Electrophoretic separation of large proteoglycans in large-pore polyacrylamide gradient gels (1.32-10.0% T) and a one-step procedure for simultaneous staining of proteins and proteoglycans.

A procedure is described for the preparation of 1.32-10% polyacrylamide gradient gels. Loose polyacrylamide gel on the top side of the gradient was stabilized with a layer of 0.4% agarose gel which also formed sample wells. The upper limit of separation achieved in these gels was estimated to be approximately 2 X 10(6) using globular protein standards. However, large aggregating proteoglycans from cartilage which have a molecular weight range of 1-4 X 10(6) penetrate and separate in these gels. A simple one-step procedure is also described for simultaneous staining of proteins and large proteoglycans in polyacrylamide gels.     

Preparative electrophoresis on agarose submerged gels of two aggregating proteoglycan monomers from articular cartilage

Analytical electrophoresis on polyacrylamide-agarose gels of aggregating proteoglycan monomers from baboon articular cartilage produces two distinct bands, corresponding to two different aggregating monomer populations. A preparative electrophoresis procedure is described for isolating the two monomers. Proteoglycans were extracted from young baboon articular cartilage in 4 M guanidinium chloride containing proteolysis inhibitors and aggregated after hyaluronic acid addition. The aggregates were separated from non-aggregated proteoglycans by isopycnic centrifugation, followed by gel chromatography on Sepharose CL-2B. The monomers of the aggregates were obtained by isopycnic centrifugation under dissociative conditions. Two monomers were separated by preparative electrophoresis on 0.8 % agarose submerged gels. Approximately 60 % of the proteoglycans were recovered from the gel using a freeze-squeeze procedure. Aliquots of the separated monomers gave single bands when submitted to analytical polyacrylamide-agarose gel electrophoresis. Their migration and appearance were similar to that of the two bands present in the non separated preparation of monomers.     

A microdetermination method for assaying glycosaminoglycans and proteoglycans

A simple and reliable method is described for the determination of glycosaminoglycans and proteoglycans in tissue extracts as well as during preparative and analytical procedures for these molecules. It is particularly useful because it requires much less starting material for the identification of glycosaminoglycans or proteoglycans and, in addition, is several fold more sensitive than the currently used uronic acid assays. The procedure allows separation of macromolecules by ion-exchange chromatography, density gradient centrifugation, or molecular sieve chromatography and involves spotting onto cellulose acetate membrane, reaction with Alcian blue, and quantitation of color in a spectrophotometer. This method is particularly appropriate to use for the analysis of proteoglycans and glycosaminoglycans in tissues which are available in limited amounts or have low levels of these macromolecules.     

Liquid chromatographic determination of diclofenac in human synovial fluid

A simple, rapid and sensitive HPLC method for the determination of diclofenac in synovial fluid is described. Special attention was paid to the procedure of sample preparation since gel formation may sometimes occur in synovial samples. With a one-step extraction procedure good and reproducible recovery of diclofenac was obtained. A subsequent HPLC assay was adjusted so as to achieve adequate sensitivity and precision needed for analysis of true samples. The results obtained by the described procedure proved the method to be suitable for monitoring concentrations of diclofenac in synovial fluid.     

Two-dimensional electrophoresis of plasma proteins without denaturing agents.

A technique of two-dimensional polyacrylamide gel electrophoresis for the separation of plasma proteins is described. Human plasma proteins were separated by isoelectric focusing followed by electrophoresis in a 4 to 21% linear gradient gel slab. No denaturing agent was used throughout the procedure, so that the analysis of native proteins is possible. Two-dimensional patterns obtained from normal human plasma samples were recorded as "staining density maps," which are similar to contour line maps, and more than 230 protein spots were counted reproducibly on each "staining density map." This technique permits the simultaneous estimation of pI's and approximate molecular weights of native proteins on the slab gel. Applications of this technique to an IgA myeloma plasma sample and a porcine serum sample are described.     

Determination of malonaldehyde in biological materials by high-pressure liquid chromatography

A new one-dimensional agarose gel electrophoresis method for the quantitation of glycosaminoglycans in biological samples has been described. In this procedure, concanavalin A, suspended in agarose gel, interacts with glycosaminoglycans such that rocket-like precipitin lines are formed. The area of the rocket is directly proportional to the glycosaminoglycan content of the sample. This procedure permits measurement of glycosaminoglycans in amounts as low as 0.5 nmol uronic acid equivalents with a coefficient of variation of only 8%. The described method has been applied to the determination of free heparan sulfate in plasma. This method can also be used to measure all high-charge glycosaminoglycans of biological interest.     

Proteoglycans of human gingival epithelium and connective tissue.

Proteoglycans extracted from separated specimens of healthy human gingival epithelium and from connective tissue have been purified. The epithelial proteoglycans fractionated as a single included peak on Sepharose 4B-CL and contained heparan sulphate and dermatan sulphate glycosaminoglycans. The connective-tissue proteoglycans separated into three major populations on Sepharose 4B-CL, one of which was excluded from this gel under associative conditions (0.5 M-sodium acetate, pH 7.4). Subsequent fractionation of the excluded material under dissociative conditions (4 M-guanidinium chloride/0.05 M-sodium acetate, pH 7.4) revealed an absence of any aggregate formation of molecules within this population. The connective-tissue proteoglycans contained heparan sulphate, dermatan sulphate and chondroitin 4-sulphate, the proportions of which varied with the molecular size of the proteoglycans. Amino acid analysis of the protein cores of gingival-epithelial and connective-tissue proteoglycans revealed differences that were similar to the differences described between other types of proteoglycans such as those from skin.     

A rapid colorimetric assay for heparinase activity.

A rapid, sensitive, assay for enzymes that degrade heparin is described. The procedure is based on the interference of heparin with color development during the interaction of protein with the dye Coomassie brilliant blue. The loss of this property when the glycosaminoglycan is degraded by heparinase can be used to quantify activity of the enzyme in pure form, or in complex biological samples such as tissue homogenates or serum. The assay is also suitable for studying dependence of heparinase activity under conditions such as varying pH and temperature.     

Extraction and purification of proteoglycans from various types of connective tissue

A new procedure for the isolation of proteoglycans has been described. Tissues are extracted with 4 M guanidinium chloride. the extracting solven is then exchanged for 7 M urea and the extract is chromatographed on a DEAE-cellulose column previously equilibrated with 7 M urea. Non-proteoglycan proteins were eluted with urea in weak salt solutions. Subsequently proteoglycans were eluted with strong salt solutions. By the procedure proteoglycans from tissues containing only small amounts of proteoglycans can be obtained virtually free from collagen in a form suitable for further fractionation.     

A sensitive GLC-method for component sugars andO-glycosidic linkage monosaccharides of cartilage proteoglycans

A method is described for the measurement ofN-acetylgalactosamine,N-acetylglucosamine, galactose, mannose and xylose present in the different carbohydrate chains of cartilage proteoglycans (PG). Bovine articular cartilage PG samples corresponding to the minimum of 1 nmol of each monosaccharide were reproducibly quantified following hydrolysis with 2 M HCl and derivatization into alditol acetates. An on-column injection mode and an OV-1701 fused silica capillary column were used for chromatography.Alkaline borohydride treatment of the PG was exploited to reduce the acid labile xylose in the base of the chondroitin sulphate chain into more stable xylitol, allowing the assay of chondroitin sulfate chain length as anN-acetylgalactosamine/xylose ratio. A novel procedure is described for the measurement of the galactosaminitol evolving from the protein linkage of oligosaccharides and of keratan sulphate.     

A simplified procedure for the reduction and alkylation of cysteine residues in proteins prior to proteolytic digestion and mass spectral analysis

A procedure for reduction and alkylation of cysteine residues in proteins was developed using the volatile reagents triethylphosphine and iodoethanol. These reagents may be used to modify proteins in solution, as well as proteins in gel slices, prior to proteolytic digestion and mass spectral analysis. The procedure eliminates several steps with both types of samples. Samples in solution do not need to be desalted following reduction and alkylation, with excess reagent being removed under vacuum. For gel slices, the procedure combines washing, destaining, reduction and alkylation into a single step. The procedure was applied successfully to samples as complex as serum, and we demonstrated alkylation of cysteines to be quantitative in purified proteins. We also were able to reduce and alkylate proteins with these reagents during the gas phase. Elimination of the need for desalting of samples after reaction raised the possibility of automation of the procedure for liquid samples, which is difficult with conventional reduction and alkylation chemistries.     

Alcian blue staining of proteoglycans in polyacrylamide gels using the "critical electrolyte concentration" approach

A method is described here for Alcian blue staining of proteoglycans in polyacrylamide gels; this is illustrated using extracts obtained from bovine corneal stroma. Other available methods for visualization of proteoglycans can produce nonspecific staining and frequently a high background in the gel; with a "critical electrolyte concentration" approach, specific staining against a clear background can be obtained.     

An investigation of plasma collection, stabilization, and storage procedures for proteomic analysis of clinical samples

In order to evaluate the critical components of the process necessary to preserve clinical plasma samples collected at research sites for proteomic analysis, various collection and preservation protocols with controlled experimentation were evaluated. The presence of a protease inhibitor cocktail (PIC) included in the blood draw tube would stabilize the plasma proteins was hypothesized. To test this hypothesis, four plasma samples from each of 14 volunteers were collected. Samples were treated following a standard protocol that included PIC or were subjected to various processing treatments that included time, temperature, different anticoagulants, and the absence of PIC. Large format two dimensional-polyacrylamide gel electrophoresis (2D-PAGE) proteomic analysis and enzyme immunoassay (EIA) were used to detect differences between the treatment groups. A novel 2D-PAGE quality scoring method was developed to determine global differences in the treatment groups, wherein a rating scale questionnaire was used to quantify the quality of each 2D-PAGE gel. The data generated from EIAs, classical 2D-PAGE image analysis and 2D-PAGE quality scoring, each generated similar results. Inclusion of protease inhibitor cocktail in the sample tubes, provided stable and reliable human plasma samples that yielded reproducible results by proteomic analysis. When PIC was included, samples retained stability under less stringent processing, such that refrigeration for several hours before processing or one freeze-thaw cycle had little detrimental effect. We demonstrated that samples without PIC, from either heparin or ethylenediaminetetraacetic acid (EDTA) plasma tubes, gave results that varied significantly from the control samples. Also, even with PIC present in blood tubes, we found it was important to quickly decant the separated plasma from the cellular components found in the blood tubes following centrifugation, as prolonged exposure again yielded different results from the standard procedure.     

Isoelectric focusing in thin-layer acrylamide geis: an improved method for sample application.

Various procedures for applying protein solutions for isoelectric focusing in thin layers of polyacrylamide gels have been described (1). In the most widely used procedure, pieces of filter paper soaked in the solution are laid on the gel surface. The major drawback of this very elegant and simple method is that some proteins will adsorb to the paper. The procedure described here, in which the samples to be analyzed can be applied directly on the gel by means of a specially designed applicator, permits a quantitative approach of isoelectric focusing separation.     

A simple and rapid procedure for removal of triton X-100 from protein solution

A simple and rapid procedure for removal of Triton X-100 from protein solutions is described. The procedure is based on the extraction of Triton X-100 with chloroform from the protein solution. By the addition of sodium dodecyl sulfate before the extraction, the procedure was improved effectively and the sample thus prepared was used directly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This method can be used for the removal of other nonionic detergents and for samples of larger size.     

Ultrafiltration-based assay for heparanase activity

Heparanase, a mammalian endoglycosidase that specifically cleaves heparan sulfate (HS), has been found in many tissues. Platelet, liver, and placenta have been abundant sources for the study of the enzyme. Notably, certain malignant cells also have been found to produce large amounts of the enzyme, the levels of which often correlate with their invasive and metastatic properties. To study roles of heparanase in various biological situations, a reliable method measuring the enzyme activity is indispensable. In the past, measurement of heparanase enzyme activity was done either by the detection of the degradation of fluorescent or radiolabeled HS chains by gel filtration procedures or by the use of radiolabeled substrate conjugated to solid matrices for the easy separation of degraded HS chains. A newly developed procedure, presented in this article, measures degradation of radiolabeled HS chains in the aqueous buffer by detecting their degradation products using an ultrafiltration device, the Centricon 30. This procedure has several advantages over previous assay procedures that involved tedious processing such as gel filtration chromatography of each sample or the preparation of substrate HS proteoglycans conjugated to a solid matrix. The simplicity of the new procedure allows a short setup time and a rapid processing of a large number of samples. Furthermore, the enzymatic reaction during the aqueous phase allows kinetic analyses in standard conditions.     

The use of micropreparative electrophoresis of protein/peptide isolations for primary structure determinations

High-performance electrophoresis chromatography (HPEC) is a recent development that features continuous-elution gel electrophoresis for isolating proteins or peptides in range of 1 to 300 microgram quantities. Column gel electrophoresis is conducted under thermostated conditions, and the field voltage can be varied within a run with a programmable power supply. Applications of this apparatus in protein purification are presented to demonstrate the utility of the (Model 230A) HPEC. These examples include on-line detection with direct analyte recovery of highly purified sample, which mimics high-performance liquid chromatography, for subsequent structure-function characterization. A method to remove salts from sodium dodecyl sulfate electrophoresed samples for subsequent sequencing or amino acid analysis is described. This desalting procedure recovers from 90%-95% of the sample and employs a low molecular weight cut-off membrane during sample centrifugation onto a polyvinylidene difluoride membrane. Subsequent washings are performed to efficiently remove salts, free amino acids and detergents that are known to interfere with sequence analysis. Sequence information such as initial recovery, repetitive yields and chromatogram comparisons are presented to demonstrate the utility of this procedure when used following isolation of sample with HPEC.     

Proteoglycans synthesized and secreted by pancreatic islet beta-cells bind amylin

Pancreatic islet amyloid deposits in type 2 diabetes are associated with decreased islet beta-cell function. They contain both amylin (islet amyloid polypeptide), the beta-cell-derived unique fibrillogenic component, and heparan sulfate proteoglycans (HSPGs). We hypothesized that beta-cell HSPGs contribute to islet amyloidogenesis. [35S]Sulfate-labeled proteoglycans from islet-derived beta-TC3 cell cultures eluted from diethylaminoethyl Sephacel at 0.35M NaCl. Chromatography on Sepharose CL-4B and SDS-PAGE analysis revealed distinct populations of proteoglycans. Medium HSPGs eluted at K(av) approximately 0.18 and 0.50 with glycosaminoglycan chains of approximately 28 and 19 kDa, respectively. A third population containing chondroitin/dermatan sulfate eluted at K(av) approximately 0.70 with glycosaminoglycan chains of approximately 10 kDa. A single size class of heparan and chondroitin/dermatan sulfate proteoglycans in the cell layer eluted at K(av) approximately 0.40 with glycosaminoglycan chains of approximately 19 kDa. Medium and cell layer proteoglycans bound exclusively to fibrillogenic amylin, as determined by gel mobility shift assays, indicating a possible role for beta-cell-derived proteoglycans in islet amyloid formation.     

A method for enzymic extraction and the measurement of chloroplast RNA

A method is described for measuring RNA associated with chloroplast thylakoid membranes. Washed thylakoids are incubated with ribonucleases A and T(1), under low Mg(2+) conditions, to release hydrolyzed RNA into solution. After removing the membranes by centrifugation, the mono- and oligonucleotides are adsorbed by Dowex 1-X2 in miniature columns made from Pasteur pipettes, and then eluted with 2 n HCl. RNA is estimated from the absorbance of the eluate at 260 nanometers, with corresponding values obtained by the orcinol reaction for pentose. Polyacrylamide gel electrophoresis patterns of extracted RNA indicate that our current procedures for preparation of thylakoids results in material containing variable and often significant levels of RNA from 80S ribosomes. Thus values for total RNA cannot be used as a valid estimate for the level of 70S ribosomes associated with these membranes, unless an additional procedure is used to estimate the per cent contamination by 80S ribosomes.Recoveries of digested RNA from the Dowex resin of 94 to 98% were obtained with 2 milliliters of HCl eluant, making possible the analysis of thylakoid samples with as little as 4 micrograms of RNA. The procedure involves small columns and only one centrifugation, so that it is useful for obtaining reliable measurements from multiple samples.