Edwin G. Piotrowski

Environmental Influences on Bovine κ-Casein: Reduction and Conversion to Fibrillar (Amyloid) Structures

The caseins of milk form a unique calcium–phosphate transport complex that provides these necessary nutrients to the neonate. The colloidal stability of these particles is primarily the result of κ-casein. As purified from milk, this protein occurs as spherical particles with a weight average molecular weight of 1.18 million. The protein exhibits a unique disulfide bonding pattern, which (in the absence of reducing agents) ranges from monomer to octamers and above on SDS-PAGE. Severe heat treatment of the κ-casein (90°C) in the absence of SDS, before electrophoresis, caused an increase in the polymeric distribution: up to 40% randomly aggregated high–molecular weight polymers, presumably promoted by free sulfhydryl groups (J. Protein Chem.17: 73–84, 1998). To ascertain the role of the sulfhydryl groups, the protein was reduced and carboxymethylated (RCM-κ). Surprisingly, at only 37°C, the RCM-κ-casein exhibited an increase in weight average molecular weight and tendency to self-association when studied at 3000 rpm by analytical ultracentrifugation. Electron microscopy (EM) of the 37°C RCM sample showed that, in addition to the spherical particles found in the native protein, there was a high proportion of fibrillar structures. The fibrillar structures were up to 600 nm in length. Circular dichroism (CD) spectroscopy was used to investigate the temperature-induced changes in the secondary structure of the native and RCM-κ-caseins. These studies indicate that there was little change in the distribution of secondary structural elements during this transition, with extended strand and κ turns predominating. On the basis of three-dimensional molecular modeling predictions, there may exist a tyrosine-rich repeated sheet-turnsheet motif in κ-casein (residues 15–65), which may allow for the stacking of the molecules into fibrillar structures. Previous studies on amyloid proteins have suggested that such motifs promote fibril formation, and near-ultraviolet CD and thioflavin-T binding studies on RCM-κ-casein support this concept. The results are discussed with respect to the role that such fibrils may play in the synthesis and secretion of casein micelles in lactating mammary gland.

Determination of flumequine and oxolinic acid in fortified chicken tissue using on-line dialysis and high-performance liquid chromatography with fluorescence detection

Abstract Isolation of the fluoroquinolones, flumequine and oxolinic acid, from fortified chicken liver was achieved using liquid–liquid extraction, aqueous on-line dialysis and trace enrichment. Dialysis, trace enrichment and column switching were performed using the ASTED system. Separation of the isolated compounds in the tissue extracts was performed using reversed-phase HPLC and fluorescence detection. This procedure yielded excellent mean recoveries at the 50, 25, 10 and 5 ng/g spiking levels for flumequine (94–96%; 4–9% R.S.D.) and at the 25 and 5 ng/g spiking levels for oxolinic acid (98–99%; 4–6% R.S.D.). Clean chromatograms were obtained, allowing detection of 5 ng/g flumequine and 2.5 ng/g oxolinic acid to be easily made. Due to its lower organic solvent consumption, automation and on-line capabilities, this method may be a suitable replacement for conventional chemical extraction techniques for drug residues in animal tissues.

SOLID-STATE, 13C, CROSS-POLARIZATION, “MAGIC-ANGLE” SPINNING, NMR SPECTROSCOPY STUDIES OF SEWAGE SLUDGE

Using 13C, solid-state NMR, we examined sludges and composts obtained from varied locations. A minimum of sample preparation is required with this technique. The samples are simply dried and ground before they are packed into the rotor. The spectra of the sewage sludges examined are composed of four broad envelopes of chemical shifts representative of carbonyl carbon, aromatic carbon, aliphatic carbon adjacent to -OH or -N, and aliphatic carbon. The intensities of the carbonyl carbon, aliphatic carbon adjacent to -OH and -N and aliphatic carbon resonances were less in the compost spectra, when compared with the intensity of the aromatic carbon resonances, than in the sewage sludge spectra. Spectra of four reference compounds (stearic acid, cellulose, lignin, and protein (keratin)) are included to assist in the interpretation of the sample spectra. Interrupted decoupling experiments were conducted to simplify these complex spectra and estimate the amount of nonprotonated (branched and carbonyl) carbon they contain. This preliminary solid-state, NMR study of sludges demonstrates that this technique is an effective tool for studying these complex systems.

Solid-phase extraction of sulfamethazine in milk with quantitation at low ppb levels using thin-layer chromatography.

Abstract A method for the solid-phase extraction and thin-layer chromatographic (TLC) quantitation of sulfamethazine (SMZ) residues in milk is presented. Sulfabromomethazine was added as an internal standard to homogenized milk samples which were then diluted and passed through C 18 solid-phase extraction columns. The C 18 columns were eluted with methanol, and interfering components in the methanol were removed by passing eluate over an acidic alumina column. The analyte was then concentrated on a small ion-exchange resin. SMZ was eluted and applied to a silica gel TLC plate. Fluorescence detection was induced with fluorescamine and quantitated with a scanning densitometer. Recoveries were 88.36–103.15% in the analysis range [0.51–15.34 ppb (μg/l)]. The average recovery over the analysis range was 96.07%, with a coefficient of variation of 12.52%.

Rapid electrodialytic clean-up of biological samples for high-performance liquid chromatography

A sample clean-up system employing electrodialysis with size-selective and charge-selective membranes is described. When applied to the treatment of 0.5-ml milk samples containing sulfamethazine, the system produced an undiluted, clear solution in 3 min and eliminated the components in untreated milk that caused column fouling and double peaks. In contrast to conventional liquid- and solid-phase extraction procedures, electrodialytic clean-up is readily automated and uses no organic solvents.

Reactions of hydroxyamino acids during hydrochloric acid hydrolysis

Chlorosubstitution reactions occur readily during HCl hydrolysis of delta- and epsilon-hydroxynorleucines (Hnle), the products of deamination of poly-L-lysine by nitrite at low pH. During amino acid analysis, chloronorleucines elute as new peaks after delta- and epsilon-Hnle. To determine if other hydroxyamino acids undergo similar changes during hydrolysis, they were subjected individually to HCl hydrolysis conditions with and without added phenol. Amino acid analyses indicated that terminal hydroxy groups on linear side chains undergo reactions during HCl hydrolysis; the products appear as new peaks which may be chloroderivatives. In contrast, no new peaks are observed in HCl hydrolysates of delta-hydroxylysine or amino acids with beta-hydroxy groups (beta-hydroxynorvaline, serine, and threonine). Phenol did not protect linear amino acids from reactions during HCl hydrolysis but did prevent loss of the cyclic amino acids tyrosine, hydroxyproline, and 3,4-dihydroxyphenylalanine. Although the gamma-hydroxy group of homoserine would be expected to undergo reaction, HCl catalyzes its cyclization to form homoserine lactone instead.

Chromatographic methods for determining the configuration of 1,2- and 1,3- (unsymmetrically substituted) dialkylglycerol ethers

Abstract The preparation of a series of 1,2- and 1,3-dialkylglycerol ethers as potential alternative substrates for biological and chemical studies is described. The absolute configurations of these compounds and their configurational purity can be determined by conversion into diastereomeric derivatives, the chromatographic properties of which are reported here. The gas—liquid chromatographic elution orders of the diastereomeric derivatives are discussed in relation to molecular size and shape.

Conversion of codeine to morphine in isolated capsules of Papaver somniferum

Abstract The biotransformation of codeine to morphine was studied in isolated capsules of Papaver somniferum . Cofactors such as nicotinamide adenine dinucleotide, adenosine 5′-triphosphate, S -acetyl coenzyme A and pyridoxal phosphate were not required in the conversion of codeine to morphine. Reducing agents such as dithiothreitol, glutathione and β-mercaptoethanol strongly promoted codeine and morphine degradation, while morphine formation remained at a constant level. Hydrogen peroxide (concentration > 0.25 mM) caused the conversion of codeine and morphine to N -oxides by non-enzymatic oxidation. Isolated capsules of P. somniferum provide a method of studying the biotransformation of codeine to morphine.

Reaction of fatty acids with 3,3′-iminobis-propylamine to produce soil hydrophobic agents

AbstractThe reaction between two moles of fatty acid and one of 3,3′-iminobis-propylamine (DPTA) is somewhat analogous to the reaction between fatty acid and diethylenetriamine (DETA) that we had reported previously, but there are significant differences. Conversion to the diamide HN(CH2CH2CH2NHCOR)2 proceeds much more rapidly but less efficiently than does the reaction of fatty acid with DETA. The former diamide is obtained in only about a 70% yield and byproducts are obtained, whereas the reaction with DETA yields the diamide almost quantitatively. Cyclization to the pyrimidine % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrpepeei0xd9vqVe0x% b9q8qqqrpe0db9pwe9Q8fs0-yqaqVepee9pg0Firpepe0de9vr0-vr% 0-vqpWqaaeaabiGaciaacaqabeaadaqaaqaaaOqaaGqaaiaa-jfaca% WFdbGaa83taiaa-5eacaWFibGaa83qaiaa-HeadaWgaaWcbaGaa8Nm% aaqabaGccaWFdbGaa8hsamaaBaaaleaacaWFYaaabeaakiaa-neaca% WFibWaaSbaaSqaaiaa-jdaaeqaaOGaa8hiaiaa-5eacaWFdbGaa8hs% amaaBaaaleaacaWFYaaabeaakiaa-neacaWFibWaaSbaaSqaaiaa-j% daaeqaaOGaa83qaiaa-HeadaWgaaWcbaGaa8NmaaqabaGccaWFobGa% a8hiaiaa-1dacaWFGaGaa83qaiaa-jfaaaa!4F73!