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S C Goheen

Publications and source records attributed to S C Goheen.

At least 19 recordsLinked to original sources
PubMed2000

Protein losses in ion-exchange and hydrophobic interaction high-performance liquid chromatography.

Protein losses in ion-exchange and hydrophobic interaction HPLC were examined. The supports were all non-porous, packed in columns of identical dimensions. Two ion-exchange chromatography (IEC), anion and cation, as well as a hydrophobic interaction chromatography (HIC) columns were tested. Proteins included cytochrome c, bovine serum albumin (BSA), immunoglobulin G and fibrinogen. Temperature effects on HIC supports were studied for cytochrome c and BSA. Both retention times and recoveries of the proteins were measured. The influence of column residence time on the recovery of proteins was also investigated. We found a linear relationship between the amount of protein recovered and the log of the molecular mass. Retention times also generally increased with temperature for both HIC and IEC. Other trends in retention behavior and recoveries are discussed.

Chromatography, High Pressure Liquid↗
PubMed1999

Cytochrome c unfolding on an anionic surface.

It is now well accepted that the adsorption of proteins to solid supports sometimes involves surface-mediated unfolding. A detailed understanding of the adsorption and surface-mediated unfolding process is lacking. We selected a well studied protein, horse heart cytochrome c, and a weakly ionic support to examine some of the characteristics of protein adsorption under near-physiological conditions. We used high-performance liquid chromatography (HPLC) to investigate the effect of temperature on surface-mediated unfolding. Samples of cytochrome c were introduced to an anionic support, and a NaCl gradient was used to desorb the protein at different times and temperatures. The profiles and retention times were monitored to examine the adhesive properties of cytochrome c to the anionic support. We found that protein retention increased with time at temperatures as low as 0 degrees C, and a significant loss of cytochrome c occurred between 55 degrees C and 70 degrees C. The loss of recovery of cytochrome c indicates irreversible surface-mediated unfolding. The changes in retention time may indicate more subtle transitions, including reversible surface-mediated unfolding of cytochrome c. These results suggest that perturbations in the structure as well as unfolding of cytochrome c can be detected at a lower temperature on an anionic surface than in solution thereby acting like a catalyst for protein unfolding.

Adsorption↗
PubMed1999

Synthesis and activity of NH2- and COOH-terminal elastase recognition sequences on cotton.

The application of peptide recognition sequences of elastase to fibers of wound dressings is a possible route to inhibiting high levels of destructive elastase in the chronic wound. For this reason we have synthesized the elastase recognition sequence Val-Pro-Val on both cotton cellulose, and carboxymethylated cellulose cotton (CMC) and prepared chromatography columns of these to examine elastase retention. The tripeptide was synthesized on cotton-based cellulose fibers both in sequence and as a tripeptide methyl ester. Glycine was employed as a linker of the recognition sequence to the cotton cellulose. Pre-treatment of cotton cellulose with cellulase improved the substitution level of glycine. The peptidocellulose conjugates were employed as a chromatographic stationary phase to assess elastase retention. The sequence Val-Pro-Val-OMe was amino-terminally anchored to carboxymethylated cotton and demonstrated retention of up to 58% of elastase when first applied to the column. Higher repetitive retention was demonstrated subsequently. Cotton gauze similarly modified with Val-Pro-Val-Gly cellulose was compared with untreated gauze for reduction of elastase activity in buffered saline. Solutions of elastase that were treated with Val-Pro-Val-Gly cellulose cotton gauze, demonstrated reduced elastase activity. This study demonstrates the use of elastase recognition sequences as sequestering agents of elastase when attached to cotton fibers and constitutes a model for the design of peptidocellulose analogs in dressing fibers for chronic wounds.

Cellulose↗
PubMed1998

High-performance ion-exchange chromatography and adsorption of plasma proteins.

Resolution and recovery are primary concerns in protein chromatography. Separations are often by size, ion exchange, or hydrophobic-hydrophilic properties of the support, eluent and protein. Adsorption of a protein to a synthetic surface plays an essential role in this complex process. In this study, we examined the adsorption properties of three representative plasma proteins (albumin, fibrinogen, and immunoglobulin G) on nonporous column materials containing either quaternary amine or sulfopropyl functional groups. The adsorption properties were studied at 37 degrees C and pH 7.4. Salt gradients were used to examine the adsorption/desorption properties of each of the proteins on each type of surface. The salt concentrations at desorption were measured and compared to the protein isoelectric points. In addition, we examined protein recoveries as a function of desorption time. Our results suggest that protein recoveries depend not only on the protein, eluent and surface, but also the residence time and overall charge concentration during the initial adsorption process. Finally, we correlated the number of charge sites on a molecule with the width of a chromatographic band at half height. The data produced as a result of this study may be used to determine the actual unfolding time of a protein, given a certain set of conditions. The data may also help in understanding the chemistry and dynamics of the protein adsorption processes in ion-exchange chromatography as well as provide key structural information about the proteins.

Adsorption↗
PubMed1986

Use of high-performance size exclusion chromatography to determine the extent of detergent solubilization of human erythrocyte ghosts.

We have studied the effects of various detergents and their concentration during human erythrocyte membrane solubilization. Detergents were selected on the basis of their low UV absorption at 280 nm, making them useful for high-performance liquid chromatography. High performance size-exclusion chromatography was then utilized to monitor the efficiency of solubilization. Sodium dodecyl sulfate solubilized more of the erythrocyte membrane proteins than any of the other detergents studied. 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfate solubilized some, but Tween 20 and reduced Triton X-100 solubilized fewer of the human erythrocyte membrane proteins. In conclusion, high-performance size exclusion chromatography provides a rapid method for determining whether membrane proteins have been effectively solubilized.

Blood Proteins↗
PubMed1986

Reversed-phase high-performance liquid chromatography of red blood cell membranes.

Three reversed-phase (Hi-Pore RP-318, Hi-Pore RP-304, and Bio-Gel TSK Phenyl-RP+) and one hydrophobic-interaction (Bio-Gel TSK Phenyl-5PW) columns were used in a Bio-Rad chromatography system to separate the membrane proteins of human erythrocytes. A linear gradient, starting with 0.05% trifluoroacetic acid and ending with 95% acetonitrile and 0.05% trifluoroacetic acid was used. The four columns demonstrated slightly different selectivities for the proteins in ghosts. These profiles were further altered when ghosts were solubilized with 0.1% sodium dodecyl sulfate. The columns with less hydrophobic packings and larger pore sizes appear to be best suited for reversed-phase analyses of erythrocyte membrane proteins. Detergent solubilization was unnecessary for good resolution of the protein components.

Blood Proteins↗
PubMed1986

Ozone and the peroxidation of polyunsaturated fatty acids in vivo.

Two experiments were carried out to determine whether ozone causes significant oxidation of pulmonary polyunsaturated fatty acids in vivo. These involved ad libitum and pair-feeding. In the first experiment, rats were fed fat-free diets and exposed to ozone for 0, 1, 2, and 4 weeks. Lung and liver fatty acids were analyzed to determine if the rats exposed to ozone lost essential fatty acids more rapidly than those exposed to filtered air. In the second experiment, rats were divided into four groups. Two of these groups were fed fat-free diets, and two were fed diets containing essential fatty acids. Rats from the two diet groups (one of each type) were exposed to ozone, while the remaining two groups were exposed to filtered air. In the second experiment, rats were pair-fed. The amounts of lung and liver fatty acids were relatively uninfluenced by breathing ozone. Results from these experiments demonstrate that in the lung, the polyunsaturated fatty acids, linoleic and arachidonic acids, appear to be oxidized by filtered air and ozone at essentially the same rate.

Animals↗
PubMed1985

Purification of human serum gamma globulins by hydrophobic interaction high-performance liquid chromatography.

Fresh, whole human serum was fractionated on a Bio-Rad Protein Chromatography System, equipped with a Bio-Gel TSK Phenyl-5PW column, by utilizing a descending linear gradient of ammonium sulfate in 0.1 M sodium phosphate buffer, pH 7.0, at 0 degree C. Two major peaks were isolated corresponding to albumin and gamma globulin. The identity of these protein peaks was substantiated by chromatography of an albumin-gamma globulin standard mixture. The purity of the individual fractions was verified by high-performance size exclusion chromatography (HPSEC) on either a Bio-Sil TSK-250 or a Bio-Gel TSK-40 column. The applicability of these HPSEC columns to the molecular weight characterization of the Bio-Gel TSK Phenyl-5PW column fractions was compared. Typically, the Bio-Gel TSK Phenyl-5PW column (75 X 7.5 mm I.D.) was used to purify gamma globulin from 100 microliter of plasma. This corresponded to approx. 1.5-2.0 mg of the globin fraction. Unidentified contaminants in this fraction had molecular weights of approx. 1000-3000 daltons and 260 000-300 000 daltons.

Chromatography, Gel↗
PubMed1983

Complexities in lipid quantitation using thin layer chromatography for separation and flame ionization for detection.

The use of thin layer chromatography (TLC) for separation (using silica gel coated quartz rods) and subsequent flame ionization for detection (FID) was examined to determine whether this method could be used for the quantitation of lipids. However, response factors (RF) for various lipids were different and depended upon several variables including the amount of material analyzed. For example, RF were 3-fold greater when 10 micrograms of tripalmitin was analyzed as compared to 1 microgram of the same material. The amount of lipid detected by FID was also dependent upon the rate at which it passed through the flame. During analysis of methylpentadecanoate, detector response increased with scan speed, while at all speeds it was completely removed from the rod. On the other hand, depending upon the amount of cholesterol or phospholipid analyzed, the response either increased, remained unchanged or decreased with scan speed. During a fast scan, detector response was reduced because some material remained on the rod. Thus, the detector response is influenced by sample volatility. In conclusion, there appears to be a complex relationship between detector response and the amount of heat available per microgram of sample. Since we could not find a direct correlation between detector response and sample quantity, it would be difficult to use TLC-FID as a tool for quantitating the components of a lipid mixture.

Cholesterol↗
PubMed1983

Severe fatty liver in rats fed a fat-free ethanol diet, and its prevention by small amounts of dietary arachidonate.

Rats were fed ethanol and a fat-free diet for 30 days to determine whether dietary fat is needed for the development of fatty liver. The severity of fatty liver was similar to that of rats fed an isocaloric diet with 35% fat. Small amounts (29 mg/day) of dietary arachidonic acid prevented alcoholic fatty liver. Rats fed either the alcohol (AF) or control (CF) fat-free diets developed essential fatty acid deficiency (EFAD) as measured by the triene/tetraene ratio of liver and plasma lipids. Rats fed arachidonic acid (AA, alcohol and CA, control diets) did not develop EFAD. Although EFAD alone did not cause the development of fatty liver, the combination of dietary ethanol and EFAD did. The ratios of 16:1/16:0 and 18:1/18:0 in liver lipids indicated that desaturase enzymes were less active and lipogenesis was reduced in rats fed the AA diet compared to those fed the AF diet. In contrast, stimulated lipogenesis appears to have been the cause of fatty liver in rats fed the AF diet.

Animals↗
PubMed1983

Morphology and fatty acid composition of erythrocytes from monkeys exposed to ozone for one year.

Monkeys (Macaca radiata) were exposed to a low dose (0.64 ppm) of ozone (O3) for 8 hr each day over a 1-year period. Control monkeys were exposed to filtered air. The morphology of the red blood cell (RBC) from these monkeys was analyzed by scanning electron microscopy. Red cells from control monkeys had normal morphology with 69% having biconcave shape (discocytes). In O3-exposed animals, blood contained only 35% discocytes. Knizocytes, stomatocytes, and spherocytes each have defined shape which are different from discocytes. The number of knizocytes and stomatocytes in O3-exposed monkeys was twice that of controls. In addition, significant levels of spherocytes were observed while they were absent in the blood of controls. The fatty acid composition of RBC from control and O3-exposed monkeys was the same. These were similar to that of human RBC. However, earlier reports of monkey RBC fatty acid composition differ from our results. These differences are discussed. We conclude that low-dose O3 exposure changes the morphology, but not the fatty acid composition, of erythrocytes in vivo.

Animals↗
PubMed1982

Morphology and fatty acid composition of reticulocytes from phenylhydrazine-treated rats.

Reticulocytosis was induced in rats by injecting phenylhydrazine, a potent oxidizing agent. Red cell morphology was analyzed by scanning electron microscopy. The majority of red cells from rats given injections of phenylhydrazine were types 2 and 3 echinocytes. Stomatocytes were also observed, but pitted lobular reticulocytes were not detected. Echinocytes have not previously been observed in reticulocyte populations. In the reticulocytes, the relative levels of 16:1 and 18:1 were significantly greater than in erythrocytes. These differences in monoenoic acids may be due to the presence of endoplasmic reticulum, the site of desaturase activity in reticulocytes. Of all the fatty acids, the polyunsaturates are the most susceptible to attack during peroxidation. However, the polyunsaturated fatty acid composition of reticulocytes was similar both to that of erythrocytes and to reported values of young erythrocytes isolated by density. Therefore, it is unlikely that lipid peroxidation caused the formation of echinocytes.

Animals↗
PubMed1981

Changes in relative levels of linoleate to arachidonate in erythrocyte phosphatidylcholine in rats fed ethanol and arachidonate.

Young male Sprague--Dawley rats were fed ad lib. for 4 weeks a liquid diet containing 34% of the calories as ethanol and 35% as fat (25 wt % linoleate). The ratio of the levels of linoleic acid to arachidonic acid (18:2/20:4) in erythrocyte phosphatidylcholine (PC) from alcohol-fed rats was greater than that from rats fed an isocaloric control diet. The increased 18:2/20:4 ratio in tissue lipids of animals ingesting ethanol is thought to be due to the reduced production of arachidonate from linoleate. In the present study we tested whether dietary arachidonate would mask the effects of alcohol on the 18:2/20:4 ratio in erythrocyte PC. Analysis of the fatty acid composition of erythrocyte PC of rats fed a diet supplemented with arachidonate showed that ethanol feeding did not alter the 18:2/20:4 ratio.

Animals↗
PubMed1980

Dietary arachidonic acid reduces fatty liver, increases diet consumption and weight gain in ethanol-fed rats.

We fed young male Sprague-Dawley rats for 4 wk ad libitum liquid diets containing 34% of the calories as ethanol and 35% as fat with (AA+) and without (AA-) arachidonic acid (20:4). Additional rats in the control groups were fed similar diets made isocaloric with dextrose with (CA+) and without (CA-) 20:4. The liver triglyceride (TG) content of rats in the AA+ group was reduced ca. 3-fold over that of rats in the AA-group. The diet consumption and body wts of rats in the AA+ group were significantly greater than those of rats fed alcohol without the 20:4 supplement (AA-). Also livers from rats in the AA+ group were as large as those from rats in control groups (CA+, CA-) and ca. twice as large as those from rats in the AA-group. The fatty acid composition of liver TG in rats fed the alcohol diet was similar to that of dietary fat. Levels of 20:4 and docosatetraenoic acid (22:4) in liver TG fatty acids from rats fed diets without arachidonate (AA-, CA-) were low (trace to 1.6%). After ingestion of arachidonic acid, 20:4 increased to ca. 10% and 22:4 to ca. 5%. The content of liver phospholipids was higher in livers of rats fed ethanol (AA-) than in those of controls (CA-).

Animals↗
PubMed1980

Preparation of radiolabled tetracosa mono- and dienoic acid methyl esters from rat erythrocyte lipids by thin layer chromatography.

An easy method of obtaining pure fatty acid methyl esters (FAME) of tetracosa mono- and dienoic acids (24:1, 24:2) using thin layer chromatography (TLC) is described. The total lipids isolated from rat erythrocytes were treated with methanolic-NaOH. Sphingomyelin was unaffected by this treatment and was separated from FAME of glycerolipids and cholesterol by TLC. FAME of sphingomyelin were then prepared by acid methanolysis. These esters migrated into 2 distinct bands on TLC. The slow moving band contained FAME of 16:0, 16:1, 18:0, 18:1, 19:0 and 20:0 wheras the fast moving band contained FAME of 22:0, 23:0, 24:0, 24:1 and 24:2. After AgNO3-TLC, the FAME of the fast moving band separated into 3 species; esters of saturated acids, 24:1 and 24:2, respectively. With erythrocyte lipids of rats fed a fat-free diet and injected with 14C-18:1, this method yielded 14C-24:1. From rats injected with 14C-18:2 and maintained on a corn oil diet, 14C-24:2 was obtained.

Animals↗