Analytical isotachophoresis of uremic blood samples

Uremic blood samples were analyzed for ionogenic substances using analytical isotachophoresis. Multicomponent separations proved that the uremic state shows significant differences from the normal state, especially with regard to anionic low-molecular-weight substances. As a quantitative parameter the ratio of anionic higher-molecular-weight substances to anionic low-molecular-weight substances is proposed: the HL ratio. Separation patterns and HL ratios were studied during nine weeks for one patient on chronic hemodialysis. The patient showed a low HL ratio due to excess of low-molecular-weight substances. Separation patterns before and after hemodialysis showed clear differences and the HL ratio increased. The method of analysis is neither time- nor sample-consuming and sample preparation is not needed. Experimental procedures are easily standardized and results are reliable.     

Monitoring of lithium levels in human serum after therapy with lithium preparations by capillary isotachophoresis

An isotachophoretic method for the evaluation of the level of lithium salts in serum samples was optimized. Use of operating systems containing polyethylene glycol permitted the separation of cationically migrating components from Li (i.e., Na, K and Ca). The pretreatment of serum samples involves only appropriate dilution with demineralized water depending on the concentration of the major components such as sodium. The lithium levels were studied both in model samples and serum from patients treated with lithium preparations.     

Analytical isotachophoresis in the analysis of synthetic peptides.

Two peptides, one undecapeptide and one decapeptide, have been synthesized by the solid phase method. Isotachophoresis has been used as an analytical tool to guide the purification of the peptides. This technique gives qualitative as well as quantitative information about the purification progress. Furthermore, the purity and identity of the final product can be established. The method is rapid, reproducible and easy to perform. Since isotachophoresis also can be used for amino acid analysis, it might have a wide use in peptide chemistry.The two peptides, having the primary sequences around the cross-linking site of fibrin, have been tested in vitro as inhibitors of the fibrin cross-linking. Both peptides were essentially inactive.     

Interaction of histones with human serum proteins.

The authors describe the interactions of whole calf thymus histone and its fractions with the human serum proteins. Immunoelectrophoretic analysis revealed two types of interactions: 1) a decrease in the electrophoretic mobility of a whole, immunochemically uniform fraction, and 2) a decrease in the electrophoretic mobility of only some molecules of such a fraction. In this association, the arginine-rich histone fraction (F3) was found to be the most active. The findings are important for interpreting the results of biological treatments.     

Analytical and preparative isotachophoresis of histoplasmin purified derivative components.

Skin test-active components of histoplasmin have been difficult to purify in quantity in the past. Analytical isotachophoresis in polyacrylamide gels was used to develop a high resolution system to separate the components of histo-plasmin. The analytical system was then expanded to preparative-scale isolation of the skin test-active components of histoplasmin.     

Determination of uric acid in serum using isotachophoresis

An operational system is described for the isotachophoretic determination of uric acid in serum, making use of column coupling. The method has been compared with a standard enzymatic procedure. With the present technique small amounts of serum (ca. 3 μl) can be applied without any pretreatment. Urate recovery was 99.0–100.5%. Under the non-physiological measuring conditions used, 12–28% of control serum uric acid was bound to macromolecules of molecular weight exceeding 25,000. The day-to-day variations of the isotachophoretic procedure were smaller than those of the enzymatic method, whereas standard deviations were comparable. The isotachophoretic procedure is less influenced by certain metabolites.     

Distribution of selenium-containing proteins in human serum

This study was undertaken to compare endogenous lithium concentrations in human blood and its components from normal donors versus bipolar patients. The patients were not on lithium therapy at the time that the blood samples were donated and had not received any lithium therapy for at least 2 yr. Blood components were separated by centrifugation. The analytical method for lithium as developed in this laboratory consists of thermal-neutron activation of freeze-dried samples. ³H is produced via the reaction ⁶Li+n=³H+⁴He, and high-sensitivity rare gas mass spectrometry is used to measure ³He formed from β-decay of ³H. Boron measurements are made concurrently using ⁴He from the reaction ¹⁰B+n=⁴He+⁷Li. Seven normal donors and seven patients with a diagnosis of bipolar disorder participated in this study. Measurements of lithium and boron were made in whole blood, plasma, and red cells. Red cell-plasma ratios R(Li) and R(B) were calculated after corrections were made for trapped plasma in the red cells. The results show that bipolar patients may have higher concentrations of lithium in blood, plasma, and red cells (p=0.08, 0.02, and 0.02, respectively) and may have higher R(Li) values than normal donors (p=0.01). No evidence was found for bipolar-normal differences in these four parameters for boron. Although our sample size is admittedly very small, the results clearly show that the endogenous red cell ratio R(Li) and plasma or red cell lithium concentrations may become useful diagnostic indicators for bipolar illness if the analytical methods are further developed. Certain commercial equipment, instruments, or materials are identified in this article to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. Prof. Clarke died unexpectedly on September 3, 2002.     

Affinity chromatography of thyroxine-binding proteins from human serum

The affinity matrix prepared by the attachment of L-thyroxine (T4) to epichlorohydrine-activated Sepharose 4B biospecifically absorbs the T4-binding globulin (TBG), 25K and 80/27K proteins, immunoglobulin G (IgG) and albumin (HSA) from human normal and retroplacental sera. The absorbed protein patterns were shown to depend on the immobilized T4 concentration, pH, temperature and incubation time. The potent eluents desorbing 85-100% of the protein are 1 mM NaOH, 3 M NH4SCN, 10(-5) M T4 or 3 mM 8-anilinonaphthalene-1-sulfonic acid (ANS) for TBG; NaOH, NH4SCN, 3 mM MgCl2 or 12mM sodium cholate for 25K protein and HSA; NaOH, NH4SCN or MgCl2 for the 80/27K and 25K proteins and IgG. Moreover, T4 desorbs small amounts (6-8%) of the 80/27K and 25K proteins, while sodium cholate elutes about 6% of TBG. The eluted from T4-Sepharose 4B and further purified TBG, 25K and 80/27K proteins display different [125I]T4-binding activities within the pH range from 2 to 9 and differ by their resistance to thermal inactivation at 50-80 degrees C. Double radial immunodiffusion analysis with the use of antisera to TBG, 25K, 80/27K, HSA and IgG demonstrated that the proteins share no common antigenic determinants. It was concluded that the novel 25K and 80/27K proteins represent endogenous components of the human blood thyroid hormone-binding protein system rather than fragments or aggregates of the known T4-binding proteins.     

Proteomic characterization of human serum proteins associated with the fat-derived hormone adiponectin

Adiponectin is a fat cell-secreted hormone with antidiabetic and anti-inflammatory activities. The reduced adiponectin levels are associated with obesity-related metabolic syndrome. Replenishment of this hormone into animal models can improve insulin sensitivity, decrease blood glucose and lipid levels, and prevent the development of atherosclerosis and fatty liver injury. Despite these findings, the underlying molecular mechanisms remain largely unknown. Here, we have used affinity chromatography to purify the protein complexes that are associated with adiponectin in human serum. The nature of these adiponectin-binding proteins was analyzed by MS/MS. Eight proteins from the adiponectin-containing protein mixtures have been identified. Many of them, including thrombospondin-1 (TSP-1), histidine-rich glycoprotein, kininogen 1, and alpha 2 macroglobulin (alpha2M), are well-known glycoproteins involved in the regulation of inflammation, angiogenesis, and tissue remodeling. Coimmunoprecipitation and radioligand competitive-binding assays confirmed the direct interactions between adiponectin and alpha2M, or TSP-1. Moreover, these specific bindings were also detected in the serum samples derived from both healthy human subjects and patients with type 2 diabetes. In summary, our study demonstrated that, in the circulation, adiponectin forms protein complexes with other serum proteins. These proteins might serve as the physiological-binding partners of adiponectin and regulate its bioavailability and biological activities.     

Preparative displacement electrophoresis (isotachophoresis) of proteins on cellulose columns

This paper describes the separation of proteins by displacement electrophoresis on columns packed with cellulose powder as a stabilizing medium. Cellulose has virtually no molecular sieving properties and thus differs from dextran, polyacrylamide, and agarose in this respect. Therefore, without the risk of unstacking, columns packed with cellulose permit conventional elution of the protein zones and the use of a counter flow (to increase the effective length of the bed). For the same reason, electroosmotic flow is less disturbing. A continuous elution-migration technique adapted to suit the special requirements of displacement electrophoresis gave better separation than was obtainable by conventional elution. Normal human serum and a fresh hemolysate from human erythrocytes were used as samples. An expression for the volume velocity of the boundaries is derived. This parameter can be used to determine the maximum duration of a run and a suitable pump speed when continuous elution or a counter flow is employed. The special advantages of displacement electrophoresis in cellulose beds are discussed as well as general disadvantages of the displacement technique, including the risk that proteins precipitate during a run.     

Inhibition of oxidation by peroxidase of human serum proteins

The oxidation of essential serum proteins, albumin and gamma globulin, by the enzyme peroxidase can be partially inhibited by compounds, such as EDTA and 2,4-pentanedione, that complex with the iron ion in peroxidase. The importance of such inhibition lies in the circumstance that the oxidations in question might be a possible causative factor in tissue aging.     

Salt-independent adsorption of human serum proteins on cyanocarbon gels

Electron donor acceptor gels based on cyanocarbons have been tested for human serum protein adsorption in the absence of salt-promotion by water-structuring salt. This phenomenon was compared with a normal adsorption process in the presence of salt. The tricyanoaminopropene–divinyl sulfone–agarose displayed unusual protein adsorption properties as binding could occur both independently or dependently of the salt-promotion. The absence of hydrophobic or ionic character of the salt-independent interaction suggests an electron donor acceptor adsorption mechanism which is shown, for the first time, to occur independently of salt-promotion in aqueous solution. Study of the protein adsorption specificity showed similar protein selectivity for the fractions adsorbed in both conditions.     

Characterization of proteins in the human serum proteome.

This paper presents a multidimensional profile of the human serum proteome, produced by a two-dimensional protein fractionation system based on liquid chromatography followed by characterization with capillary electrophoresis (CE). The first-dimension separation was done by chromatofocusing over a pH range from 8.5 to 4.0, where proteins were separated by their isoelectric points (pI). In this dimension, fractions were collected based on pH. The first-dimension pI fractions were then resolved in the second dimension by high-resolution, reversed-phase chromatography with a gradient of trifluoroacetic acid (TFA) in acetonitrile and TFA in water. A selected protein fraction collected from the second dimension by time was characterized by CE for molecular-weight estimation and for presence of isoforms. Molecular-weight estimation was done by sodium dodecyl sulfate capillary gel electrophoresis, where proteins were separated in the range of 10,000-225,000 Da. Detection of isoforms was done by capillary isoelectric focusing over a pH range of 3-10. A selected second-dimension fraction that contained the putative serum iron-binding protein transferrin was analyzed by these two CE techniques for molecular-weight determination and the presence of isoforms. The combination of two-dimensional protein fractionation and CE characterization represents an advanced tool for proteomics.