Investigation of genetic variants of α-1 acid glycoprotein by ultra-performance liquid chromatography–mass spectrometry

Genetic variants of human plasma alpha-1 acid glycoprotein (AGP) have been studied in cancer, compared with a group of healthy control. AGP has four genetic variants: AGP F1, F2, and S variants correspond to the ORM1 gene whereas AGP A corresponds to the ORM2 gene. The proportion of ORM1 and ORM2 variants were studied in plasma using a novel UPLC–MS method. Plasma total AGP level was 0.5 ± 0.2 g L⁻¹ and the proportions of the ORM1 and ORM2 variants were 76.3 ± 8.2% and 23.7 ± 8.2%, respectively. In cancer plasma AGP levels increased fourfold and the proportion of ORM1 variants increased to 88.7 ± 6.8%. Changes in the proportion of genetic variants due to cancer were clearly significant, as shown by statistical analysis. Three different cancer types have been studied, lymphoma, melanoma, and ovarian cancer. The results did not show any difference depending on cancer type. The results indicate that, in accordance with prior expectations, the ORM1 variant is predominantly responsible for the acute-phase property of AGP.     

pH titration curves of the desialylated human alpha 1-acid glycoprotein variants by combined isoelectrofocusing-electrophoresis: utilization in the development of a fractionation method for the protein variants by chromatography on immobilized metal affinity adsorbent.

Using a two-dimensional isoelectrofocusing (IEF)-electrophoresis technique, the pH titration curves of the three main desialylated variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG) were studied to assist in the development of a fractionation method for the AAG variants. For this purpose, different AAG samples, each corresponding to one of the three main phenotypes of the protein (F1S/A, F1/A and S/A), were first purified by chromatographic separation of individual human plasma samples on immobilized Cibacron Blue F3G-A. The purified AAG samples were then disialylated and their heterogeneity was checked by analytical IEF. The pH-mobility curves of the desialylated AAG samples were displayed in polyacrylamide gel slabs, under a constant set of experimental conditions, by carrying out electrophoresis of the protein samples perpendicularly to two stationary pH gradients: a large gradient (pH 3.5-9.5) and a narrow gradient (pH 5-8). The curves showed that all the desialylated variants of AAG exhibited small charge differences and moved closely together between about pH 3.5-5.5 and pH 7.5-9.5. However, the variants were found to show microheterogeneity in their total charge between about pH 5.5 and 7.5 due to the titrated ionizable groups involved along this pH zone. This microheterogeneity was assumed to be accounted for by the existence of differences between the titratable histidyl residues of the AAG variants. Consequently, the interactions of the variants with immobilized transition metal ions were studied at pH 7, using affinity chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the A variant was strongly bound by immobilized Cu(II) ions, whereas the F1 and S variants interacted non-specifically with the immobilized ligand. This finding allowed the development of a rapid and effective fractionation method for desialylated AAG into its A and F1 or S variants, depending on the AAG phenotype, by chromatography on an immobilized affinity Cu(II) adsorbent. The quantitative relationships between immobilized Cu(II) ions and desialylated AAG (the apparent association constant and gel protein-binding capacity) were also determined using a non-chromatographic equilibrium binding technique.     

High-performance anion-exchange chromatographic study of desialylated human alpha 1-acid glycoprotein variants. Development of a fractionation method for the protein slow variants

The three main desialylated variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG), a serum acute-phase reactant, were analysed by high-performance anion-exchange chromatography in order to determine their optimum separation conditions. The analysis consisted of three steps, as follows: (1) A desialylated commercial AAG was separated into one "fast"- and one "slow"-migrating fraction by preparative isoelectrofocusing. The "fast" and "slow" fractions were shown to contain the F1 variant and a mixture of the S and A variants, respectively. (2) The pH titration curves of these two fractions were then measured by strong anion-exchange chromatography with several buffer systems of increasing pH. From the data obtained, it was not possible to select the optimum conditions to separate the "fast" variant F1 from the "slow" variants A and S. However, the S and A variants were shown to ionize very differently. (3) The specific fractionation of the S and A variants was therefore carried out by anion-exchange chromatography under operating conditions based on the data obtained from the study of their pH titration curves. This was performed both with the "slow"-migrating fraction obtained by preparative isoelectrofocusing of commercial AAG and with an AAG (containing only variants S and A) purified from an individual serum on immobilized Cibacron Blue F3G-A. Identification of the fractionated proteins was achieved by analytical isoelectrofocusing.     

Chiral high-performance liquid chromatographic separations of vinca alkaloid analogues on alpha 1-acid glycoprotein and human serum albumin columns.

Separations of the stereoisomers of a series of tetracyclic and pentacyclic vinca alkaloid analogues having two or three chiral centres were performed on Chiral-AGP and Chiral-HSA high-performance liquid chromatographic columns. Phosphate buffers with pH 5-7 containing 5-35% acetonitrile or 2-propanol were used as mobile phases. The results were in accordance with previous binding data obtained with native AGP and on an HSA-Sepharose column. Whereas on Chiral-AGP the retention of the trans isomers having 1(R),12b(S)-indolo[2,3-a]quinolizidine or the corresponding 3(S),16(R)-eburnane absolute configurations was exceedingly high, on Chiral-HSA the trans isomers, independently of their absolute configurations, were more retained. Eburnane-type compounds could also be separated according to the configuration of the chiral centre at position 14. A comparison of the chromatographic properties of the vinca alkaloids on the Chiral-AGP and Chiral-HSA columns demonstrates that these compounds are bound with higher affinity to the AGP phase. The AGP column resolves a very broad range of vinca alkaloids compared with the HSA column. Higher stereoselectivity and a much better chromatographic performance were also obtained on the Chiral-AGP column.     

Lipophilicity and bio‐mimetic properties determination of phytoestrogens using ultra‐high‐performance liquid chromatography

This paper presents lipophilicity and bio‐mimetic property determination of 15 phytoestrogens, namely biochanin A, daidzein, formononetin, genistein, genistein‐4,7‐dimethylether, prunetin, 3,4,7‐trihydroxyisoflavon, 4,6,7‐trihydroxyisoflavon, 4,6,7‐trimethoxyisoflavon, daidzin, genistin, ononin, sissotrin, coumestrol and coumestrol dimethylether. High‐performance liquid chromatography with fast gradient elution and Caco‐2 cell line were used to determine the physicochemical properties of selected phytoestrogens. Lipophilicity was determined on octadecyl‐sylane stationary phase using pH 2.0 and pH 7.4 buffers. Immobilized artificial membrane chromatography was used for prediction of interaction with biological membranes. Protein binding was measured on human serum albumin and α‐1‐acid‐glycoprotein (AGP) stationary phases. Caco‐2 assay was used as a gold standard for assessing in vitro permeability. The obtained results differentiate phytoestrogens according to their structure where aglycones show significantly higher lipophilicity, immobilized artificial membrane partitioning, AGP binding and Caco‐2 permeability compared with glucosides. However, human serum albumin binding was very high for all investigated compounds. Furthermore, a good correlation between experimentally obtained chromatographic parameters and in silico prediction was obtained for lipophilicity and human serum albumin binding, while the somewhat greater difference was obtained for AGP binding and Caco‐2 permeability.     

Water soluble extended naphthalene diimides as pH fluorescent sensors and G-quadruplex ligands

Extended naphthalene diimides (NDIs) fused to 1,4-dihydropyrazine-2,3-dione, containing two solubilizing moieties, have been synthesized. Fluorescence spectra of the new NDIs were remarkably affected by pH, as the second deprotonation of the dihydropyrazinedione moiety (pK(a) 6.9) switched off the emission. Binding to a G-quadruplex folded oligonucleotide and stoichiometry were evaluated by FRET melting assay and CD analysis. G-quadruplex binding was strongly enhanced shifting from pH 7.4 to pH 6.0 as a consequence of the dihydropyrazinedione moiety protonation. Cytotoxicity studies using two human telomerase-positive cell lines (HT29 and A549) revealed that the best G-quadruplex ligand was very active against the colon cell line, with an EC(50) of 300 nM.     

Biointeraction analysis of carbamazepine binding to α1‐acid glycoprotein by high‐performance affinity chromatography

Interactions of the drug carbamazepine with the serum protein α₁‐acid glycoprotein (AGP) were examined by high‐performance affinity chromatography. Frontal analysis studies with an immobilized AGP column and control column indicated carbamazepine had both low‐affinity interactions with the support and high‐affinity interactions with AGP. When a correction was made for binding to the support, the association equilibrium constant measured at pH 7.4 and 37°C for carbamazepine with AGP was 1.0 (±0.1)×10⁵ M^?1, with values that ranged from 5.1 to 0.58×10⁵ M^?1 in going from 5 to 45°C. It was found in competition studies that these interactions were occurring at the same site that binds propranolol on AGP. Temperature studies indicated that the change in enthalpy was the main driving force for the binding of carbamazepine to AGP. These results provide a more complete picture of how carbamazepine binds to AGP in serum. This report also illustrates how high‐performance affinity chromatography can be used to examine biological interactions and drug–protein binding in situations in which significant interactions for an analyte are present with both the chromatographic support and an immobilized ligand.     

Synthesis and Guest-Binding Properties of pH/Reduction Dual-Responsive Cyclophane Dimer

A water-soluble cyclophane dimer having two disulfide groups as a reduction-responsive cleavable bond as well as several acidic and basic functional groups as a pH-responsive ionizable group 1 was successfully synthesized. It was found that 1 showed pH-dependent guest-binding behavior. That is, 1 strongly bound an anionic guest, 6-p-toluidinonaphthalene-2-sulfonate (TNS) with binding constant (K/M⁻¹) for 1:1 host-guest complexes of 9.6 × 10⁴ M⁻¹ at pH 3.8, which was larger than those at pH 7.4 and 10.7 (6.0 × 10⁴ and 2.4 × 10⁴ M⁻¹, respectively), indicating a favorable electrostatic interaction between anionic guest and net cationic 1. What is more, release of the entrapped guest molecules by 1 was easily controlled by pH stimulus. Large favorable enthalpies (ΔH) for formation of host-guest complexes were obtained under the pH conditions employed, suggesting that electrostatic interaction between anionic TNS and 1 was the most important driving force for host-guest complexation. Such contributions of ΔH for formation of host-guest complexes decreased along with increased pH values from acidic to basic solutions. Upon addition of dithiothreitol (DTT) as a reducing reagent to an aqueous PBS buffer (pH 7.4) containing 1 and TNS, the fluorescence intensity originating from the bound guest molecules decreased gradually. A treatment of 1 with DTT gave 2, having less guest-binding affinity by the cleavage of disulfide bonds of 1. Consequently, almost all entrapped guest molecules by 1 were released from the host. Moreover, such reduction-responsive cleavage of 1 and release of bound guest molecules was performed more rapidly in aqueous buffer at pH 10.7.     

Spectroscopic characterizations of bridging cysteine ligand variants of an engineered Cu2(Scys)2 CuA azurin

Bridging cysteine ligands of the Cu(A) center in an engineered Cu(A) azurin were replaced with serine, and the variants (Cys116Ser and Cys112Ser Cu(A) azurin) were characterized by mass spectrometry, as well as UV-vis and electron paramagnetic resonance (EPR) spectroscopic techniques. The replacements resulted in dramatically perturbed spectroscopic properties, indicating that the cysteines play a critical role in maintaining the structural integrity of the Cu center. The replacements at different cysteine residues resulted in different perturbations, even though the two cysteines are geometrically symmetrical in the primary coordination sphere with respect to the two copper ions. The Cys112Ser variant contains two distinct type 2 copper centers, while the Cys116Ser variant has one type 1 copper center with slight tetragonal distortion. Both the UV-vis and EPR spectra of the Cys116Ser variant change with pH, and the pK(a) of the transition is 6.0. A type 1 copper EPR spectrum with A(||) = 26 G was obtained at pH 7.0, while a type 2 copper EPR spectrum with A(||) = 140 G was found at pH 5.0. Interestingly, lowering the temperature from 290 to 85 K resulted in conversion of the Cys116Ser variant from a type 1 copper center to a type 2 copper center, suggesting rearrangement of the ligand around the copper or binding of an exogenous ligand at low temperature. This difference in mutation effects at different cysteines may be due to different constraints exerted on the two cysteines by hydrogen-bonding patterns in the ligand loop.     

Comparison of Dechlorane Plus Concentrations in Sequential Blood Samples of Pregnant Women in Taizhou,China

To develop an appropriate sampling strategy to assess the intrauterine exposure to dechlorane plus (DP), we investigated DP levels in sequential maternal blood samples collected in three trimesters of pregnancy, respectively, from women living in Taizhou. The median concentration of DPs (sum of syn-DP and anti-DP) in all samples was 30.5 pg g⁻¹ wet-weight and 5.01 ng g⁻¹ lipid-adjusted weight, respectively. The trimester-related DP concentrations were consistently strongly correlated (p < 0.01), indicating that a single measurement of DP levels could represent intrauterine exposure without sampling from the same female repeatedly; however, the wet-weight levels significantly increased across trimesters (p < 0.05), while the lipid-adjusted levels did not significantly vary. Notably, whether lipid-adjusted weight or wet-weight levels, the variation extent of DP across trimesters was found to be less than 41%, and those for other persistent organic pollutants (POPs) reported in the literature were also limited to 100%. The limitation in variation extents indicated that, regardless of the time of blood collection during pregnancy and how the levels were expressed, a single measurement could be extended to screen for exposure risk if necessary. Our study provides different strategies for sampling the maternal blood to serve the requirement for assessment of in utero exposure to DP.     

SnO2负载Au和M-Au(M=Pt,Pd)催化剂及其低温催化氧化CO性能

以SnO₂为载体, 采用沉积沉淀法(DP)、共沉淀法(CP)和浸渍法(IM)制备了金负载Au/SnO₂催化剂, 同时采用沉积沉淀法制备了M-Au/SnO₂(M=Pd, Pt)双金属负载催化剂. 通过X射线衍射(XRD)、BET比表面积测定、透射电镜(TEM)和X射线光电子能谱(XPS)等技术对样品进行表征, 并测定其对CO的催化活性. 结果表明: 与CP法和IM 法相比, DP法制备的Au/SnO₂-DP 催化剂, Au 颗粒(<5 nm)较小, 分布均匀; Au/SnO₂-DP 中的Au 是以金属态Au0存在, 而Au/SnO₂-CP 和Au/SnO₂-IM 中, 金以Au⁰和Au³⁺的混合价态存在, 在Au/SnO₂-DP和M-Au/SnO₂中的Au、Pt、Pd和SnO₂之间存在相互作用; Au/SnO₂-DP 催化性能明显优于Au/SnO₂-CP 和Au/SnO₂-IM. Au与Pt 和Pd的双金属复合催化剂催化活性明显提高. 不同方法制备Au/SnO₂催化活性的差别主要是由于Au颗粒大小和Au氧化态的不同而产生. 而M-Au/SnO₂活性提高, 可能是由于Au与Pt 和Pd之间的相互作用.     

In silico analysis of nonsynonymous single nucleotide polymorphisms of the human adiponectin receptor 2 (ADIPOR2) gene

Polymorphisms of the ADIPOR2 gene are frequently linked to a higher risk of developing diseases including obesity, type 2 diabetes and cardiovascular diseases. Though mutations of the ADIPOR2 gene are detrimental, there is a lack of comprehensive in silico analyses of the functional and structural impacts at the protein level. Considering the involvement of ADIPOR2 in glucose uptake and fatty acid oxidation, an in silico functional analysis was conducted to explore the possible association between genetic mutations and phenotypic variations. A genomic analysis of 82 nonsynonymous SNPs in ADIPOR2 was initiated using SIFT followed by the SNAP2, nsSNPAnalyzer, PolyPhen-2, SNPs&GO, FATHMM and PROVEAN servers. A total of 10 mutations (R126W, L160Q, L195P, F201S, L235R, L235P, L256R, Y328H, E334K and Q349H) were predicted to have deleterious effects on the ADIPOR2 protein and were therefore selected for further analysis. Theoretical models of the variants were generated by comparative modeling via MODELLER 9.16. A protein structural analysis of these amino acid variants was performed using SNPeffect, I-Mutant, ConSurf, Swiss-PDB Viewer and NetSurfP to explore their solvent accessibility, molecular dynamics and energy minimization calculations. In addition, FTSite was used to predict the ligand binding sites, while NetGlycate, NetPhos2.0, UbPerd and SUMOplot were used to predict post-translational modification sites. All of the variants showed increased free energy, though F201S exhibited the highest energy increase. The root mean square deviation values of the modeled mutants strongly indicated likely pathogenicity. Remarkably, three binding sites were detected on ADIPOR2, and two mutations at positions 328 and 201 were found in the first and second binding pockets, respectively. Interestingly, no mutations were found at the post-translational modification sites. These genetic variants can provide a better understanding of the wide range of disease susceptibility associated with ADIPOR2 and aid the development of new molecular diagnostic markers for these diseases. The findings may also facilitate the development of novel therapeutic elements for associated diseases.     

The influence of alpha1-acid glycoprotein on collagenase-3 activity in early rheumatoid arthritis

The concentration and glycosylation of alpha(1)-acid glycoprotein (AGP) alter significantly during inflammation. A definitive physiological role for AGP remains elusive and is the subject of extensive investigation. This study investigated the influence of AGP on the activity of collagenase-3, an important mediator of cartilage destruction in rheumatoid arthritis. AGP was isolated from normal and rheumatoid plasma. Fucosylation was determined by high pH anion-exchange chromatography; sialylation was assessed following enzymatic digest. Rheumatoid AGP displayed elevated fucosylation and sialylation compared with normal. The influence of each sample on collagenase-3 activity was measured fluorometrically. AGP influenced collagenase-3 catalysis and collagen binding, with catalytic activity correlating with fucosylation. Rheumatoid AGP exhibited less efficient inhibition than normal plasma AGP. It is hypothesized that AGP within rheumatoid synovial fluid may be inadequate to prevent excessive cartilage destruction and hence may exacerbate the disease process.     

The group specific component/vitamin D binding protein (GC/DBP) system in the analysis of disputed paternities

The group-specific component (GC) was discovered in 1959, and in the same year a vitamin D binding protein (DBP) in human plasma was found; however, their identity was established as late as 1975. In the GC/DBP system three common alleles, GC*1F, GC*1S, and GC*2, determine six GC phenotypes: 1F, 1S, 2, 1F-1S, 2-1F and 2-1S, these common alleles having been found in all human populations studied. In addition, more than 120 GC variants have been discovered, with varying frequencies in different populations. The distribution of the common GC phenotypes and the presence of rare GC variant phenotypes render the GC/DBP system useful for the analysis of disputed paternities.     

Interaction of the vitamin D-binding protein (group-specific component) and its ligand 25-hydroxy-vitamin D3: binding differences of the various genetic types disclosed by isoelectric focusing

The three common variants of the vitamin D binding protein, also known as group specific component (Gc), namely types 1S, 1F and 2, as well as some rare variants were studied by thin-layer polyacrylamide gel isoelectric focusing in a pH 4.5-5.4 carrier ampholyte generated pH gradient, additionally containing N-(2-acetamido)-2-aminoethanesulfonic acid (ACES). Prior to isoelectric focusing, whole serum or purified preparations of the vitamin D binding protein were incubated with 25-hydroxycholecalciferol at various ligand/protein ratios. Binding differences were found for the anodal and cathodal isoforms of Gc 1 variants and also for various allelic types. Isoforms with higher isoelectric points generally had a lower affinity for the ligand than the variants with lower isoelectric points.     

Effect of pH on the interaction of vitamin B12 with bovine serum albumin by spectroscopic approaches

The interaction mechanism between vitamin B12 (B12, cyanocobalamin) and bovine serum albumin (BSA) has been investigated by fluorescence, synchronous fluorescence, ultraviolet-vis (UV) absorbance, and three-dimensional fluorescence. The intrinsic fluorescence of BSA was strongly quenched by the addition of B12 in different pH buffer solutions (pH 2.5, 3.5, 5.0, 7.4, and 9.0) and spectroscopic observations are mainly rationalized in terms of a static quenching process at lower concentration of B12 (C(B12)/C(BSA)<5) and a combined quenching process at higher concentration of B12 (C(B12)/C(BSA)>5). The structural characteristics of B12 and BSA were probed, and their binding affinities were determined under different pH conditions. The results indicated that the binding abilities of B12 to BSA in the acidic and basic pH regions (pH 2.5, 3.5, 5.0, and 9.0) were lower than that at simulating physiological condition (pH 7.4). In addition, the efficiency of energy transfer from tryptophan fluorescence to B12 was found to depend on the binding distance r between the donor and acceptor calculated using F?rster's theory. The effect of B12 on the conformation of BSA was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results showed that the binding of B12 to BSA causes apparent change in the secondary and tertiary structures of BSA.     

On-line immunoaffinity capillary electrophoresis based on magnetic beads for the determination of alpha-1 acid glycoprotein isoforms profile to facilitate its use as biomarker

An immunoaffinity purification method coupled on-line to capillary electrophoresis (IACE) which allows the determination of several isoforms of intact alpha-1 acid glycoprotein (AGP) in serum samples using UV detection is developed. The immunoaffinity step is based on anti-AGP antibodies (Abs) covalently bound to magnetic beads (MBs) which are captured at the inlet end of the capillary using permanent magnets placed inside the cartridge of the CE instrument. The on-line method includes injection of the MBs with the Ab bound (MBs–Ab) and their trapping by the magnets at the entrance of the separation column, injection of serum sample and capture of AGP by the Abs, release of captured AGP, focus of desorbed protein, separation of AGP isoforms, and removal of MBs–Ab. The optimization of the different factors involved in each step allowed purification, separation and detection of AGP isoforms in a single electrophoretic analysis in about 1 h. Automation, sample and reagents consumption as well as analysis time was improved compared to off-line alternatives which use purification of AGP in an immunochromatographic column and CE separation of AGP isoforms in two independent operations. The analytical methodology developed allows the separation of 10 AGP isoforms in serum samples from a healthy donor. For a serum sample, precision (expressed as relative standard deviation) in terms of corrected area percentage was better than 0.5% for each peak accounting for more than 10% of total AGP and it was better than 4.0% in terms of relative migration time of each AGP isoform considering the whole process.     

Interaction of albumin with polysaccharides containing ionic groups

It is very important to clarify the mechanisms of the interaction between components of organisms. In this report, the interaction between bovine serum albumin (BSA) and ionic polysaccharides were discussed. The fluorescence spectrum of tryptophan (Trp) in BSA changed as its conformation changed. On adding polysaccharide containing sulfonic acid residues (sulfonic‐type) at pH 7.4, a remarkable blue shift of the emission maximum (λ_em) of Trp was observed. This blue shift was decreased by adding NaCl. In contrast, polysaccharide containing carboxylic acid residues (carboxy‐type) scarcely changed Trp fluorescence at the same pH. At a pH lower than the isoelectric point (PI = 4.7–4.9) of BSA, a remarkable blue shift was observed not only by adding sulfonic‐type polysaccharide but also by adding carboxy‐type polysaccharide. Moreover, using the energy transfer method, in the pH region higher than the PI of BSA, carboxylic‐type polysaccharides may interact relatively weakly with the binding site II of BSA, but sulfonic‐type ones can selectively interact with binding site I weakly and with binding site II strongly. And in the pH region lower than the PI of BSA, carboxylic‐type polysaccharides interact with binding site II strongly. On the other hand, sulfonic‐type polysaccharides are bound to both binding site I and binding site II very strongly. Copyright © 1999 John Wiley & Sons, Ltd.     

Competitive reactions in solutions of poly-L-histidine, calf thymus DNA, and synthetic polyanions: determining the binding constants of polyelectrolytes

The physicochemical characteristics of a nonviral gene delivery system will govern its functional bioactivity; however, empiricism dominates the literature in this field, and a significant deficiency of quantitative investigation and evaluation of nonviral gene delivery vehicles remains. Herein, we derive a physical model and experimental method to quantitatively determine the binding constants between a model polycationic nonviral gene delivery vehicle poly-L-histidine (PLH) and calf thymus DNA. The approach has utility to a variety of systems and is not limited to the described polymer model. The interaction of PLH with DNA was monitored by fluorescence quenching of an ethidium bromide probe in the pH range 4 to 8. The interaction increased with pH decrease with the most pronounced change between pH 6 and 7. The obtained pH-dependence of fraction of salt bonds formed between PLH and DNA was used to estimate pK(a) of PLH in the presence of DNA, which equaled 6.24. The interaction of PLH with DNA in the presence of added synthetic polyanions was studied by the same approach and found to be controlled by pH, nature of the charge groups of the polyanion, and its degree of polymerization. In the mixture with sodium poly(styrenesulfonate) the interaction was negligible in the whole studied pH range, whereas in the mixtures with sodium poly(acrylate) (PA) or sodium poly(methacrylate), DNA was able to compete effectively for the binding with PLH. For PA samples with degree of polymerization higher than degree of polymerization of PLH, DP(PA) > DP(PLH), the fraction of polycation bound to DNA was constant regardless of DP(PA.) In contrast, at DP(PA) < DP(PLH), a pronounced increase in the bound fraction was observed. It substantiates the notion that the binding energy of two polymers is mainly controlled by the DP of the shorter component of polyelectrolyte complex. The data on PLH distribution between DNA and added polyanion with different values of DP were treated according to the developed procedure to yield the effective binding constants of PLH with DNA and polyanion-competitor, calculated both per mole of interacting units K(1) and mole of interacting chains K(n). In all cases, K(1) had similar numerical values reflecting common type of interaction stabilizing the complexes, i.e., electrostatics. Slight variation of K(1) yielded in drastic changes in K(n) and alteration of dominance of PLH interaction with DNA or synthetic polyanion. The results of the study can have a high impact in deriving the correlation between the binding constant of a polycation to DNA and its ability to serve as gene delivery vehicle.     

Isoelectric focusing in immobilized pH gradients with carrier ampholytes added for high-resolution phenotyping of bovine beta-lactoglobulins: characterization of a new genetic variant

The genetic variants of bovine beta-lactoglobulin (beta-lg) from the "Murnau-Werdenfelser" breed were analyzed in three different isoelectric focusing (IEF) systems. While carrier ampholyte IEF with a pH gradient of 0.2 pH/cm did not resolve the new variant W from the B variant and IEF in immobobilized pH gradients (IPG) with 0.1 pH/cm only partially resolved it, adequate separation was achieved with IPG-IEF in a pH 5.25-pH 5.7 gradient, in presence of 0.8 % w/v carrier ampholytes, both over a 10 and 17 cm separation distance. Apparent isoelectric points (pI's) and genetic frequencies (f) were as follows: beta-lg A, pI = 5.370, f = 0.364; beta-lg B, pI = 5.485, f = 0.480; beta-lg W, pI = 5.492, f = 0.076; and beta-lg D, pI = 5.610, f = 0.080. The small difference of delta pI = 0.007 between beta-lg B and beta-lg W respectively, seems to originate from a "silent" substitution of neutral amino acid residues as compared to the larger delta pI's of the other genetic variants of beta-lg, which result from substitution of charged amino acids.