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Abstract: In chromatography and electrophoresis S/N is increased by time or frequency domain filtering, multiple injections or sample stacking (in case of CZE). Bottom line is noise reduction without peak distortion. Isotachophoresis (ITP) using conductivity detection is also relevant because of its entirely different information content and the importance in CZE stacking. Several time- and frequency domain filters (Fourier & Walsh transform) were compared. Results were: [•] Time domain filters can be equally applied to either integral or differential signal; [•] Fourier and Walsh transforms are likewise suitable, whereas Walsh better preserves sharp boundaries; [•] Disturbance by detector transfer function can only be partly un-done with de-convolution; conditions are critical. [Copyright &y& Elsevier]

Pentocin 31-1, an anti-Listeria bacteriocin produced by Lactobacillus pentosus 31-1 from the traditional Chinese fermented Xuan-Wei ham, was successfully purified by the pH-mediated cell adsorption-desorption method and then purified by gel chromatography with Sephadex G-10. The purification resulted in a 1,381.9-fold increase in specific activity with a yield of 76.8% of the original activity. Using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular mass of the purified peptide was found to be between 3,500 and 6,400 Da, and bacteriocin activity was confirmed by overlayer techniques. When subjected to mass spectrometry analysis, the protein was highly pure and its molecular mass was 5,592.225 Da. The partial N-terminal sequence of pentocin 31-1 was the following: NH2-VIADYGNGVRXATLL. Compared with the sequence of other bacteriocins, pentocin 31-1 has the consensus sequence YGNGV in its N-terminal region, and therefore it belongs to the class Ha of bacteriocins. [ABSTRACT FROM AUTHOR]

Abstract: The removal efficiency of algae and microcystins (MCs) of a pilot setup based on biological degradation of enrichment microbes by artificial media was studied. The results showed that when chlorophyll-a in Lake Taihu was 3.76–266.1μgL−1, 62.8% of chlorophyll-a could be removed within a hydraulic retention time of 6–7 days. High-performance liquid chromatography (HPLC) analysis for the detection of MCs was applied. When total microcystins RR and LR (TMC-RR and TMC-LR) and extracellular microcystins RR and LR (EMC-RR and EMC-LR) were 0.23–8.93, 0.14–4.73, 0.12–1.15, 0.02–0.17μgL−1, respectively, and in source water, the average removal efficiencies of TMC-RR, TMC-LR, EMC-RR, EMC-LR were 67.0%, 40.5%, 40.0% and 66.0%, respectively. The polymerase chain reaction (PCR) electrophoresis chart revealed that there was a large amount of algae-lysing bacteria such as Pseudomonas spp. and Bacillus spp. on the artificial medium. The protozoa number in the assembled medium was higher than in lake water. Enrichment microbes on the artificial medium could effectively degrade algae and microcystins in Lake Taihu. [Copyright &y& Elsevier]

αS1-Casein was isolated from Haflinger mare's milk by hydrophobic interaction chromatography and displayed great micro-heterogeneity by 2-dimensional electrophoresis, probably because of a variable degree of phosphorylation and alternative splicing events. The aim of the present work was to investigate the complexity of the mare's αS1-casein. The different isoforms present in milk were submitted to a double treatment of dephosphorylation, first by using alkaline phosphatase and then acid phosphatase to achieve complete dephosphorylation. The apoforms were then analyzed by electrospray ionization mass spectrometry. The results revealed the existence of a full-length protein and 7 variants resulting from posttranscriptional modifications; that is, exon skipping involving exon 7, exon 14, or both and use of a cryptic splice site encoding a glutamine residue. The determination of the different phosphorylation degrees of the native isoforms of αS1-casein was finally achieved by electrospray ionization mass spectrometry analysis after fractionation of the isoforms by ion-exchange chromatography. Thus, 36 different variants of equine αS1-casein were identified with several phosphate groups ranging from 2 to 6 or 8 depending on whether exon 7 was skipped. [ABSTRACT FROM AUTHOR]

Because of variable degrees of phosphorylation, alternative splicing, and probable instability resulting from nonenzymatic deamidation, equine β-casein presents a complex pattern by 2-dimensional electrophoresis that needs clarification. β-Casein prepared from Haflinger mare's milk by hydrophobic interaction chromatography was fractionated by ion-exchange chromatography according to the degree of phosphorylation. Isoforms were identified by mass spectrometry; they corresponded to the full-length protein having 3 to 7 phosphate groups and to the splicing variant involving exon 5 and containing 4 to 7 phosphate groups. Investigations of nonenzymatic deamidation showed that β-casein did not deamidate spontaneously in stored milk and during the different steps of chromatography, but deamidation could occur when 2-dimensional electrophoresis was performed, increasing the β-casein pattern complexity. This phenomenon was strongly minimized when the first dimension step was carried out at 10°C instead of at room temperature. Finally, spot attribution on 2-dimensional pattern of β-casein was achieved by mixing each phosphorylation isoform in its native state with the whole β-casein fraction. [ABSTRACT FROM AUTHOR]

Abstract: Electrophoresis in a concentrated suspension of charged porous spheres is investigated theoretically, taking into account of the polarization effect of double layer in particular. The double layer thickness and fixed charge density of the porous spheres are arbitrary and double layer overlapping is considered as well. A pseudo-spectral method based on Chebyshev polynomial is used to solve the resulted general electrokinetic equations. Local extrema are observed in the mobility profile as the double layer thickness varies, which is absent in previous theoretical studies neglecting polarization effect but agrees well with the experimental observations in the literature, indicating the importance of polarization effect in correctly interpreting the electrophoretic experimental results. A simple mutual transform of mobility predictions between Levine–Neale and Shilov–Zharkikh boundary conditions is established and confirmed by direct calculations, which facilitates the dialogue between theoretical and experimental researches. In general, the higher the fixed charge density, the more significant the polarization effect is. [Copyright &y& Elsevier]

Abstract: A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase. [Copyright &y& Elsevier]

Objectives: (1) To investigate the effects of exercise and osteochondral (OC) injury on synovial fluid (SF) chondroitin sulfate (CS) and hyaluronan (HA) concentration and chain length, (2) to compare SF and cartilage CS data from joints with OC fragmentation, and (3) to compare SF CS and HA profiles with those seen in serum from the same horses.Methods: Serum and SF were obtained from (1) normal horses after 8 weeks rest, (2) the same horses after 9 months treadmill training, and (3) horses with OC injury from racing. Articular cartilage was also collected from group 3 horses. Concentrations and chain lengths of CS and HA were determined by gel chromatography and fluorophore-assisted carbohydrate electrophoresis.Results: SF CS peak chain length in the OC injury group increased significantly (18.7kDa) when compared to rested horses (11.6kDa), with exercise producing an intermediate chain length (15.6kDa). Cartilage and serum from the OC injury group had the abnormally long CS chains seen in SF from these horses. Total SF HA was significantly lower in the OC injury group compared to the rested group. Both the OC injury group and the exercised group had significant decreases in SF HA chain length compared to the rested group.Conclusions: Chain length of SF CS was increased by exercise and OC injury. Exercise resulted in a modest increase, whereas OC injury caused a marked increase. In contrast to CS, SF HA chain length was decreased by OC injury, and to a lesser extent by exercise. Chain length analysis of SF CS and HA may provide a useful tool for evaluation of joint health. [ABSTRACT FROM AUTHOR]

Abstract: Humic acids (HAs) extracted from soils of different origins were fractionated using size exclusion chromatography-polyacrylamide gel electrophoresis set-up. For each HAs, three fractions showing distinct electrophoretic mobilities and molecular sizes (MS) were obtained. Bulk HAs and their fractions (25mgL−1) were then compared for their ability to photosensitize the degradation of 2,4,6-trimethylphenol upon irradiation at 365nm. Rate coefficients were deduced from pseudo-first order kinetics. For five of the six series, the rate coefficient varied in the order: high MS

The objective of this study was to develop an economical, simple, and large-scale separation method for IgY from egg yolk. Egg yolk diluted with 9 volumes of cold water was centrifuged after adjusting the pH to 5.0. The supernatant was added with 0.01% charcoal or 0.01% carrageenan and centrifuged at 2,800 × g for 30 min. The supernatant was filtered through a Whatman no. 1 filter paper and then the filtrate was concentrated to 20% original volume using ultrafiltration. The concentrated solution was further purified using either cation exchange chromatography or ammonium sulfate precipitation. For the cation exchange chromatography method, the concentrated sample was loaded onto a column equilibrated with 20 mM citrate-phosphate buffer at pH 4.8 and eluted with 200 mM citrate-phosphate buffer at pH 6.4. For the ammonium sulfate precipitation method, the concentrated sample was twice precipitated with 40% ammonium sulfate solution at pH 9.0. The yield and purity of IgY were determined by ELISA and electrophoresis. The yield of IgY from the cation exchange chromatography method was 30 to 40%, whereas that of the ammonium sulfate precipitation was 70 to 80%. The purity of IgY from the ammonium sulfate method was higher than that of the cation exchange chromatography. The cation exchange chromatography could handle only a small amount of samples, whereas the ammonium sulfate precipitation could handle a large volume of samples. This suggests that ammonium sulfate precipitation was a more efficient and useful purification method than cation exchange chromatography for the large-scale preparation of IgY from egg yolk. [ABSTRACT FROM AUTHOR]

Objective: Loss of articular cartilage and intervertebral disc function in arthritis or disc degeneration is associated with degradation of the proteoglycan (PG) aggregates by either proteolysis of aggrecan or hyaluronan (HA) degradation. The aim of this work was to determine whether degradation of HA in PG aggregate degradation is influenced by link protein (LP) stabilization of the PG aggregates.Methods: Aggrecan and LP were prepared from fetal bovine epiphyseal cartilage, and PG aggregates were formed in the presence or absence of LP. The PG aggregates were exposed to hyaluronidase or free radicals to promote HA degradation. Degradation of HA, aggrecan and LP were assessed by gel filtration chromatography and polyacrylamide gel electrophoresis.Results: High concentrations of hyaluronidase cleaved both PG aggregates between each aggrecan molecule, whereas low concentrations gave much less cleavage of the LP-stabilized aggregate. High free radical concentrations gave extensive cleavage of all components of both PG aggregates, whereas low concentrations are more selective for HA damage and to a much lesser extent in the LP-stabilized aggregates. Thus the presence of LP caused a diminution in the capacity of both catabolic agents to degrade HA as long as levels of the degradative agents were not excessive.Conclusion: In addition to stabilizing the PG aggregates towards dissociation, LP may also help protect the PG aggregates from degradation under conditions where tissue catabolism is promoted. [ABSTRACT FROM AUTHOR]

Abstract: Several approaches for proteome analysis and the generation of proteome subsets rely on engineered chemical probes that are tailored towards the detection of different protein classes. The concepts are presented in this review covering the literature until mid-2005. [Copyright &y& Elsevier]

Abstract: Bioseparation techniques exploit the differences of physicochemical or thermodynamic properties between the product and the contaminants. Rapid development of a downstream process, therefore, requires physicochemical and thermodynamic characterization of the components to be separated. In this paper, we investigate whether a generalized thermodynamic interrelation exists among different parameters. For instance activity coefficients, osmotic virial coefficients and the solubility of macromolecules are interrelated to each other. Experimental determination of any one of these parameters can be translated across the boundaries of different separation techniques. A number of downstream separation processes, including size-exclusion chromatography, hydrophobic-interaction chromatography, reversed-phase chromatography, aqueous two-phase separation, crystallization and precipitation, are found to be explained and designed using this generalized thermodynamics. This generalization of thermodynamic properties together with high-throughput experimentation provides a systematic and high-speed approach to bioseparation process development and optimization. The applicability of this approach for bioseparation process design was investigated by a case study on nystatin, a medium-sized biomolecule. The distribution coefficients of nystatin in reversed-phase chromatography showed straightforward relationship with the solubilities at various solvent compositions and the experimental data supported the trend of the relationship. [Copyright &y& Elsevier]

Abstract: Affinity chromatography represents a promising technique for decoding the proteomics universe. While conventional affinity purification is being used in conjunction with two-dimensional electrophoresis (2D-PAGE) and mass spectrometry (MS) for the study of proteomes and subproteomes, scientists are still confronted with the need for specific and tailor-made affinity ligands to target desired groups and families of proteins. Evidence has shown that, in many situations, synthetic affinity ligands can circumvent inconveniences associated with the utilisation of biological ligands for the chromatography-based purification of biomolecules. This review will highlight the potential applications of affinity chromatography and synthetic de novo designed ligands as separation tools for proteomics. [Copyright &y& Elsevier]

Abstract: Four cytosolic glutathione S-transferase (GST) classes were isolated and characterized from juvenile winter run Chinook salmon (Oncorhynchus tshawytscha) liver. Two techniques were used: (1) gel electrophoresis/immunoblotting against a polyclonal striped bass GST antibody and (2) high-pressure liquid chromatography (HPLC). Nanospray liquid chromatography–tandem mass spectrometry (LC–MS/MS) was used to elucidate peptide sequences and the proteins were identified as π, θ, μ and α, by searching against the NCBI non-redundant database (nrDB). Catalytic activity of the cytosolic GSTs towards 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETHA) were determined to be 0.3±0.05U/mg cytosolic protein and 0.06±0.02U/mg cytosolic protein, respectively. [Copyright &y& Elsevier]

Abstract: Purified Trematomus bernacchii bile IgM analysed by SDS-PAGE under reducing and non-reducing conditions consisted essentially of tetramers of the basic structure H2L2. The relative molecular mass of the glycosilated H chain was 76kDa, while that of L chain was 25kDa. In addition, the presence in the liver of IgM and μ chain-specific mRNA was demonstrated. Immunohistochemistry detected IgH- and IgL-reactivity in perisinusoidal cells, bile canaliculi and pre-ductules. In the anterior intestine, the intraluminal mucus retained a significant Ig-immunoreactivity, while the mucosa housed a limited density of Ig-producing cells. These findings strongly indicate that Ig could be transported across the hepatocytes to be secreted into the bile and protect the intestinal epithelium. In addition, extravasated plasma cells accumulated within liver portal tracts and close to the capsule that, in turn, was evenly coated by Ig molecules at the peritoneal surface. [Copyright &y& Elsevier]

The aim of the present study was to assess the antiatherogenic properties of Pinus pinaster (maritime pine) seed oil. To this end, the effects of P. pinaster oil supplementation on lipoprotein levels and atherosclerotic lesions were compared to those of lard or sunflower oil in apolipoprotein E-deficient mice. Plasma total cholesterol (P < 0.0001) and VLDL + intermediary density lipoprotein (IDL)-cholesterol (P < 0.0001) levels were lower in mice fed P. pinaster and sunflower oil than in those fed the lard diet. In contrast, triglycerides (P < 0.0001) and VLDL + IDL-triglycerides (P < 0.0001) levels were higher in mice fed P. pinaster oil than sunflower oil or lard. The VLDL + IDL lipid composition of apolipoprotein E-deficient mice fed P. pinaster oil was intermediate between that of lard-fed transgenic mice and that of wild-type mice fed nonpurified diet. Using the Triton WR1339 method, the fractional catabolic rate of plasma triglycerides was found to be lower in mice fed P. pinaster oil (P < 0.0001) than sunflower oil or lard diet, suggesting a defective clearance of triglycerides in the P. pinaster group. Finally, the susceptibility of triglyceride-rich lipoproteins to in vitro lipoprotein lipase-mediated lipolysis was lower in the P. pinaster oil-fed group than in the lard-fed group. Despite the differences in VLDL + IDL level and lipid composition, the surface areas of aortic atherosclerotic lesions were not significantly different among mice fed P. pinaster, sunflower or lard diets. In conclusion, the results of the present study indicated that feeding P. pinaster oil had no better preventive effect on aortic atherosclerotic lesion extension in apolipoprotein E-deficient mice than other saturated or polyunsaturated fats. [ABSTRACT FROM AUTHOR]

The proteoglycans (PGs) in pooled normal scars and pooled hypertrophic scars were extracted with 4 M guanidinium chloride and isolated by DEAE-cellulose chromatography. The PG samples were then fractionated further by dissociative CsCl density gradient sedimentation. Following cleavage of the density gradient PG fractions with alkaline NaB3H4, the glycosaminoglycan (GAG) constituents were isolated and analyzed by quantitative cellulose acetate electrophoresis. In addition, single samples of normal skin and a keloid scar were also analyzed. The results showed that the hypertrophic scars had a higher average content of extractable and also residual PGs than did the normal scars but a wide range of values was obtained for each type of scar. Some differences were noted in the amounts and distribution of the GAGs in CsCl gradient fractions, in the different types of scar tissue. The PGs in tissues were distributed in low-, medium-, and high density fractions and are, therefore, heterogeneous. Dermatan sulfate (DS) was the major GAG in each tissue and smaller quantities of chondroitin sulfate (CS), heparan sulfate (HS), and heparin (HP) were also present. In addition, 2 other GAG constituents were also detected. Based on the susceptibility of these GAGS to enzymes and nitrous acid treatments, one was a HS or HP while the second was a DS. The major differences in the PG composition of the scar tissues were the higher proportions of low-density CS-PGs in the keloid scar and of low density DS-PGs in hypertrophic and keloid scars. [ABSTRACT FROM AUTHOR]

An aminoendopeptidase isolated from 2-day-old rat epidermis was purified to apparent homogeneity by the procedures of ammonium sulfate fractionation, DE-52 column chromatography, Sephadex G-200 gel filtration, and CM-52 and DEAE-Sepharose 6B column chromatography. Enzymatic activity was exhibited only in the presence of sulfhydryl compounds and further enhanced by addition of 5 mM EDTA. It was inhibited by p-chloromercuribenzoate, other sulfhydryl blocking reagents, and o-phenanthroline. The monomer form of the enzyme is Mr = 52,000 ± 2,300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, but a native form was considered to be Mr = 400,000 ± 26,000 having an isoelectric point of pH 5.25. Among synthetic substrates the enzyme hydrolyzed amino acid 2-naphthylamide derivatives and L-leucine amine (L- LeuNH2) most effectively. N-α-benzoyl-DL-arginine-2-naphthylamide (BANA) was the only endopeptidase substrate for the enzyme and a competitive inhibitor for its aminopeptidase activity. Protein substrates have not yet been found. The pH optimum is 7.5 and in a range of pH 6.5-7.5 it is stable at 37°C for 30 min but loses about 50% of its activity at 50°C. [ABSTRACT FROM AUTHOR]

Candida Albicans was able to produce a keratinolytic proteinase (KPase) when cultivated in a medium containing human stratum corneum as a nitrogen source. The KPase was purified to 108.5-fold by ion-exchange chromatography and gel filtration. The molecular weight of the enzyme was estimated to be 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration through Sephacryl S-200, while the isoelectric point was determined to be at pH 4.5. The enzyme had an optimum pH of 4.0 and was "inactive" below pH 2.5 and above pH 6.0. The activity of KPase after preincubation at various temperatures was stable up to 50°C. The keratinolytic activity was not affected by the addition of nonionic detergents and divalent cations. The enzyme was a glycoprotein and contained a high content of aspartic acid residues (172/1000) Pepstatin and chymostatin inhibited the activity in a dose-dependent manner; however, neither the other group specific inhibitors tested nor the pepsin specific inhibitors, DAN or EPNP, showed any effect on the enzyme. From these inhibitory profiles, this enzyme was determined to be a carboxyl proteinase such as cathepsin D. Among the various substrates for proteolytic enzymes, KPase digested human stratum corneum as much as albumin and hemoglobin. In the three fractions (water soluble, keratin filamentous, and membranous) prepared from human stratum corneum, the keratin filamentous fraction was more susceptible to degradation by KPase than the other two fractions were. KPase also digested much less human fingernail (13%) than human stratum corneum, but did not show any signs of there being any digestion of human scalp hair. These studies suggest that KPase from C. albicans may play an important role in superficial infection by affecting the human stratum corneum of the skin and nail. [ABSTRACT FROM AUTHOR]

A new papain inhibitor was purified from psoriatic epidermal scales using gel chromatography and anion exchange chromatography. The purified protein inhibited papain and ficin but not cathepsin B, cathepsin H, trypsin, or chymotrypsin. Isoelectric focusing revealed 3 major inhibitor variants with pI's of 7.3, 6.9, and 6.5. A Mr of 38,000 was obtained by a gel chromatographic method for the crude inhibitor. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr values of the isoelectric variants were: 43,000 for the variant pI 7.3, 43,000 and 35,000 for the variant pI 6.9, and 34,000-35,000 for the variant pI 6.5. An antiserum of the inhibitor was used to locate the inhibitor in the psoriatic and normal epidermis. In psoriatic epidermis, the inhibitor was found in the peripheral cytoplasm of spinous cells and in the scale. In normal epidermis, the staining was seen only in orifices of hair follicles. An inhibitor with similar size and antigenic properties to that isolated from the psoriatic scales was demonstrated in extracts made from the whole-thickness epidermis but not in extracts from the healthy epidermal scales, the dermis, the liver, the spleen, or the blood serum. [ABSTRACT FROM AUTHOR]

The biochemical composition of blister fluid was compared with serum and with blister fluid from erythematous lesions induced by ultraviolet irradiation. By using glass capillary gas chromatography--mass spectrometry over 100 metabolites were determined and with the aid of two-dimensional high resolution electrophoresis (the ISO-DALT system) several hundred protein spots were seen. The results show that the suction blister fluid qualitatively have a serum-like pattern but that the concentration of each compound was smaller than in serum. Also in suction blisters raised on erythematous reactions induced by ultraviolet light the same pattern was seen. The content of sodium, potassium and chloride was the same in suction blisters raised on erythematous and normal skin as that of serum. [ABSTRACT FROM AUTHOR]

A kinin-forming-enzyme in human skin extract was further purified by successive column chromatography on DEAE-cellulose, Hydroxylapatite-cellulose and Sepharose-4B. By these procedures, 2.7 mg of purified enzyme was obtained from 10 gin of original skin. The purified material was homogeneous as ascertained by cellulose acetate membrane electrophoresis, sodium dodecyl sulfate polyacrylamide gel disc electrophoresis and ultra-centrifugation. It had an S20,w, value of 4.3 and an apparent molecular weight of 104,000 as measured by gel filtration on Sephadex G-200. The purified enzyme was comparatively heat-stable, but was unstable below pH values of 5 and above pH 9. It possessed arginine or lysine esterolytic activity, but not tyrosine or tryptophane esterolytic activity and denatured proteolytic activity. This enzyme was not affected by metal ion, cystein, glutathion or ρ-chloromercuribenzoate, but was strongly inhibited by α-N-ρ-tosyl-L-lysine chioromethyl ketone or soybean-trypsin inhibitor. It was also inhibited by &alph;1-antitrypsin, but not by α2-macroglobulin. This enzyme was confirmed to be immunologically distinct from human plasma, urinary or pancreas kallikrein. [ABSTRACT FROM AUTHOR]

Tyrosinase was dissolved from the melanosome fraction of Harding-Passey mouse melanoma and purified further by Sephadex G-100 gel filtration and DEAE-cellulose column chromatography. The specific activity of the purified preparation was 763 times as high as that of the original melanosome. Tyrosinase thus isolated showed only a single activity in both the electrophoresis of a polyacrylamide gel and a cellulose acetate. The Km value is the same as those of the original melanosome and the smooth-surfaced-membrane. The relationship between particle bound tyrosinase and solubilized tyrosinase is discussed in this paper. [ABSTRACT FROM AUTHOR]

Objective: To determine whether dimethylmethylene blue (DMMB) analysis, when combined with agarose gel filtration chromatography (Superose 6), can be performed instead of fluorophore-assisted carbohydrate electrophoresis (FACE) to determine chondroitin sulfate (CS) chain length in synovial fluid (SF).Methods: SF was obtained from (1) normal horses after 8 weeks of rest, (2) the same horses after 9 months of treadmill training, and (3) horses with osteochondral (OC) injury from racing. SF CS concentrations and chain lengths were determined by gel chromatography and DMMB analysis and compared with previous results determined by FACE analysis on the same samples.Results: DMMB analysis showed that SF CS peak chain length in the OC injury group increased significantly (18.7 kDa) when compared to rested and exercised normal horses (15.6 kDa). The assay had a positive predictive value of 71% and a negative predictive value of 75% for discriminating between normal and injured joints.Conclusions: We report a simple and inexpensive DMMB analysis of SF CS chain length, which, when coupled with Superose 6 chromatography, discriminates between normal and post-injury joints. Similar to our previous FACE analysis results [Brown MP, Trumble TN, Plaas AHK, Sandy JD, Romano M, Hernandez J, et-al. Exercise and injury increase chondroitin sulfate chain length and decrease hyaluronan chain length in synovial fluid. Osteoarthritis Cartilage 2007;15], our DMMB results show an increase in the chain length of the CS in the SF of injured joints. [ABSTRACT FROM AUTHOR]