Dye Affinity Chromatography Research Articles

Purification, properties, and kinetic studies of cytoplasmic malate dehydrogenase from Taenia solium cysticerci

Malate dehydrogenase (L: -malate: NAD oxidoreductase, EC 1.1.1.37) from the cytoplasm of Taenia solium cysticerci (cMDHTs) was purified 48-fold through a four-step procedure involving salt fractionation, ionic exchange, and dye affinity chromatography. cMDHTs had a native M (r) of 64,000, while the corresponding value per subunit, obtained under denaturing conditions, was 32,000. The enzyme is partially positive, with an isoelectric point of 8.7, and had a specific activity of 2,615 U mg(-1) in the reduction of oxaloacetate. The second to the 21st amino acids from cMDHTs N-terminal group were P G P L R V L I T G A A G Q I A Y N L S. This sequence is 100% identical to that of Echinococcus granulosus. Basic kinetic parameters were determined for this enzyme. The optimum pH for enzyme reaction was at 7.6 for oxaloacetate reduction and at 9.6 for malate oxidation. K (m) values for oxaloacetate, malate, NAD, and NADH were 2.4, 215, 50, and 48 microM, respectively. V (max) values for the substrates and cosubstrates as described above were 1,490, 87.8, 104, and 1,714 micromol min(-1) mg(-1). Several NAD analogs, structurally altered in either the pyridine or purine moiety, were observed to function as coenzymes in the reaction catalyzed by the purified malate dehydrogenase. cMDHTs activity was uncompetitive inhibited by arsenate for both the forward (Ki = 8.2 mM) and reverse (Ki = 77 mM) reactions. The mechanism of the cMDHTs reactivity was investigated kinetically by the product inhibition approach. The results of this study are qualitatively consistent with an Ordered Bi Bi reaction mechanism, in which only the coenzymes can react with the free enzyme.

Antineutrophil cytoplasmic antibodies (ANCA) directed against cathepsin G in ulcerative colitis, Crohn's disease and primary sclerosing cholangitis.

Autoantibodies directed against polymorphonuclear neutrophils (PMN) have been observed in serum from patients with ulcerative colitis (UC), Crohn's disease (CD) and primary sclerosing cholangitis (PSC) using indirect immunofluorescence and fixed granulocyte ELISA. Our study demonstrates the presence in the serum of these patients of autoantibodies which bind to an azurophilic granule component distinct from proteinase 3, elastase and myeloperoxidase. These autoantibodies thus belong to the ANCA family, but their antigen specificity differs from the already characterized ANCA antigens. We have found that the same ANCA antigen target, named UC-antigen, was recognized by serum IgG from patients with UC, CD and PSC. It was purified by Matrex Gel Orange A dye affinity chromatography and subsequent immunoabsorption of contaminant proteinase 3 with immobilized anti-proteinase 3 MoAb. The identity between this UC antigen and cathepsin G was demonstrated by their coelution from Matrex Gel Orange A column and the parallel titration of cathepsin G-specific enzymatic activity and UC-ANCA binding, both in partially purified UC antigen and in highly pure cathepsin G. Furthermore, the use of cathepsin G ELISA confirmed that UC, CD and PSC patients' IgG did indeed bind to cathepsin G. Comparison of the results obtained with azurophilic granule- and cathepsin G-ELISA as well as inhibition of ANCA binding by anti-cathepsin G polyclonal antibodies, revealed that in some patients cathepsin G is the main azurophilic granule target of ANCA while others have other ANCA specificities. The fact that UC, CD and PSC are frequently associated with cathepsin G ANCA, while rarely occurring in other types of vasculitis, is intriguing but suggests that these diseases may have a common pathogenetic mechanism.

Thiacarbocyanine as ligand in dye‐affinity chromatography for protein purification

A thiacarbocyanine has been post-grafted onto beaded cellulose by a curing method envisioning their use as a biomimetic ligand in dye-affinity chromatography. The immobilization of this dye was performed based on a previously brief derivatization study where the analogous microcrystalline cellulose was alternatively used and some of the curing reaction conditions were varied. The grafted beaded cellulose so obtained was qualitatively and quantitatively characterized by SEM, SEM-EDS and EA. The immobilized dye-affinity interaction with the standard proteins bovine serum albumin, alpha-chymotrypsin and lysosyme was analysed. The influence of the mobile phase composition on the chromatographic behaviour of these standard proteins was also studied. A selective interaction was observed allowing the separation of all the three proteins from an artificial mixture.

Endogenous peptides from biophysical and biochemical fractionation of serum analyzed by matrix-assisted laser desorption/ionization and electrospray ionization hybrid quadrupole time-of-flight

Blood peptides can be concentrated, extracted, and analyzed with strong signal-to-noise ratios by precipitation in organic solvents followed by extraction in water. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) hybrid quadrupole time-of-flight (Qq–TOF) were used to analyze the precipitated and extracted endogenous peptides from fetal calf serum. C18 solid-phase extraction with or without prior precipitation in ammonium sulfate, size exclusion chromatography, dealbuminization, dye affinity chromatography, ultrafiltration, and differential precipitation in organic solvents were compared. Hundreds of different ions could be observed by MALDI in the various fractions. It appeared that some peptides were freely dissolved and that not all peptides in blood were obliged to remain bound to albumin or other high-molecular-mass proteins. Mass spectra with high signal-to-noise ratios were obtained from polypeptides precipitated with organic solvents followed by extraction of the peptides from the pellet with water. The peptides extracted from organic precipitates were analyzed by nano liquid chromatography (LC)–ESI–Qq–TOF. In addition to many commonly abundant serum proteins, apparent low-abundance peptides associated with cancer biology from proteins such as insulin-like growth factor II, thymosin β4 and β9, plasminogen, coagulation factors, and extracellular matrix protein 1 were observed.

Preparation and characterization of a potent, long-lasting recombinant human serum albumin-interferon-α2b fusion protein expressed in Pichia pastoris

A long-lasting recombinant human serum albumin-interferon-α2b fusion protein (rHSA/IFNα2b) was prepared and its structure and biological activities were studied. rHSA/IFNα2b was expressed in methylotrophic yeast Pichia pastoris with HSA’s natural signal peptide and purified by dye affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography and Sephadex G25. Purity of the prepared rHSA/IFNα2b was greater than 97% analyzed by non-reduced SDS–PAGE and RP-HPLC. Structure and biological activities of the prepared rHSA/IFNα2b were characterized by physical, chemical and biological methods. Its p I was 5.3 and showed a single band on IEF gel. Molecular weight determined by MALDI-TOF was 86004.3 ± 29.2. Amino-terminal and carboxyl-terminal amino acid sequences were identical to predicted sequence. Its specific activity in vitro was 6.3 ± 0.8 × 10 5 IU/mg fusion protein, retaining about 1.4% of that of unmodified rIFNα on a molar basis. After administered in monkeys, significant increases of 2′,5′-oligoadenylate synthetase activity relative to IFN-α were maintained for 14 days in serum and the rHSA/IFNα2b showed more potent biological activity than IFN-α on a molar basis. Therefore, markedly improved in vivo biological activity of rHSA/IFNα2b could exhibit more potent antiviral activity than IFNα2b in future clinical trials.

Affinity chromatographic separation of secreted alkaline phosphatase and glucoamylase using reactive dyes

A single-step dye affinity chromatographic separation method was developed to separate secreted alkaline phosphatase (SEAP) and glucoamylase produced in CHO cell culture and Aspergillus niger fermentation, respectively. The reactive dye, Procion ® Green H-E4BD, was found to have a good binding capacity for SEAP, whereas Procion ® Blue H-ERD was the best dye ligand for glucoamylase. However, these dyes have a relatively low selectivity for the target protein. Consequently, elution of the adsorbed proteins by KCl solution resulted in a product with many impurity proteins as evident by the multiple protein bands on SDS-PAGE. However, elution of SEAP by its substrate, phosphate, produced a relatively pure protein with a high specific enzyme activity because of the competition for active site between the substrate and the dye ligand. Also, a high-purity glucoamylase product was obtained by elution with a borate solution. The relatively inexpensive dye affinity chromatography thus can be used for purifying enzymes from cell culture and fermentation broths. The adsorption of SEAP on the dye-ligand affinity resin followed the Langmuir isotherm. An axial dispersion model with external mass transfer limitation was developed to simulate the breakthrough curve in the chromatographic column. This mathematical model can be used to scale up the protein adsorption process.

Improved performance of column chromatography by arginine: Dye-affinity chromatography

Arginine has been effectively used in various column chromatographies for improving recovery and resolution, and suppressing aggregation. Here, we have tested the effectiveness of arginine as an eluent in dye-affinity column chromatography using Blue-Sepharose, which binds enzymes requiring adenyl-containing cofactors (e.g., NAD). A common eluent, NaCl, showed a broad elution peak with low recovery of lactate dehydrogenase, at most ∼60% using 2 M salt. The recovery decreased as the NaCl concentration was either decreased or increased; i.e., the recovery was maximum at 2 M. On the contrary, addition of arginine to the eluent resulted in more than 80% recovery above 0.5 M and the recovery was nearly independent of the arginine concentration. The elution peak was much sharper with arginine, leading to elution of more concentrated protein solution. Successful elution of proteins bound to the ATP–agarose resins by arginine was also described.

Purification and kinetics of a thermostable laccase from Pycnoporus sanguineus (SCC 108)

Pycnoporous sanguineus was identified as a laccase producer when grown on diluted molasses. The single laccase was purified with a purification factor of 967 by ammonium sulphate precipitation, ion exchange and dye affinity chromatography, to a specific activity of 32.9 U/mg. The molecular mass of 58,000 Da, the optimum pH range between 3 and 5 with ABTS, DMP and guaiacol as substrates and the isoelectric point of 6.7, was similar to some other laccases of filamentous fungi. The optimum temperature was 55 °C and the enzyme displayed enhanced thermal stability with a half-life of 170 min at 75 °C. In terms of k cat/ K m values for ABTS, syringaldazine and DMP, DMP was the best substrate. Kinetic analysis of fifteen more compounds revealed that, the enzyme was a true laccase with a requirement for a free –OH group with an adjacent free or derivatised –OH. The o-diphenols were preferred above their p-counterparts. Compounds with three adjacent –OH groups displayed higher binding affinities but phloroglucinol, an m-substituted phenol was unreactive. The apparent higher stability of this laccase makes it a good candidate for further investigation into it possible application in biotechnology.

Molecular cloning and characterization of a plant α1,3/4-fucosidase based on sequence tags from almond fucosidase I

Our work with almond peptide N-glycosidase A made us interested also in the α1,3/4-fucosidase which is used as a specific reagent for glycoconjugate analysis. The enzyme was purified to presumed homogeneity by a series of chromatographic steps including dye affinity and fast-performance anion exchange chromatography. The 63 kDa band was analyzed by tandem mass spectrometry which yielded several partial sequences. A homology search retrieved the hypothetical protein Q8GW72 from Arabidopsis thaliana. This protein has recently been described as being specific for α1,2-linkages. However, cDNA cloning and expression in Pichia pastoris of the A. thaliana fucosidase showed that it hydrolyzed fucose in 3- and 4-linkage to GlcNAc in Lewis determinants whereas neither 2-linked fucose nor fucose in 3-linkage to the innermost GlcNAc residue were attacked. This first cloning of a plant α1,3/4-fucosidase also confirmed the identity of the purified almond enzyme and thus settles the notorious uncertainty about its molecular mass. The α1,3/4-fucosidase from Arabidopsis exhibited striking sequence similarity with an enzyme of similar substrate specificity from Streptomyces sp. (Q9Z4I9) and with putative proteins from rice.

Structural Basis for Activation of the Thiamin Diphosphate-dependent Enzyme Oxalyl-CoA Decarboxylase by Adenosine Diphosphate

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate-dependent enzyme that plays an important role in the catabolism of the highly toxic compound oxalate. We have determined the crystal structure of the enzyme from Oxalobacter formigenes from a hemihedrally twinned crystal to 1.73 A resolution and characterized the steady-state kinetic behavior of the decarboxylase. The monomer of the tetrameric enzyme consists of three alpha/beta-type domains, commonly seen in this class of enzymes, and the thiamin diphosphate-binding site is located at the expected subunit-subunit interface between two of the domains with the cofactor bound in the conserved V-conformation. Although oxalyl-CoA decarboxylase is structurally homologous to acetohydroxyacid synthase, a molecule of ADP is bound in a region that is cognate to the FAD-binding site observed in acetohydroxyacid synthase and presumably fulfils a similar role in stabilizing the protein structure. This difference between the two enzymes may have physiological importance since oxalyl-CoA decarboxylation is an essential step in ATP generation in O. formigenes, and the decarboxylase activity is stimulated by exogenous ADP. Despite the significant degree of structural conservation between the two homologous enzymes and the similarity in catalytic mechanism to other thiamin diphosphate-dependent enzymes, the active site residues of oxalyl-CoA decarboxylase are unique. A suggestion for the reaction mechanism of the enzyme is presented.

Metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, in mouse liver by alcohol dehydrogenase Adh1 and aldehyde reductase AKR1A4

The reductive metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, was studied in mouse liver. Using an HPLC-based stopped assay, the primary reduced metabolite was identified as 6-hydroxy-trans, trans-2,4-hexadienal (OH/CHO) and the secondary metabolite as 1,6-dihydroxy-trans, trans-2,4-hexadiene (OH/OH). The main enzymes responsible for the highest levels of reductase activity towards trans, trans-muconaldehyde were purified from mouse liver soluble fraction first by Q-sepharose chromatography followed by either blue or red dye affinity chromatography. In mouse liver, trans, trans-muconaldehyde is predominantly reduced by an NADH-dependent enzyme, which was identified as alcohol dehydrogenase (Adh1). Kinetic constants obtained for trans, trans-muconaldehyde with the native Adh1 enzyme showed a Vmax of 2141+/-500 nmol/min/mg and a Km of 11+/-4 microM. This enzyme was inhibited by pyrazole with a KI of 3.1+/-0.57 microM. Other fractions were found to contain muconaldehyde reductase activity independent of Adh1, and one enzyme was identified as the NADPH-dependent aldehyde reductase AKR1A4. This showed a Vmax of 115 nmol/min/mg and a Km of 15+/-2 microM and was not inhibited by pyrazole.

Enzymatic Clarification of Fruit Juices by Fungal Pectin Lyase

The activity of pectic enzymes was measured in culture filtrates of four selected fungal strains. Pectin lyase (PL) activity was produced by all fungi, but pectinesterase activity was not detected in culture filtrates of the strain Penicillium expansum F16. The use of crude enzymes resulted in good clarification of apple juice as evaluated by an increase in transmittance at 660 nm. PL activities were partially purified by dye-affinity chromatography on Blue Sepharose. The enzyme was bound to the column and eluted in a single peak, free of PE activity, and not coinciding with the major amount of protein. Apple, grape, and passion fruit juices were effectively clarified by these partially purified enzymes. Methanol content was determined by gas-liquid chromatography, being undetectable in the clarified juices. These pectic enzymes demonstrate potential for use in fruit juice processing.

Rapid neutral protease purification by dye-affinity membrane chromatography

Dye-affinity hollow fibres were prepared by radiation-induced grafting of polymers with different hydrophilicity degree on to the membranes and dye attachment after reaction of the grafted epoxy groups with ammonia to produce amino groups. Membranes grafted with glycidyl methacrylate/dimethyl acrylamide (GMA/DMAA) 1/3 showed better chromatographic properties than those grafted with GMA. The dye Yellow HE-4R was selected for purification of a neutral protease from Flavourzyme™ on the basis of the maximum capacity of the membranes obtained. No leakage of the dye from the membranes was evidenced after incubation in 8 M urea in 0.5 M NaOH for 2 months. From isotherms developed with membranes grafted with GMA/DMAA 1/3, a maximum capacity of 24220 U/ml membrane and a dissociation constant of 91 U/ml was calculated. Elution with 2 M NaCl at pH 4 allowed quantitative recovery of the adsorbed protease. A yield of 72% with a purification factor of 3.7 was achieved by working at 5 ml/min for the load, wash and regeneration steps and 1 ml/min for the elution step. Under these conditions, productivity was 1828 U/h ml, far higher than that attained with the same dye attached to a soft gel: 304 U/h ml [Iannucci NB, Navarro del Cañizo AA, Cascone O. Purification of neutral protease by dye-affinity chromatography. Appl Biochem Biotechnol 2003;104:173–84].

An Integrated Process for Separation of Major and Minor Proteins From Goat Serum

Analysis of minor proteins in animal sera is of considerable clinical significance. To be able to detect these proteins, depletion of major proteins (albumin and immunoglobulin G [IgG]) is necessary. Many of these proteins are also required in pure form for a variety of biochemical applications. The present work uses goat serum as the system and describes the separation and purification of both major and several minor proteins. This was carried out by judicious adaptation and combination of separation technologies such as immobilized metal ion affinity chromatography (on a somewhat novel matrix), dye affinity chromatography, and lectin affinity chromatography. Albumin, IgG, alpha2-macroglobulin, alpha1-proteinase inhibitor, and transferrin were obtained from the serum. The purified preparations were found to be homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Metabolic Control Analysis of Aspergillus nigerl‐Arabinose Catabolism

A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography, and their kinetic properties were characterized. For the other enzymes of the pathway the kinetic data were available from the literature. The metabolic model was used to analyze flux and metabolite concentration control of the L-arabinose catabolic pathway. The model demonstrated that flux control does not reside at the enzyme following the intermediate with the highest concentration, L-arabitol, but is distributed over the first three steps in the pathway, preceding and following L-arabitol. Flux control appeared to be strongly dependent on the intracellular L-arabinose concentration. At 5 mM intracellular L-arabinose, a level that resulted in realistic intermediate concentrations in the model, flux control coefficients for L-arabinose reductase, L-arabitol dehydrogenase and L-xylulose reductase were 0.68, 0.17 and 0.14, respectively. The analysis can be used as a guide to identify targets for metabolic engineering aiming at either flux or metabolite level optimization of the L-arabinose catabolic pathway of A. niger. Faster L-arabinose utilization may enhance utilization of readily available organic waste containing hemicelluloses to be converted into industrially interesting metabolites or valuable enzymes or proteins.

Improved hollow-fibre membranes for dye-affinity chromatography

Hollow-fibre membranes with different degrees of surface hydrophilicity were obtained by grafting mixtures of glycidyl methacrylate (GMA) and dimethyl acrylamide (DMAA) in various proportions, and Cibacron Blue F3G-A was attached to them through ammonia or glucamine spacers. Membrane hydrophilicity increased with the amount of dimethyl acrylamide in the grafted polymer. As the hydrophilicity increased the permeability decreased from 352 mL/cm2 min MPa for membranes grafted with GMA with ammonia spacer to 12.7 mL/cm2 min MPa for membranes grafted with GMA/DMAA 1/3 with glucamine spacer. Membranes grafted with GMA/DMAA 1/3 with ammonia spacer showed the best performance for BSA and lysozyme adsorption: maximum capacity was 15.3 +/- 2.2 mg BSA/mL membrane and 58.3 +/- 6.6 mg lysozyme/mL membrane while dissociation constants were 0.27 +/- 0.16 and 0.13 +/- 0.12 mg/mL, respectively. Over 80% of adsorbed proteins could be eluted with 2 M NaCl + 20% isopropanol in 20 mM sodium phosphate buffer, pH 7.0.

Poly(ethylene dimethacrylate-glycidyl methacrylate) Monolith as a Stationary Phase in Dye-Affinity Chromatography

Porous monolith was obtained by the bulk polymerization of ethylene dimethacrylate (EDMA) and glycidyl methacrylate (GMA) conducted in a glass tube. Poly(EDMA-GMA) monolith had a specific surface area of 98.7 m2/g. Poly(EDMA-GMA) monolith was characterized by swelling studies, FTIR, scanning electron microscopy, and elemental analysis. Poly(EDMA-GMA) monolith with a swelling ratio of 48%, and containing 45.7 μmol Cibacron Blue F3GA/g, were used in the adsorption/desorption of human serum albumin (HSA) from aqueous solutions and human plasma. The nonspecific adsorption of HSA was very low (0.8 mg/g). The maximum amount of HSA adsorption from aqueous solution in phosphate buffer was 22 mg/g at pH 5.0. Higher HSA adsorption value was obtained from human plasma (up to 53.2 mg/g) with a purity of 92%. Desorption of HSA from Cibacron Blue F3GA-attached poly(EDMA-GMA) monolith was obtained using 0.1 M Tris/HCl buffer containing 0.5 M NaCl. It was observed that HSA could be repeatedly adsorbed and desorbed with poly(EDMA-GMA) monolith without significant loss in the adsorption capacity.

Purification of post-translationally modified proteins from bacteria: homologous expression and purification of histidine-tagged pilin from Neisseria meningitidis

Until recently, glycosylation of proteins in prokaryotes was regarded as uncommon and thought to be limited to special cases such as S-layer proteins and some archeal outer membrane proteins. Now, there are an increasing number of reports of bacterial proteins that are glycosylated. Pilin of pathogenic Neisseria is one of the best characterised post-translationally modified bacterial proteins, with four different types of modifications reported, including a novel glycosylation. Pilin monomers assemble to form pilus fibres, which are long protein filaments that protrude from the surface of bacterial cells and are key virulence factors. To aid in the investigation of these modifications, pure pilin is required. A number of pilin purification methods have been published, but none are appropriate for the routine purification of pilin from many different isolates. This study describes a novel, rapid, and simple method of pilin purification from Neisseria meningitidis C311#3, which facilitates the production of consistent quantities of pure, native pilin. A 6× histidine tag was fused to the C-terminus of the pilin subunit structural gene, pilE, via homologous recombination placing the 6× histidine-tagged allele in the chromosome of N. meningitidis C311#3. Pilin was purified under non-denaturing conditions via a two-step process using immobilised metal affinity chromatography (IMAC), followed by dye affinity chromatography. Analysis of the purified pilin confirmed that it retained both of the post-translational modifications examined. This novel approach may prove to be a generally applicable method for purification and analysis of post-translationally modified proteins in bacteria.

Expression, purification and preliminary crystallographic analysis of human sorbitol dehydrogenase.

Human sorbitol dehydrogenase (SDH) was expressed in Escherichia coli BL21 cells and purified using ammonium sulfate precipitation and anion-exchange and dye-affinity chromatography. Purified SDH was crystallized from polyethylene glycol solutions using the hanging-drop vapour-diffusion method. X-ray data were collected to 2.75 A resolution. The crystals belong to the monoclinic C2 space group, with unit-cell parameters a = 145.9, b = 52.3, c = 169.0 A, beta = 101.8 degrees. This is the first crystallization report of human sorbitol dehydrogenase.

A 60-66 kDa protein with gonadotrophin surge attenuating factor bioactivity is produced by human ovarian granulosa cells.

We aimed to confirm the ovarian site of gonadotrophin surge-attenuating factor (GnSAF) production and produce granulosa/luteal cell-conditioned medium (G/LCM) containing GnSAF for purification studies. Blue dye affinity chromatography followed by pseudochromatofocusing of G/LCM yielded bioactive fractions at pH 5.74 and 5.77. The former had a major 60-66 kDa band with an internal amino acid sequence of EPQVYVHAP following tryptic digestion. A rat polyclonal antiserum (rPAb) raised against this band completely blocked in-vitro GnSAF bioactivity in human follicular fluid, serum and G/LCM. GnSAF bioactivity was localized to a 64 kDa band of serum-free G/LCM and following 2D gel electrophoresis, one of the spots recognized by Western blotting with the GnSAF rPAb had an N-terminal amino acid sequence of NH-XVPQGNAXXN. Neither amino acid sequence had significant homology with proteins in the human genome database. When ovarian tissues from spontaneously cycling women were cultured under serum-free conditions, neither theca- nor stroma-conditioned media contained GnSAF bioactivity. However, granulosa cell-conditioned medium significantly reduced GnRH-induced LH secretion, an effect that was reversed by incubation with the GnSAF rPAb. In conclusion, we have confirmed that human granulosa cells produce GnSAF within the ovary and have two candidate amino acid sequences for GnSAF. We have also demonstrated that serum-free granulosa cell culture constitutes the method of choice for the characterization of GnSAF since recovery of bioactivity is superior in the presence of fewer serum proteins.