities copurified to give a protein indistinguishable from commercial beta-amylase by electrophoretic and immunological methods, and the two activities showed parallel responses in pH, temperature, and inhibitor sensitivity tests. The amylolytic pattern of the inhibitor corresponded to that of beta-amylase and its inhibitory effect toward starch phosphorylase was due to neither deprivation of starch, the primer for the phosphorylase assay, nor the inhibitory effect of amylolytic products.

Oven-drying method for polyacrylamide gel slab packed in cellophane sandwich.

Polyacrylamide gel slabs can be dried quickly without elaborate tools and the results are similar or even better than those obtained with a commercial drying apparatus. The discontinuous, sodium dodecyl sulfate, and gradient polyacrylamide gel slabs yielded similar results regardless of the staining methods, e.g., Coomassie blue, periodate-Schiff's reagent, or ammoniacal silver.