Development of poly(methacrylate)-grafted Sepharose FF for cation-exchange chromatography of proteins.

Polymer-grafted media have been a focus of recent development for ion exchange chromatography (IEC) because of their capacity and uptake kinetics that can lead to high dynamic capacity in protein purification. This work is devoted to developing novel cation exchangers of high adsorption performance by grafting polymerization of sodium methacrylate (MA) onto a commercial agarose gel Sepharose FF (FF). Five polyMA (pMA)-grafted FF gels were prepared with the same grafting density but different chain lengths (i.e., different ionic capacities, ICs), and named as FF-pMA-IC (IC denotes IC value in mmol/L). The effects of chain length (IC) and ionic strength (IS) on protein adsorption and chromatographic behaviors were examined using lysozyme (at pH 8.0) and γ-globulin (at pH 5.0) as model proteins. It was found that lysozyme adsorption capacity increased with increasing IC till reaching a plateau (390 mg/mL) over IC=540 mmol/L (FF-pMA-540), while there was an optimum IC (320 mmol/L, FF-pMA-320) at which γ-globulin adsorption capacity reached the highest (208 mg/mL). With increasing chain length (IC), the uptake rates of both the proteins presented decreasing trends due to the steric hindrance caused by the polymer chains. At the same IC, however, the uptake rate of lysozyme was much higher than that of γ-globulin because of the different sizes of the two proteins. Increasing salt concentration obviously promoted the uptake rates of the proteins, which led to the increase of dynamic binding capacities (DBCs) in different salt concentration ranges. The DBC value of lysozyme on FF-pMA-540 kept as high as 108-198 mg/mL in the salt concentration range of 0-150 mmol/L, and the DBC of γ-globulin on FF-pMA-320 increased to 27 mg/mL with increasing salt concentration from 100 mmol/L. This work clearly indicated the presence of optimal IC values (chain lengths) for different sized proteins, and IS was also crucial for reaching a high DBC for a specific protein. The findings provided insight into the selection of FF-pMA-n gels and operational conditions (e.g., IS) for the purification of a target protein of defined size.

Evaluation of steric exclusion chromatography on cryogel column for the separation of serum proteins.

Steric exclusion chromatography (SXC) is a new mode of protein chromatography, in which large proteins are retained on hydrophilic stationary phase surface due to the steric exclusion of polyethylene glycol (PEG) in the mobile phase, and thereafter the retained proteins can be eluted by reducing PEG concentration. In this work, SXC was evaluated on a polyacrylamide cryogel monolith. Microscopic observation of γ-globulin precipitates on the gel surface in SXC was reported for the first time. Due to the compact packing of protein precipitates on the stationary phase surface, the dynamic retention capacity of the cryogel monolith for γ-globulin reached 20 mg/mL bed volume, much higher than those of cryogel beds in adsorption-based chromatography. The effect of molecular weight and concentration of PEG, solution pH and salt concentration on protein retention capacity was in agreement with the earlier work on SXC. Because the cryogel monoliths with interconnected macropores (10-100 µm) allow much easy flow-through of viscous PEG buffer, the SXC can be operated at low back pressure. Hence, the cryogel monoliths are more suitable for SXC than other monoliths of narrow pores reported previously. In the separation of bovine serum proteins, albumin was recovered in the breakthrough fraction with high purity, and globulin was over eight times concentrated in the elution pool. This work has, thus, demonstrated the rapid serum protein separation and concentration by SXC on the cryogel monolith columns.

Activated carbons and carbon-containing poly(vinyl alcohol) cryogels: characterization, protein adsorption and possibility of myoglobin clearance.

Adsorption of myoglobin (Mb), bovine serum albumin (BSA) and γ-globulin (GG) onto activated carbons (ACs) with different pore size distributions, and poly(vinyl alcohol) (PVA) monolithic cryogels containing AC particles was studied. The highest initial rate of Mb adsorption was observed for AC having the largest specific surface area (1939 m(2) g(-1)) and pore volume (1.82 cm(3) g(-1)). The adsorption kinetics of proteins was characterized by a bimodal shape of the distribution f(D) function of an effective diffusion coefficient. Adsorption isotherms of Mb and GG were of Freundlich type within the studied range of equilibrium concentrations (10-150 µg mL(-1)). The distributions of free energy of protein adsorption were bimodal and reflected both interactions with carbon surfaces and self-association of proteins. Adsorbed amounts of Mb were the highest among the proteins studied (up to 700 mg g(-1) carbon), which was attributed to the higher fraction of pores accessible for Mb. Incorporation of carbon particles into PVA-based cryogel resulted in macroporous monolithic composite materials (AC-PVA) exhibiting good flow-through properties. Scanning electron microscopy of the composites showed macroporous aggregates of carbon particles held together by films and bridges of PVA. The rates of adsorption and adsorbed amounts of proteins on AC-PVA were reduced compared to the pristine carbon and depended on the carbon content in the composites. Nevertheless, adsorption of Mb on AC-PVA took place even in the presence of 500-fold higher concentration of BSA. This indicated a possibility of Mb clearance from blood plasma using the PVA-carbon monoliths.

A one-step exclusion-binding procedure for the purification of functional heavy-chain and mammalian-type gamma-globulins from camelid sera.

A new approach has recently been proposed for the purification of 'mammalian-type' IgG, consisting of exclusion binding. The technique uses a gel ('Melon gel'; Pierce) that binds to all plasma proteins, but not to IgGs, thus allowing IgGs to be recovered in the FT (flow-through) fraction. Here, the technique was applied to camelid IgGs, which are known to be composed of not only classic mammalian-type IgGs (IgG1) but also HC-IgGs (heavy chain IgGs). Both mammalian type and HC-IgGs can be purified in the FT fraction of dromedary (Camelus dromedarius) plasma samples with less than 8.5% contamination, by making minor improvements to the conditions recommended by the manufacturer. The contaminant proteins, as determined by LC-MS/MS (liquid chromatography-tandem MS), are mainly transferrin and albumin. The recovery rate is elevated for both types of IgGs (95+/-14% and 88+/-25% for IgG1 and HC-IgGs respectively). IgGs thus purified maintain their ability to bind to their antigen, as measured by surface plasmon resonance and Western ligand blotting. Furthermore, IgGs can be purified from plasma samples of all camelid species in a similar manner, although the ratio of HC-IgGs to total IgGs was lower for Lama (llama) and Vicugna (vicuña) than for Camelus species. The 'Melon gel' technique can thus be used to satisfactorily purify IgG1 and HC-IgGs from all camelid species.

Analysis of gamma-globulin mobility on routine clinical CE equipment: exploring its molecular basis and potential clinical utility.

A study was conducted on the variability of gamma-globulin mobility in serum protein electrophoresis and its molecular basis. We found that the migration time of gamma-globulins can be reproducibly determined (CV=1.1%) on clinical CE equipment. Moreover, we found a significant difference (p<0.001) in the migration of gamma-globulins between chronic liver disease patients (n=98) and a healthy reference group (n=47). Serum immunoglobulins were purified from these patients' sera using protein L-agarose and their glycosylation was studied using CE on a DNA sequencer. This glycomics approach revealed that several non-sialylated N-glycans show a moderate Pearson correlation coefficient (r=0.2-0.4) with the migration time of gamma-globulins. Their sialylated structures correlate negatively (r=-0.2 to -0.3). Immunoglobulins are significantly more sialylated in the healthy reference group compared with the patients (p<0.001). We estimated that sialylation heterogeneity contributes about 36% to the molecular variance (carbohydrates and amino acid composition) that affects the electrophoretic mobility of immunoglobulins. This is the first report on the migration time of gamma-globulins on a clinical CE instrument and its potential clinical value to the routinely analyzed serum protein CE profiles.

Analysis of immune complexes by capillary electrophoresis.

A simple method for immune complexes (IC) analysis by CE is described. This method combines the ease of precipitation of the IC by polyethylene glycol with the separation power of CE. The advantage of this method is a better quantitation of the IC, since it corrects and eliminates the interferences from other serum proteins. It also reveals the composition (monoclonality) of the precipitate. Three types of IC have been detected in this method: monoclonal, polyclonal and mixed (mono-polyclonal) IC. Furthermore, the method is rapid and simple.

[Kidney vasculitis connected to cryoglobulinemia IIA and hepatitis B]. / Vascularite rénale dans le cadre d'une cryoglobulinémie mixte associée au virus de l'hépatite B.

We report the case of a 70 years old patient hospitalized for renal insufficiency and deterioration of the general state. The electrophoresis of serum proteins on freezing of agarose reveals the presence of a discrete peak of monoclonal pace on the level of the gammaglobulines identified by serum immunofixation like IgM of the kappa type. The research of the cryoglobulinemia carried out in a laboratory of city was made positive and typified like a monoclonal cryoglobulinemia IgM kappa, thus directing the diagnosis towards a disease of Waldenström. However, the result of the biopsy medullary made exclude any lymphoprolifératif syndrome. The positivity of the serology of hepatitis B justified a second request for study of the cryoglobulinemia, carried out within our laboratory. The cryoglobuline was typified like mixed (IgM kappa monoclonal and IgG polyclonales). This result associated with the immunological assessment and the renal biopsy made retain for our patient the diagnosis of a kidney vasculitis connected to an infection chronicle by the virus of hepatitis B. This observation points out the interest of the preanalytic, analytic and post analytic phases in the study of the cryoglobulinemias. A good technical control is today the only guarantee of the quality of the result of this examination which has large importance in internal medicine in the etiologic assistance with the diagnosis of certain clinical demonstrations.

[Preparation of placental-eluted gamma globulin and its immunosuppressive effect in vitro and in vivo].

The aim of this study was to establish a simple, convenient and efficient method of producing placental-eluted gamma globulin (PEGG) from human placenta, explore its inhibitory effect on the function of T lymphocyte in vitro and graft-versus-host disease (GVHD) in vivo. PEGG was prepared by elution at acid pH from human placental tissues that were extensively washed. Its effects on T lymphocyte proliferation induced by PHA and mixed lymphocyte reaction (MLR) were analysed by BrdU ELISA, its effect on the CD25 and CD69 expression on T cells was observed by flow cytometry, and the interferon gamma (IFN-gamma) and interleukin-4 (IL-4) quantification in MLR supernatant were assayed by ELISA. A murine GVHD model was established, the effect of PEGG on the manifestation and pathologic change of GVHD and 45-day survival rate were observed. The results showed that considerable level of immunoglobulin could be eluted from placenta at acid PH, of which the main components were IgG checked by SDS-PAGE analysis. In vitro study indicated that PEGG significantly inhibited both the proliferative response of T cells to PHA and the MLR, down-regulated the expression of CD25 and CD69 on T cells stimulated by PHA, and decreased the secretion of IFN-gamma but increased the production of IL-4 in MLR supernatant. In vivo, recipient mice treated with PEGG had a markedly increased survival rate with less histopathological evidence of GVHD. It is concluded that PEGG can inhibit the proliferation and activation of T cells, regulate the direction of T helper cells differentiating towards Th2 type, and effectively prevent GVHD in a murine model. In short, PEGG may be a potent therapeutic agent for GVHD.

Novel prefractionation method can be used in proteomic analysis.

For the first time, a novel prefractionation method used in proteomic analysis was developed, which is performed by a novel aqueous two-phase system (NATPS) composed of n-butanol, (NH(4))(2)SO(4), and water. It can separate proteomic proteins into multigroups by one-step extraction. The phase-separation conditions of n-butanol solutions were studied in the presence of commonly used inorganic salts. The NATPS was subsequently developed. Using human serum albumin, zein, and gamma-globulin as model proteins, the separation effectiveness of the NATPS for protein was studied under affection factors, i.e., pH, n-butanol volume, protein, or salt concentration. The model and actual protein samples were separated by the NATPS and then directly used for gel electrophoresis without separating the target proteins from phase-forming reagents. It revealed that the NATPS could separate proteomic proteins into multigroups by one-step extraction. The NATPS has the advantages of rapidity, simplicity, low cost, biocompability, and high efficiency. It need not separate target proteins from the phase-forming reagents. The NATPS has great significance in separation and extraction of proteomic proteins, as well as in methodology.

[Comparison of hepatic protein synthesis function between Banna Minipig Inbred Line and human].

OBJECTIVE: This is a research aimed at comparing the hepatic protein synthesis function of Banna Minipig Inbred Line (BMI) with that of human so as to evaluate the possibility of porcine liver replacement of human counterpart. METHODS: The venous blood samples from BMI and volunteer participants were collected. The albumin and total protein in the separated sera were tested using Beckman delta CX7 autoanalyzer, and serum protein electrophoresis was performed. RESULTS: No significant differences in albumin and in the percentage and concentration of globulins synthesized in liver (alpha1-, alpha2- and beta-globulin) between BMI and human were observed. The percentage and concentration of gamma-globulin synthesized in the lymphocytes of BMI were significantly higher than those of human. CONCLUSION: The above findings suggested the similarity between BMI liver and human liver in respect to albumin and alpha, beta-globulin synthesis.

Preparative separation of proteins using centrifugal precipitation chromatography based on solubility in ammonium sulfate solution.

A preparative modification of the centrifugal precipitation chromatography (CPC) is described. The sample-loading capacity is improved in the present system by the use of convoluted tubing containing dialysis tubing instead of a dialysis membrane placed between a pair of disks equipped with mirror-imaged spiral grooves as in the original design. The system uses, basically, the same principle of as the original CPC, in that a concentration gradient of precipitant is generated under a centrifugal force field. The protein sample injected into the CPC column is exposed to an increasing concentration of the precipitant where it precipitates at various portions of the column according to its solubility. The gradient is then gradually lowered so that the sample undergoes dissolution and precipitation many times within the column; the proteins finally elute from the column according to their solubilities. A basic study was performed using this machine to separate human albumin and 3-globulin using ammonium sulfate (AS) as precipitant. Preliminary results indicate that this method can separate 500 mg of protein.

Direct quantification of human serum albumin in human blood serum without separation of gamma-globulin by the total internal reflected resonance light scattering of thorium-sodium dodecylbenzene sulfonate at water/tetrachloromethane interface.

A direct quantification of human serum albumin (HSA) in blood serum samples without separation is proposed based on the measurements of total internal reflected resonance light scattering (TIR-RLS) at water/tetrachloromethane (H(2)O/CCl(4)) interfaces. In the pH range of 6.37-6.59, the coadsorption of the binary complex of HSA-Th(IV) with sodium dodecylbenzene sulfonate occurs at the H(2)O/CCl(4) interface, forming an amphiphilic layer and displaying greatly enhanced TIR-RLS signals with the maximum peak located at 340-370 nm. The enhanced TIR-RLS intensity is in proportion to the HSA concentration in the range 0.15-1.0 micro gml(-1). The limit of detection is 14.4 ngml(-1). The contents of HSA in blood serum samples were determined with the recovery of 97.1-102.3% and RSD of 0.6-2.9%, which are identical to those obtained according to the spectrofluorimetric method using chrome azurol S.

Gamma-globulins prepared from sera of multiparous women bind anti-HLA antibodies and inhibit an established in vivo human alloimmune response.

It has previously been shown that sera from multiparous women have increased levels of anti-idiotypic antibodies specific for anti-HLA molecules. gamma-Globulins prepared from these sera may be superior to commercial preparations of intravenous gamma-globulin (IVIg) for inhibiting HLA alloimmunization. To test this, F(ab')2 fragments prepared from either commercial IVIg or from the sera of men or multiparous women were coupled to CNBr-Sepharose and tested for their ability to bind F(ab')2 fragments derived from polyspecific anti-HLA sera. As determined by flow cytometry, compared with columns coated with F(ab')2 derived from commercial IVIg or sera from men, columns coated with F(ab')2 prepared from the sera of multiparous women bound significantly more anti-HLA. In addition, intact IgG molecules prepared from the sera of multiparous women significantly neutralized the reactivity of the anti-HLA F(ab')2 fragments. To determine whether the intact IgG molecules or their corresponding F(ab')2 fragments could affect in vivo alloimmunity, they were tested for their ability to inhibit an established IgG human alloimmune response in humanized severe combined immunodeficient (SCID) mice. Compared with commercial IVIg, when intact IgG or F(ab')2 fragments derived from multiparous women were administered to SCID mice making human anti-HLA antibodies, a significant reduction in anti-HLA reactivity was observed. The findings suggest that IgG molecules prepared from the sera of multiparous women have increased anti-idiotypic reactivity against anti-HLA antibodies, which can significantly inhibit an established human IgG alloimmune response in an Fc-independent manner.

[Purification of proteins in human plasma with new nylon affinity membranes].

A series of affinity membranes were prepared by using microporous nylon membrane as matrix and activation method with s-triazine and 1,4-butanediol diglycidyl ether, separately. Three proteins with clinic value, gamma-globulin, plasminogen and thrombin, were purified from human plasma by one step with affinity membrane chromatography. The purities of gamma-globulin purified were higher than 83%, and purification folds were higher than 5. The purity of gamma-globulin obtained was higher than that of standard human gamma-globulin. The efficiencies of gamma-globulin purification with affinity membranes prepared by two activation methods were equal. Plasminogen purified with affinity membranes prepared by s-triazine activation was higher than that by 1,4-butanediol diglycidyl ether activation. And SDS-PAGE analysis showed that the purities of gamma-globulin and plasminogen obtained were higher than that of the commercial product. In addition, the purification of thrombin from human plasma by one step with trypsin activation column and affinity membrane was studied. Thrombin with specific activity of 42 NIH unit/mg and 42.5 NIH unit/mg could be obtained from human plasma through affinity membranes prepared by s-triazine and 1,4-butanediol diglycidyl ether methods, respectively.

Reference values for the five electrophoretic serum protein fractions in Caucasian children by capillary zone electrophoresis.

We report age-related reference intervals for capillary zone electrophoresis for children between 1 and 14 years of age.

Centrifugal precipitation chromatography: principle, apparatus, and optimization of key parameters for protein fractionation by ammonium sulfate precipitation.

A novel chromatographic system introduced here internally generates a concentration gradient of ammonium sulfate (AS) through a long separation channel under a centrifugal force field. Protein samples are exposed to a gradually increasing AS concentration and precipitated along the channel. Then, chromatographic elution is initiated by gradually decreasing the AS concentration in the gradient which causes the proteins to repeat dissolution and precipitation through the channel. Consequently, they are eluted out in the order of their solubility in the AS solution. The separation column consists of a pair of disks equipped with mutually mirror-imaged spiral grooves. A dialysis membrane is sandwiched between the disks to form two identical channels partitioned by the membrane. The disk assembly is mounted on the sealless continuous-flow centrifuge. When a concentrated AS solution is eluted through one channel and water through the other channel in an opposite direction, an exponential AS gradient is formed through the water channel. A series of basic experiments was performed to study the rates of AS transfer and osmosis through the membrane, and the operational parameters including elution time, revolution speed, inclination of gradient, and sample size were optimized using stable protein samples. Preliminary applications were successful in purification of monoclonal antibody from cell culture supernatant and an affinity separation of recombinant ketosteroid isomerase from a crude Escherichia coli lysate.

Direct sedimentation analysis of interference optical data in analytical ultracentrifugation.

Sedimentation data acquired with the interference optical scanning system of the Optima XL-I analytical ultracentrifuge can exhibit time-invariant noise components, as well as small radial-invariant baseline offsets, both superimposed onto the radial fringe shift data resulting from the macromolecular solute distribution. A well-established method for the interpretation of such ultracentrifugation data is based on the analysis of time-differences of the measured fringe profiles, such as employed in the g(s*) method. We demonstrate how the technique of separation of linear and nonlinear parameters can be used in the modeling of interference data by unraveling the time-invariant and radial-invariant noise components. This allows the direct application of the recently developed approximate analytical and numerical solutions of the Lamm equation to the analysis of interference optical fringe profiles. The presented method is statistically advantageous since it does not require the differentiation of the data and the model functions. The method is demonstrated on experimental data and compared with the results of a g(s*) analysis. It is also demonstrated that the calculation of time-invariant noise components can be useful in the analysis of absorbance optical data. They can be extracted from data acquired during the approach to equilibrium, and can be used to increase the reliability of the results obtained from a sedimentation equilibrium analysis.