Steven C. Goheen

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.

Mass spectrometry of low molecular mass solids by matrix-assisted laser desorption/ionization

Matrix-assisted laser desorption/ionization combined with time-of-flight mass spectrometry (MALDI/TOF-MS) was used for the analysis of low molecular mass compounds. Three classes of molecules were studied: organic acids, salts of oxyanions and amine-based chelating compounds. Mass spectra from samples of citric, propionic, butyric, oxalic and stearic acid; ethylenediaminetetraacetic acid (EDTA),N-(2-hydroxyethyl) ethylenediaminetriacetic acid (HEDTA), ethylenediamineN,N′-diacetic acid (EDDA) and nitrilotriacetic acid (NTA); and sulfate, nitrate, nitrite and phosphate salts were obtained. These species were analyzed alone and as mixtures in both the positive and negative ion modes. The organic acids and oxyanion salts displayed much stronger signals in the negative ion detection mode whereas chelating compounds, which contain basic amine functional groups, yielded stronger signals in the positive ion mode. This implies that detection sensitivity is often better for a particular ion mode in the analysis of small molecules containing limited classes of functional groups. In all analyses, the presence of high concentrations of sodium was found to quench the MALDI signals. To increase the detection sensitivity, some samples were processed through an ion-exchange column to remove sodium ions. This step was found to enhance the signal by two orders of magnitude over untreated samples.

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.

Modified Fibers with Medical and Specialty Applications

The research and development of chronic wound dressings, which possess a mechanismbased mode of action, has entered a new level of understanding in recent years based on improved definition of the biochemical events associated with pathogenesis of the chronic wound. Recently, the molecular modes of action have been investigated for skin substitutes, interactive biomaterials, and some traditional material designs as balancing the biochemical events of inflammation in the chronic wound to improve healing. The interactive wound dressings have activities including up-regulation of growth factors and cytokines and down-regulation of destructive proteolysis. Carbohydrate-based wound dressings have received increased attention for their molecular interactive properties with chronic and burn wounds. Traditionally, the use of carbohydrate-based wound dressings including cotton, xerogels, charcoal cloth, alginates, chitosan, and hydrogels have afforded properties such as absorbency, ease of application and removal, bacterial protection, fluid balance, occlusion, and elasticity. Recent efforts in our lab have been underway to design carbohydrate dressings that are interactive cotton dressings as an approach to regulating destructive proteolysis in the non-healing wound. Elastase is a serine protease that has been associated with a variety of inflammatory diseases and has been implicated as a destructive protease that impedes wound healing. The presence of elevated levels of elastase in non-healing wounds has been associated with the degradation of important growth factors and fibronectin necessary for wound healing. Focus will be given to the design, preparation, and assessment of a type of cotton-based interactive wound dressing designed to intervene in the pathophysiology of the chronic wound through protease sequestration.

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.

Performance of Bioactive Molecules on Cotton and Other Textiles

Four types of biologically active molecules were examined for their structure/activity relationships as applied to textile functionalization. Bio-molecules including enzymes, peptides, carbohydrates, and lipids have been found to retain their activity when linked to cotton fabrics. Wound dressing protection against the protease destruction caused by human neutrophil elastase was examined with cellulose conjugates and formulations of peptides, carbohydrates, and lipids attached with various chemistries to cotton dressings. These serve as a model for protective textiles at the surface of the skin. Additional biological activities that were explored included antibacterial and haemostatic fabrics related to wound healing, and neurotoxin neutralization related to decontamination. Lysozyme was found to have robust antibacterial activity when conjugated to cotton. Peptide conjugates of cellulose have been explored as enzyme substrates, antimicrobial agents, and cell adhesion promoters on textiles for wound healing. Carbohydrates ranging from low molecular weight monosaccharides to high molecular weight polysaccharides have both molecular and functional activity when crosslinked or grafted onto cotton with numerous textile performance properties. Textile bound lipids have been explored for a variety of applications including antibacterial, hygienic function, and enzyme inhibition. A lipid: albumin complex that serves as a carrier transfer agent involved in enzyme inhibition is given as an example.

Modification of polyurethane to reduce occlusion of enteral feeding tubes

Feeding tubes are used to supply nutritional formula to immobilized patients. The most common cause for failure of enteral feeding tubes is their occlusion. The purpose of this study was to examine whether occlusion of enteral feeding tubes could be minimized using an additive. An open, intermittent enteral feeding system was simulated in the laboratory and data were collected over a period ranging from 2 to 6 days. Feeding formula was cycled through a feeding tube in either the presence or absence of simulated gastric acid in an effort to generate a reproducible occlusion. Pressures in the tube were measured frequently throughout these cycles. We observed pressure spikes with each cycle, but never a complete occlusion. Pressure spikes formed only when simulated gastric acid was mixed with the feeding solution. Large amounts of feeding formula adsorbed onto polyurethane (PU) surfaces in the presence of gastric acid. Also, this subtle change in surface chemistry significantly affected the number of pressure spikes observed. The maximum pressure required to maintain flow in the tube was reduced by about half from 2.0 psi to 0.8 psi when polyvinyl alcohol (PVA) was added. The addition of PVA to PU also reduced the contact angle from 83 degrees (untreated) to approximately 64 degrees in the presence of PVA. Furthermore, when formula was added to PU in the presence of PVA the thickness of the layer that remained on the surface was almost 10 times greater in controls than on PVA-treated surfaces. These results suggest that a treatment that increases the hydrophilicity of the feeding tube may help minimize clogging.

Application of derivatization gas chromatography/mass spectrometry for the identification and quantitation of pinitol in plant roots.

Abstract Pinitol (D-3-O-methyl-chiro-inositol), a cyclitol, has a variety of roles in plant biology, and is being used as a nutritional supplement. These applications increase the importance of finding new methods for determining the pinitol content in plant tissues. A reliable method for the identification and quantitation of pinitol using trimethylsilyl imidazole (TMSI) derivatization and gas chromatography/mass spectrometry (GC/MS) was developed. One major ion fragment, m/z 260, was used to quantify pinitol in three plant species. Soybeans contained approximately 1.9 mg/g wet weight pinitol while levels in sugar beets and snap beans were below detection limits.

Nature and Nanotechnology

Scientists and engineers are rapidly developing techniques to produce nanostructures for various applications: nano-devices include electronic components, catalysts, and mechanical systems such as levers and motors. Biomedical applications include nanobots intended to function in various body fluids, pumps and drug delivery products, and implantable biosensors. Nature has been developing processes and products at the nano scale throughout the evolutionary process. There are several biochemical processes that involve energy-producing reactors, synthesis, and various mechanical processes. The relationships between the recent accomplishments of scientists and engineers in nanotechnology and natural processes are the subject of this paper. Numerous examples are provided in which the structure of a biological system at the nano-scale relates to some elaborate biological function. The structure and function for biological systems and biochemicals can in some cases be correlated to the structure and function of man-made nanomaterials. In this paper, we focus on proteins, but similar insight can be attained from other biomolecules and a wide variety of biological processes.

Analysis of Hanford-Related Organics Using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry

Matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI/TOF-MS) was used for the analysis of low-molecular phosphate compounds found in Hanford tank wastes. The mass spectra of these compounds indicate protonated peaks as well as sodium adducts. Analytical methods presently utilized for the analysis of the phosphate-related organics are both time consuming and labor intensive. A promising alternative is MALDI/TOFMS. The MALDI process produces both positive and negative ions directly and very little sample is required. In addition, there is limited sample preparation and minimal hazardous waste production.