High-performance molecular sieve chromatography of proteins on agarose columns: the relation between concentration and porosity of the gel

Curves showing the relation between log (molecular weight) and distribution coefficient are presented for proteins subjected to molecular sieve chromatography on crosslinked and non-crosslinked agarose gels of different concentrations. These curves, which facilitate selection of the gel concentration that gives optimal resolution in any particular separation problem, show that the exclusion limit of 5, 9, 12, and 20% agarose gels correspond to protein with molecular weights above 1,000,000, 600,000, 450,000, and 280,000, respectively. Plate numbers have been determined for columns of 20% agarose at different flow rates and bead sizes. Separations of model proteins by high-performance molecular sieve chromatography on agarose beads are shown.     

Urinary protein profiling by high-performance gel permeation chromatography

We describe a new application of high-performance aqueous gel permeation chromatography for the analysis of human proteinuria. Separations of urinary proteins from normal subjects and patients with renal impairment were performed with TSK G 3000 SW columns. The effects of pH and icnic strength of the eluent on the separation of urinary proteins were investigated. Albumins were selectively separated from urine by affinity chromatography on Blue Sepharose CL-6B. According to the results of clinical investigations, urinary protein pattern derived from gel permeation chromatography revealed a good prediction of the site of renal involvement. Predominant excretion of proteins with lower molecular weight than albumin correlated with tubular damage. Albumin and higher molecular weight protein patterns were associated with glomerular disease. Absorbance measurements of the eluent at 280 nm were used for quantitative determination of total urinary protein. Gel permeation chromatography was compared to sodium dodecyl sulfate—polyacrylamide gel electrophoresis and the resulting protein patterns are in good agreement.     

Purification and characterization of proteins,including procollagen type III,in salt extracts of fetal bovine skin

The nature of high-molecular-weight proteins in salt extracts of fetal bovine skin was investigated. A series of DEAE cellulose ion-exchange columns separated the mature collagen from the high molecular weight proteins and also separated the high molecular weight proteins from each other. The following proteins were isolated: (a) a very high molecular weight protein which appears to be aggregated mature collagen; (b) two high molecular weight proteins of slightly faster mobility on SDS polyacrylamide gels, one of which is collagen-like and one of which is not; and (c) a type III procollagen, purer than those previously reported in the literature. These latter three proteins were characterized by amino acid analysis, SDS polyacrylamide gel electrophoretic mobility, collagenase sensitivity, and CNBr peptide patterns from SDS-PAGE.     

Purification of four gelatin-binding proteins from bovine seminal plasma by affinity chromatography

Bovine seminal plasma contains three similar acidic proteins, which we have previously designated as BSP-A1, BSP-A2, and BSP-A3. These proteins contain two homologous domains that are similar to type II structures present in the gelatin-binding domain of fibronectin. The present data have revealed that these proteins, like fibronectin, also form complexes with gelatin, a denatured collagen. Based on this property, a single step affinity purification method has been developed. In addition to these three proteins BSP-A1, -A2 and -A3, another protein with an apparent molecular weight of 30,000 dalton (named BSP-30-kDa) also bound to the gelatin-agarose column. Elution of these proteins from affinity columns using a linear gradient of either urea or arginine gave essentially the same pattern with a high yield of 90–95%. The purified proteins were homogeneous by SDS-polyacrylamide gel electrophoresis, amino acid composition and HPLC. Chromatography of bull seminal vesicular fluid also exhibited an elution pattern similar to that obtained for bull seminal plasma. The availability of these purified proteins should aid in understanding the physiology of these gelatin-binding proteins.     

Molecular weight determination of peptides by high-performance gel permeation chromatography

A method for molecular weight determination of small peptides using Bio-Sil TSK 20 and Bio-Gel TSK 125 columns is described. The TSK 20 column provided a good separation of the standard peptides in the range from 1000-10,000 with an accuracy of less than 5% from the calculated regression line. Two combined TSK 125 columns allowed a reliable molecular weight determination in the range from 800 to 3500.     

Ryanodine-affinity chromatography purifies 106 kD Ca release channels from skeletal and cardiac sarcoplasmic reticulum

A 106 kD protein was isolated from skeletal sarcoplasmic reticulum (SR) vesicles and shown to have the properties of SR Ca2+ release channels, including blockade by 5 nM ryanodine. In view of extensive reports that the ryanodine-receptor complex consists of four 565 kD junctional feet proteins (JFPs) and is the 'physiological' Ca2+ release channel, we prepared ryanodine-affinity columns to isolate its receptor site(s). Conditions known to maximize the association and dissociation of ryanodine to SR proteins were respectively used to link, then elute, the receptor(s) from ryanodine-affinity columns. The method purified a protein at about 100 kD from both rabbit skeletal and canine cardiac SR vesicles. The skeletal and cardiac proteins isolated by ryanodine-affinity chromatography were identified as the low molecular weight Ca2+ release channel through their antigenic reaction with an anti-106 kD monoclonal antibody. Upon reconstitution in planar bilayers, both skeletal and cardiac proteins revealed the presence of functional SR Ca2+ release channels. Surprisingly, ryanodine-affinity columns did not retain JFPs but purified 106 kD Ca2+ release channels which are a minor component (0.1-0.3%) of SR proteins.     

Isolation of early viral proteins from poxvirus-infected chick embryo fibroblasts by DNA-cellulose chromatography and inhibition of their synthesis by chicken interferon

Up to seven early poxvirus-specific proteins have been isolated from vaccinia-WR-infected and cowpox-virus-infected chick embryo fibroblasts by affinity chromatography on native DNA-cellulose columns. The proteins have been characterized by one-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis and by nonequilibrium pH-gradient electrophoresis. The molecular weights of the viral proteins were determined by comparison with proteins of known molecular weight and are comparable to several of the vaccinia-WR-specific DNA-binding proteins isolated previously from infected L-929 cells by Solosky J. M., Esteban M. and Holowczak J.A. [J. Virol. 25, 263-273 (1978)]. The viral proteins binding reversibly to native DNA have been classified as immediate early viral gene products. Synthesis of cowpox-virus-induced early DNA-binding proteins is inhibited in chick cells pretreated with homologous interferon at a concentration of 500--1000 units/ml.     

Preferential affinity of high molecular weight high mobility group non-histone chromatin proteins for single-stranded DNA.

We have subjected proteins dissociated from chicken erythrocyte or calf thymus chromatin by 0.35 M NaCl to sequential chromatography on columns containing immobilized double-stranded DNA and single-stranded DNA. At 0.2 M NaCl, 1 mM Tris . Cl (pH 7.5), the high molecular weight, high mobility group proteins (HMG-1, HMG-2, and HMG-E), were not retained by double-stranded DNA columns, but were retained by single-stranded DNA columns. Thus, in that solvent, those proteins exhibit selective affinity for single-stranded DNA. This suggests that the functions of the high molecular weight, high mobility group proteins might involve destabilizing the DNA double helix by virtue of their preferential affinity for single-stranded DNA.     

Isolation and purification of lactoferrin and immunoglobulin G from bovine colostrum with serial cation-anion exchange chromatography

High-value dairy proteins such as lactoferrin (LF) and immunoglobulin (IgG) were separated from bovine colostrum. The whey was initially adjusted to pH 6.8 with 1 mol/L NaOH and then went through centrifugation, precipitation, and filtration to eliminate the fat and caseins in bovine colostrum. The treated whey was further ultra-filtrated to partially remove both other proteins and carbohydrates under 50 kD molecular weight. Then the ultra-filtrated whey was passed through cation and anion exchange columns in series. The LF and IgG were adsorbed on cation and anion exchanger, respectively, due to their different pI. Both the cation and anion exchange columns were washed with de-ionized water followed by successive elution with sodium chloride solutions of increasing molarities (0.27 and 0.85 mol/L; 17 and 51 mmol/L) in a stepwise manner, respectively. After desalted, the elution was freeze-dried. Finally, the LF and IgG with respective purities of 95.0% and 96.6% were obtained.     

Isolation of an organic anion binding protein from rat liver plasma membrane fractions by affinity chromatography

As part of a study of hepatic organic anion transport, solubilized liver plasma membrane proteins were subjected to affinity chromatography on bilirubin- and sulfobromophthalein-labeled agarose columns. Both columns retained a Sudan Black and PAS negative protein of molecular weight 60,000 daltons, which cochromatographed with [³⁵S]sulfobromophthalein on Sephadex G-75, and reversibly bound [³⁵S]sulfobromophthalein in vitro with high affinity (K_a ? 10⁷ M^?1) and a valence of 2. Erythrocyte ghost membranes did not contain this protein. Sulfobromophthalein-agarose retained two additional smaller proteins which did not cochromatograph with [³⁵S]sulfobromophthalein. Their significance is unclear. This study supports the hypothesis that liver cell plasma membranes participate in the hepatic transport of organic anions.     

The use of high-performance liquid chromatography for the determination of size and molecular weight of proteins: a caution and a list of membrane proteins suitable as standards

We propose a list of 15 water-soluble globular proteins and 13 forms of detergent-soluble membrane proteins of known Stokes radii for the calibration of high-performance liquid chromatography columns. It is shown that it is advisable to use different sets of standards for these two types of proteins as the detergent-solubilized membrane proteins may behave differently, being excluded or retarded, depending upon the gel support. A smooth, although nonlinear, relationship between Stokes radii and erf-1(1--KD) is observed while a large scatter of points exists if the calibration is expressed as the molecular weight as a function of KD.     

Isolation of neuronal parvalbumin by high-performance liquid chromatography. Characterization and comparison with muscle parvalbumin

Neuronal parvalbumin has been isolated from rat brain and purified to homogeneity by high-performance liquid chromatography (HPLC) on reverse-phase supports. This procedure includes four consecutive chromatographic steps with an overall protein recovery of 74% and a 26 400-fold purification. The concentration of parvalbumin was found to be approximately 10 mg/kg wet weight in brain tissue, which is about 100 times lower than that in rat muscle. The physical properties of brain parvalbumin are described and compared with those of the muscle counterpart. These proteins were identical in their molecular weights (12 000), isoelectric points (4.9), retention times on C-18 reverse-phase HPLC columns, Ca2+ content (two per molecule), amino acid compositions, and immunological properties. A comparison of the tryptic peptide maps of brain and muscle parvalbumin by analytical HPLC also revealed identity and showed that the isolation method described here did not alter the chemical structure of the protein.     

Polymorphism of blood serum transcortin during column chromatography

Serum protein bound progestins, androgens, estrogens, and cortexolone, were fractionated on a number of chromatographic systems. Contrary to earlier suggestions of a homogeneous unit by competition binding and Scatchard analysis, polymorphism and heterogeneity in the molecular nature of the corticosteroid binding globulin (CBG) were evident with progesterone on Sephadex A-25 columns. Components in the 55 000 and 67 000 molecular weight regions were obtained with cortexolone, estradiol, progesterone and testosterone on Ultrogel columns. Separation on DEAE-cellulose-52 resin revealed a fraction in the low ionic prewash followed by another, highly charged entity eluted only with 0.06 M phosphate buffers. Thus, under these conditions, serum protein bound sex steroids eluted in the same position as transcortin glucocorticoid complexes. These results are presented as a caution against the universal use of association dissociation assays to study steroid ligand binding and biological response. The techniques here detailed may fruitfully be employed to explore the hydrodynamic characteristics of protein ligand interactions. In addition, they support the model, earlier proposed with tissue specific steroid receptors, that calls for the various subunits of the ligand as a prerequisite for the expression of all grades of agonist and antagonist activity of a given hormone.     

Variables in the high-pressure cation-exchange chromatography of proteins

The use of cation-exchange high-pressure liquid chromatography for the separation of proteins has been investigated. Several factors, including solvent composition, pH, flow rate, and temperature, were examined for their effects on the resolution of protein standards (insulin, β-lactoglobulin, and carbonic anhydrase B; molecular weight range, 6000 to 30,000 and pI range, 5.3 to 6.5). An initial comparison was made of the recovery of these proteins from three commercially available columns (Whatman Partisil SCX, Separation Industry CM silica, and MCB Reagents Lichrosorb KAT). In general, under the conditions employed, the SCX column gave the highest recovery of applied protein. Based on this recovery data, the Partisil SCX column was chosen for subsequent examination of chromatographic parameters that would optimize protein resolution. An increase in temperature decreased retention and resolution but increased recovery, with some proteins being affected more than others. A decrease in pH in the final eluant or an increase in pH in the initial eluant caused an increase in retention times. For some proteins, the decrease in pH resulted in a greater total recovery of protein. This information has been applied to the purification by cation-exchange high-pressure liquid chromatography of transforming growth factors from a human tumor cell line.     

GO associates with another 40 kDa brain protein

Guanine nucleotide binding proteins (G proteins) mediate a variety of cellular responses to external stimuli. Pure G protein, receptor, and effector are sufficient to reconstitute hormonal activation of an effector in phospholipid vesicles, but other components may be important for specificity or localization in vivo. If another protein associates with GO, the molecular weight of GO solubilized from membranes would be larger than the molecular weight of GO after purification. We find that GO solubilized from bovine brain membranes by Triton X-100 behaves as a single population of molecules on sucrose density gradients and gel filtration columns. Its molecular mass is about 40 kDa larger than pure GO. Association of GO with the other protein is fragile as the proteins dissociate on further purification. There was no difference in ADP-ribosylation or tryptic cleavage of GO in larger and smaller form. These studies provide a basis for future experiments to stabilize the interaction and identify the protein.     

A requirement for albumin as carrier for low molecular weight leukocyte chemotactic factors

Using a micropore filter assay, low molecular weight leukocyte chemotactic factors such as fatty acids, formyl-methionyl peptides, and peptides purified from dextran-activated plasma attract leukocytes suspended in albumin-containing media, but not cells suspended in media lacking in protein or containing non-albumin proteins. These factors become bound to albumin and can be eluted with it from Sephadex columns. The albumin-chemotactic factor complex attracts leukocytes more strongly than either the albumin or the chemotactic factor alone. It is suggested that albumin presents the chemotactic factors at the cell membrane in a form suitable to initiate a response which is not achieved by the same factors in free, low molecular weight form.     

Purification of chicken testicular müllerian inhibiting substance by ion exchange and high-performance liquid chromatography

A highly purified protein molecule was obtained from the secretory proteins of 8-week-old chicken testes using ion-exchange column chromatographic procedures, including DEAE Bio-Gel A, CM Bio-Gel A, wheat germ lectin columns, and high-performance liquid chromatographic (hplc) separation techniques. This protein molecule has a molecular weight of 74,000 Da (74K protein). The isolated 74K protein induces regression of chicken Müllerian ducts grown in vitro. The 74K protein does not cause regression of cultured embryonic intestine or Wolffian duct. When the total testicular secretory proteins are resolved in a two-dimensional gel electrophoresis, approximately 120 polypeptides are obtained. The purified 74K protein has a pI of 6.1. Analysis of amino acid composition indicates that the 74K protein is relatively acidic in nature with a ratio of acidic to basic amino acids of 1.93.     

Determination of the molecular weight of microbial polysaccharides using high performance exclusion chromatography

An automatic method of determining the molecular weight parameters (Mw, Mn) of microbial polysaccharides such as dextran, pullulan was developed based on the use of high performance size-exclusion chromatography on the two types of columns: Zorbax PSM 60 + 300 + 1000 and SynChropack GPC 100 + 500 + 1000. The Mw and Mn values were determined for a number of domestic and foreign dextran preparations. Changes in the molecular weight of pullulan and hydroxyethylstarch resulted from acid and enzymatic hydrolysis were estimated.     

Proteins associated with tubulin.

The distribution of proteins on SDS-urea polyacrylamide (7.5%) disc gel electrophoresis is studied from rat brain tubulin purified by three different procedures, including ammonium sulfate precipitation followed by DEAE cellulose chromotography, three cycles of polymerization-depolymerization and colchicinecontaining agarose affinity columns. Three tubulin-associated proteins other than the principal tubulin dimer are identified and characterized with respect to molecular weight, behavior on gel filtration chromatography and method of tubulin purification. One of these proteins (H₁) is released from the tubulin complex when colchicine is irreversibly bound to tubulin. These proteins may participate in processes related to microtubule assembly or function.     

High-performance size-exclusion chromatography of peptides

Gel filtration on soft gels has been employed for over 40 years for the separation, desalting and molecular weight estimation of peptides and proteins. Technical improvements have given rise to high-performance size-exclusion chromatography (HPSEC) on rigid supports, giving more rapid run times and increased resolution. Initially, these packings were more suitable for the separation of proteins than of peptides, but supports that operate in the fractionation range <10,000 Daltons (Da) are now available. In this report, HPSEC is described in relation to its application to peptides, especially regarding purification, estimation of molecular weight and study of molecular associations.