Rapid fractionation of collagen chains and peptides by high-performance liquid chromatography

A strategy was developed, using a Pharmacia Fast Protein Liquid chromatography (FPLC) system, for the rapid preparation of the alpha-chains, cyanogen bromide peptides and tryptic peptides of type I collagen obtained from tissues and cultured fibroblasts. Collagen alpha-chains were prepared using a C18 PEP-RPC reverse-phase column and volatile solvents. Preliminary Superose 6 gel permeation chromatography was used to separate the crosslinked beta- and gamma-chains from the alpha-chains of tissue collagen samples. A Mono S cation-exchange column was used to resolve all of the major type I collagen cyanogen bromide peptides including the alpha 1(I)CB7 and CB8 peptides, which have not been well resolved by previously published methods. Collagen tryptic peptides were chromatographed on the PEP-RPC reverse-phase column.     

The effect of some antiinflammatory drugs on collagen of rat fibrous cartilage

The use of SDS-polyacrylamide gel electrophoresis of the cyanogen bromide derived peptides from fibrous cartilage collagens enabled to calculate type I to type II collagen ratio in this tissue. Some of the investigated drugs (acetylsalicylic acid, colchicine) changed this ratio without having a significant effect on total collagen content in fibrous cartilage.     

A simplified method for quantitation of the relative amounts of type I and type III collagen in small tissue samples

A method is described for the quantitation of the relative amounts of types I and III collagens in rabbit lung tissue. This involved (i) repeated homogenization in the presence of 2% sodium dodecyl sulfate and the production of an acetone dried powder, (ii) reaction with cyanogen bromide, (iii) polyacrylamide gel electrophoresis, and (iv) densitometric scanning of proteins stained by Coomassie blue R-250. Several features of this procedure were shown to offer advantages over methods previously employed. First, the sodium dodecyl sulfate solution was shown to remove the bulk of noncollagen proteins leaving an insoluble residue which could then be reacted with cyanogen bromide without further purification. Second, cyanogen bromide was shown to solubilize essentially all of the collagen in the residue leaving an insoluble pellet with an amino acid analysis similar to elastin. Finally, to facilitate accurate quantitation, types I and III collagen standards were included with each gel so that a standard curve of protein versus staining density could be constructed. This method is assessed to be simpler and more accurate than those employed previously for the quantitation of collagens and can be applied to small tissue samples (<100 mg) such as would be obtained by lung biopsy.     

Optimization and scale up of the adsorption fractionation of cod pyloric caeca deoxyribonuclease using axial and radial flow columns.

The key step in the purification of a deoxyribonuclease (DNase) from extracts of cod (Gadus morhua L.) pyloric caeca, is the selective retention of the enzyme by anion exchange chromatography. The cod DNase purification on Q-Sepharose Fast Flow (Pharmacia) was optimized, using a 60 ml fixed-bed column. In combination with titration curve analysis, we have screened the effect of buffer pHs, feed conductivity and protein loading, on the product recovery and purity. We have developed elution conditions which allow effective separation of the cod DNase from bounded impurities, such as proteinases and nucleic acids. Low levels of these impurities were regarded as essential for the desired product quality. The optimum resolution and maximum purification (ca. 20-fold increase in specific activity) of DNase, was, however, achieved at low protein loading (2.6 mg ml-1 gel), corresponding to less than 4% of the dynamic bed capacity. Scale-up to a 2.5 l pilot scale column (axial flow) and a 0.25 l radial flow column showed that the separation and yield obtained at laboratory scale was retained, and was independent of column geometry and bed height. The implications for a production scale scenario of 100 g of fractionated protein, are also discussed, as well as process hygiene. The optimization described herein adds further knowledge to the treatment of fish waste and the downstream processing of valuable biochemicals from marine raw material.     

The distribution of different molecular species of collagen in fibrous, elastic and hyaline cartilages of the pig.

The distribution of type II collagen, considered to be characteristic of cartilaginous tissues, was determined in various specialized cartilages of the mature pig. The tissues examined were: (1) fibrocartilage of the semilunar meniscus of the knee; (2) elastic cartilage of the external ear; (3) hyaline cartilage of (a) the synovial joint (b) the thyroid plate of the larynx, and (c) the nasal septum. The predominant species of collagen in each tissue, whether type I or type II, was appraised semi-quantitatively by analysis of purified collagen solubilized by pepsin and of peptide fragments produced by cyanogen bromide. Cyanogen bromide-derived peptides were characterized by column chromatography on CM-cellulose and by electrophoresis in sodium dodecyl sulphate-polyacrylamide gels. The proportion of each type of collagen was determined precisely by isolating the homologous small peptides alpha1(II)CB6 [nomenclature of Miller (1973) Clin. Orthop. 92, 260-280], by column chromatography on phosphocellulose and determining their relative proportions by amino acid analysis. Thus collagen of the fibrocartilage of the meniscus proved to be all type I; type II was not detected. In contrast, collagen of elastic cartilage of the outer ear, after rigorous exclusion of perichondrium, was type II. Similarly, type II was the only collagen detected in all the mature hyalline cartilages examined.     

Separation of β22 dimer from bovine bone collagen

The precise role of the α₂-chain in collagen type I is of considerable scientific interest. Our recent studies demonstrated that the most noticeable difference between type I collagens, which were obtained from bovine hard tissues (bone, dentine) and soft tissues (tendon, skin), was presented in the position of β chain dimers using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The additional band observed both in the bone and dentine collagen was putatively identified as β₂₂ dimer (made of by an intermolecular cross-linking between two α₂-chains). Further investigations carried out on bovine bone and skin collagen, corresponding to hard tissue and soft tissue collagen respectively, confirmed this hypothesis. Successful separation of individual β₂₂ dimer from bone collagen was achieved. The procedure involves molecular-sieve chromatography on a Sephacryl S-400 column followed by differential acetone precipitation. Identification was done by the widely used methods, such as SDS-PAGE and cyanogen bromide (CNBr)-cleaved peptide analysis. It was proposed that the dimer and consequently α₂-chains may play important roles in the morphological and biological differences between hard and soft tissues.     

Purification, stabilization, and characterization of rat hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was purified to near homogeneity from heparin-containing rat liver perfusates with the following column chromatography steps: heparin-Sepharose affinity chromatography, anion-exchange chromatography on DEAE-Sephacel, and gel filtration on Ultrogel AcA 34. A final specific activity of 45,000 μmol fatty acid/mg/h was obtained with an overall 31% recovery of catalytic activity. The heparin-Sepharose step resulted in a 20-fold purification, while the DEAE and gel filtration steps led to further purification with complete recovery of activity. An extensive survey of various detergents as potential stabilizers of H-TGL activity led to the selection of Triton N-101 for use in the column buffers of the DEAE and gel filtration steps. Relative to initial H-TGL activity upon dilution in buffer without detergent, recoveries between 90 and 100% were consistently obtained with Triton N-101-containing buffers following a 24-h incubation at 20°C. In contrast after a 24-h incubation at 20°C those control samples lacking detergent were at least 95% inactivated. The highly purified H-TGL exhibited a single major band by sodium dodecyl sulfate-electrophoresis. The use of DEAE chromatography and stabilization of H-TGL with Triton N-101 are the improvements in purification that resulted in an 8-fold enhancement in specific activity relative to the highest previous report of purification from rat liver perfusates.     

A method for quantitative analysis of ratios of types I and II collagen in small samples of articular cartilage

Currently available methods for quantitative analysis of type II collagen in studies of articular cartilage repair either require much larger samples than are available or are inaccurate and unreliable. A method of determining the percentage of type II collagen in small samples of articular cartilage (100 to 200 micrograms) by measuring the spectrophotometric densities of specific cyanogen bromide peptide bands from mixtures of types I and II collagen on sodium dodecyl sulfate-polyacrylamide gels has been developed and found to be accurate and very reliable. The ratio of the area under the alpha 1(II)CB10 peak to the area under the alpha 1(I)CB7,8 + alpha 1(II)CB11 peak was function of the proportion of type II collagen in the sample. Since the ratio was independent of the quantity of sample loaded onto the gel, it was not affected by moderate losses of sample. This method should therefore be useful in the fields of collagen research and particularly valuable to those investigating the repair and regeneration of articular cartilage.     

Purification of phospholamban from bovine cardiac muscle with organic solvents

Phospholamban (PLB), an integral membrane protein of cardiac sarcoplasmic reticulum, was extracted from bovine cardiac muscle with an acidic chloroform/methanol mixture. A combination of gel permeation and ion-exchange chromatographies in organic solvents allowed the purification of PLB. The intensive use of organic solvents throughout the isolation yielded a highly purified and intact protein that can be phosphorylated by cAMP protein kinase. The ease of purification and the high yield obtained (2.5 mg/100 g of fresh tissue) show that organic solvents can be very useful in the extraction and purification of hydrophobic membrane proteins.     

Evaluation and Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.     

Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples.

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.     

Purification and measurement of calpromotin, the cytoplasmic protein which activates calcium-dependent potassium transport

A simple procedure is described for the purification of calpromotin, a protein from the cytoplasm of red blood cells which is capable of activating calcium-dependent potassium transport. The purification steps involve a salt gradient elution from an anion exchange column (Whatman DE-52) followed by a potassium phosphate gradient elution from a column of hydroxyapatite (HA Ultrogel). These steps result in a 54% yield with a 161 fold purification. The calpromotin is estimated to be 99% pure as determined by densitometry of the protein profile on an SDS polyacrylamide gel. A competitive enzyme-linked immunosorbent assay (ELISA) using rabbit anti-human calpromotin antibodies, is described for measuring levels of calpromotin in the 5 to 100 ng range.     

The staining of type V collagen obtained from FBJ virus-induced osteosarcoma with concanavalin A

Type V collagen from FBJ virus-induced osteosarcoma of mice has a high content of saccharide as has been noted for type V collagens from different sources. In the present study, this collagen was found to contain significant amounts of mannose and hexosamine. Three alpha chains of this collagen were electrophoretically separated and cleaved by cyanogen bromide. The cyanogen bromide peptides, following their separation by SDS-polyacrylamide gel electrophoresis, were transferred onto nitrocellulose paper and stained with concanavalin A. Several bands derived only from alpha 3(V) stained positively, but this was inhibited by the presence of alpha-methylmannoside. Thus, at least one of these three chains appears to have an asparagine-linked oligosaccharide.     

Purification of recombinant brain derived neurotrophic factor using reversed phase displacement chromatography

This work investigates the utility of RPLC displacement chromatography for the purification of recombinant brain derived neurotrophic factor (rHu-BDNF) from its variants and E. coli. protein (ECP) impurities. The closely associated variants (six in total) differ by one amino acid from the native BDNF and thus pose a challenging separation problem. Several operational parameters were investigated to study their effects on the yield of the displacement process. The results indicated that the concentration of trifluoroacetic acid (TFA) in the buffer was a key factor in achieving the desired purification. Displacement chromatography on an analytical scale column resulted in extremely high purity and yield in a single chromatographic step. The process was successfully scaled-up with respect to particle and column diameter. The production rate of a pilot scale RPLC displacement process was shown to be 23 times higher than the combined production rates of the current preparative ion exchange and hydrophobic interaction gradient elution steps that are used to remove variant and ECP impurities, respectively.     

Analysis of histidine-containing dipeptides, polyamines, and related amino acids by high-performance liquid chromatography: application to guinea pig brain.

A procedure is described for coupling wheat germ agglutinin to cyanogen bromide-activated Sepharose to yield a lectin affinity column of high capacity. Covalent linkage of the lectin to the insoluble matrix is carried out in the presence of a mixture of β-1,4-linked N-acetylglucosamine oligosaccharides prepared from chitin. The lectin-affinity column specifically recognizes glycoproteins containing N-acetylglucosamine residues with the capacity of binding 0.6–1.0 mg of ovomucoid per milliliter of gel. The affinity column is stable (as determined by ovomucoid binding) and shows little loss in binding capacity or specificity after repeated usage. Important characteristics for the use of this column to purify glycoproteins are described.     

A three-step method for isolating a few to several hundred milligrams of protein

An operationally simple general protein isolation method was devised from three previously available separation tools, and was tested by application to two demanding fractionation problems and for yield. One test system was the isolation by gel electrofocusing of two model proteins with pI values of 4.6 and 4.8, bovine serum albumin and ovalbumin, with a load of 220 mg each. The other test was the isolation of 10 mg of human growth hormone isohormone B from a mixture of closely migrating other isohormones. The three-step procedure comprises of: (1) separation into zones of homogeneous protein by gel electrofocusing; (2) excision of the zones of homogeneous protein from the gel followed by concentration of the protein to a small volume of solution by means of Steady-State Stacking; (3) purification from polyacrylamide-like contaminants and non-volatile buffers by gel filtration followed by lyophilization. The average overall recovery was 70--80%.     

Increased yield of high purity recombinant human interferon-gamma utilizing reversed phase column chromatography

Increasing therapeutic applications for recombinant human interferon-gamma (rhIFN-gamma), an antiviral proinflammatory cytokine, has broadened interest in optimizing methods for its production and purification. We describe a reversed phase chromatography (RPC) procedure using Source-30 matrix in the purification of rhIFN-gamma from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-gamma monomer from the RPC column is refolded in Tris buffer. Optimal refolding occurs at protein concentrations between 50 and 100 microg/ml. This method yields greater than 90% of the dimer form with a yield of 40 mg/g cell mass. Greater than 99% purity is achieved with further purification over a Superdex G-75 column to obtain specific activities of from 2 x 10(7) to 4 x 10(7)IU/mg protein as determined via cytopathic antiviral assay. The improved yield of rhIFN-gamma in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.     

Characterization of two possible forms of type IV collagen from human kidney cortex

Type IV collagen was isolated from human kidney cortex according to the purification of mouse kidney type IV collagen (MKIVC) with minor modifications as described previously [Oikawa, T., et al. (1986) Chem. Pharm. Bull. 34, 789-797]. Chromatography of human kidney type IV collagen (HKIVC) on a column of DEAE-cellulose resulted in its separation into two fractions, i.e., one (HKIVC-1) passed through and the other (HKIVC-2) adsorbed to the column, similar to MKIVC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Bio-Gel A-5m chromatography revealed that both HKIVC-1 and -2 differed from each other in the ratios of major and minor components. Amino acid analyses also demonstrated that there was a significant difference in the contents of several amino acid residues between both type IV collagens and their 80-kDa components, one of the major ones. These results indicate the possibility that there exist two forms of type IV collagen in human kidney.     

Quantitation of Type I to type III collagen ratios in small samples of human tendon,blood vessels,and atherosclerotic plaque

A method is described whereby the ratio of the major interstitial collagens (Types I and III) can be measured in biopsy specimens of human tissue weighing as little as 25 mg. Marker peptides are solubilized from the tissue by digestion with cyanogen bromide. These peptides which are not known to be involved in collagen crosslinking are isolated and quantified by a combination of carboxymethyl-cellulose chromatography and polyacrylamide gel electrophoresis. The peptides used are α1(I)-CB7 and α1(III)-CB5. The use of the method is illustrated by analyzing the collagen type ratio in small specimens of tendon, aorta, and vena cava.     

Isolation and characterization of insoluble collagen of dog hearts.

A procedure for isolating insoluble heart collagen has been developed. The method involves the use of defined optimal conditions of sonication that yield no thermal denaturation of the triple-helical structure nor disruption of the primary structure of the collagen molecules; this is followed by extraction of isolates with nondenaturing agents. The amino acid residues of the isolates are then reacted with dansyl chloride to allow determination of amino-terminal residues and quantification of the collagen. The method has several advantages over existing procedures: (i) There is no other method available for isolation of undenatured insoluble heart collagen in almost pure form (consists of 96% of type I collagen) and in a good yield. Sonication of tissue at or below 4 degrees C for a total of 120 s (15 s sonication repeated 8 times at 120-s intervals) yielded insoluble collagen fibers with 90% yield and a 20-fold purification as determined by the increase in Hyp content of the isolates. Extraction of these isolates with 0.6 M KCl and 1 M NaCl at 4 degrees C resulted in a 22-fold purification with 70% yield, while the classical extraction method with nondenaturing reagents yielded only 5-fold purification. (ii) There has been little study of the derivatization of an insoluble protein (collagen) with dansyl chloride. The Lys residues of collagen could be recovered as epsilon-Dns-Lys in 84% yield from a reverse-phase C-18 column by high-performance liquid chromatography. This assay allows measurement of 0.1-100 nmol epsilon-Dns-Lys. (iii) The method generates direct information concerning the quantity of collagen and its nature with respect to amino groups.