Kinetics of Hyal-1 and PH-20 hyaluronidases: comparison of minimal substrates and analysis of the transglycosylation reaction

The availability of recombinant expression systems for the production of purified human hyaluronidases PH-20 and Hyal-1 facilitated the first detailed analysis of the enzymatic reaction products. The human recombinant enzymes, both expressed by Drosophila Schneider-2 (DS-2) cells, were compared to bovine testicular hyaluronidase (BTH), a commercially available hyaluronidase preparation, which has long been considered a prototype of mammalian hyaluronidases. The conversion of low molecular weight hyaluronic acid (HA) fragments was detected by a capillary zone electrophoresis (CZE) method. Surprisingly, the HA hexasaccharide, which is generally accepted to be the minimum substrate of BTH, was not a substrate of recombinant human PH-20 and Hyal-1. However, HA octasaccharide was converted efficiently by both enzymes, thus representing the minimum substrate for human PH-20 and Hyal-1. Additionally, BTH was shown to catabolize the HA hexasaccharide at pH 4.0 mainly by hydrolysis, while at pH 6.0 transglycosylation prevailed. Human PH-20 was found to catalyze both hydrolysis and transglycosylation of the HA octasaccharide. On the contrary, human Hyal-1 converted the HA octasaccharide mainly by hydrolysis with transglycosylation products occurring only at high substrate concentrations (> or = 500 microM). The differences between the hyaluronidase subtypes and isoenzymes were much more prominent than expected. Obviously, the different hyaluronidase subtypes have evolved into very specialized enzymes with respect to their catalytic mechanism of action.     

Syntheses of the 4-nitrophenyl glycosides of hyalobiuronic acid and chondrosine

4-Nitrophenyl [sodium beta-D-glucopyranosyluronate]-(1-->3)-2-acetamido-2-deoxy-beta-D-glucopyranoside (1) and 4-nitrophenyl [sodium beta-D-glucopyranosyluronate]-(1-->3)-2-acetamido-2-deoxy-beta-D-galactopyranoside (2) were prepared from the zwitterions hyalobiuronic acid [beta-D-glucopyranuronic acid-(1-->3)-2-amino-2-deoxy-D-glucopyranose] and chondrosine [beta-D-glucopyranuronic acid-(1-->3)-2-amino-2-deoxy-D-galactopyranose], respectively. Compounds 1 and 2 were not hydrolysed by bovine testicular hyaluronidase.     

Determination of uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection

A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K₃[Fe(CN)₆]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 × 10⁻⁴ M luminol added to the CE running buffer and 1.0 × 10⁻⁴ M K₃[Fe(CN)₆] in 0.2 M NaOH solution introduced postcolumn. The proposed CE-CL assay showed good repeatability (relative standard deviation [RSD] = 3.5%, n = 11) and a detection limit of 3.5 × 10⁻⁷ M UA (signal/noise ratio [S/N] = 3). A linear calibration curve ranging from 6.0 × 10⁻⁷ to 3.0 × 10⁻⁵ M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.     

Chain-length dependence of the kinetics of the hyaluronan hydrolysis catalyzed by bovine testicular hyaluronidase

Hyaluronan (HA) has various biological functions that are strongly dependent on its chain length. In some cases, as in inflammation and angiogenesis, long and short chain-size HA effects are antagonistic. HA hydrolysis catalyzed by hyaluronidase (HAase) is believed to be involved in the control of the balance between longer and shorter HA chains. Our studies of native HA hydrolysis catalyzed by bovine testicular HAase have suggested that the kinetic parameters depend on the chain size. We thus used HA fragments with a molar mass ranging from 8x10(2) g mol(-1) to 2.5x10(5) g mol(-1) and native HA to study the influence of the chain length of HA on the kinetics of its HAase-catalyzed hydrolysis. The initial hydrolysis rate strongly varied with HA chain length. According to the Km and Vm/Km values, the ability of HA chains to form an efficient enzyme-substrate complex is maximum for HA molar masses ranging from 3x10(3) to 2x10(4) g mol(-1). Shorter HA chains seem to be too short to form a stable complex and longer HA chains encounter difficulties in forming a complex, probably because of steric hindrance. The hydrolysis Vm values strongly suggest that as the chain length decreases the HAase increasingly catalyses transglycosylation rather than hydrolysis. Finally, two HA chain populations, corresponding to HA chain molar masses lower and higher than approximately 2x10(4) g mol(-1), are identified and related to the bi-exponential character of the model we have previously proposed to fit the experimental points of the kinetic curves.     

Capillary electrophoretic determination of methotrexate, leucovorin and folic acid in human urine

A simple, rapid and sensitive procedure using capillary zone electrophoresis (CZE) to measure methotrexate, folinic acid and folic acid in human urine has been developed and validated. Optimum separation of methotrexate, folinic acid and folic acid was obtained on a 60 cm x 75 microm capillary using a 15 mM phosphate buffer solution (pH 12.0), temperature and voltage 20 degrees C and 25 kV, respectively and hydrodynamic injection. Under these conditions the analysis takes approximately 9.0 min. Good results were obtained for different aspects including stability of the solutions, linearity, accuracy and precision. Before CZE determination, the urine samples were purified and enriched by means of a solid phase extraction step with a preconditioned C(18) cartridge and eluting the compound with a mixture 1:1 of methanol:water. A linear response over the urine concentration range 1.0-6.0 mgL(-1) for MTX and 0.5-6.0 mgL(-1) for folinic acid and folic acid was observed. Detection limits for the three compound in urine were 0.35 mgL(-1). CZE was shown to be a good method with regard to simplicity, satisfactory precision, and sensitivity.     

Simultaneous determination of aminomethylbenzoic acid,cefminox sodium and etamsylate in human urine by capillary electrophoresis

A sensitive and selective capillary electrophoresis method is developed, for the first time, for effective separation and simultaneous determination of aminomethylbezoic acid (PAMBA), cefminox sodium (CMNX) and etamsylate (ETM). The electrophoresis conditions were investigated and optimized. A 25 mM phosphate solution (pH 8.5) was used as a buffer and the peak area was determined with UV detection at 216 nm wavelength under 18 kV separation voltage. Under optimal conditions, the three drugs can be separated effectively. Good linearity was achieved in 3.13–150 μg/mL for PAMBA, 6.25–150 μg/mL for CMNX and 3.13–150 μg/mL for ETM, with the correlation coefficients of >0.999. The limit of detection (LOD) for PAMBA, CMNX and ETM was 1.04, 2.08 and 1.04 μg/mL, respectively. Their recoveries in human urine were in the range from 90.2% to 101% with the RSD (n = 5) of 0.7–3.1%. The proposed method is simple, rapid and accurate, and provides the sensitivity and linearity necessary for analysis of the test drugs in human urine at clinically relevant concentrations.     

Separation and detection of vitellogenin in fish plasma by capillary zone electrophoresis

A method for coupling capillary zone electrophoresis (CZE) with rapid membrane chromatography purification (RMCP) was established for the analysis of vitellogenin (VTG) in male fish plasma induced with 17ss-estrodiol. CZE analyses of purified VTG were performed in a buffer containing 25 mM sodium borate (pH 8.4). A 50 microm i.d. fused-silica capillary was used for separation and the detection was carried out by UV-diode array at 214 nm. Inter- and intra-assay variabilities of the proposed method were less than 10.06 and 1.95%, respectively. The method has good linear relationship over the scope of 15-2250 microg/ml with a correlation coefficient of R2 = 0.9965 and a detection limit of 7.0 microg/ml. The established CZE method was also applied to directly separate and identify VTG from fish plasma. The results indicated this method could minimize interferences from plasma proteins, allowing the detection of at least 62.5 microg/ml of VTG proteins in total proteins. This is a rapid and easy method to determine the quantity and purity of VTG compared to Bradford method and SDS-PAGE.     

Uptake of circulating hyaluronic acid by the rat liver

Summary The present study deals with the localization and development of S-100 protein-like immunoreactivity in the retina, ciliary body and iris of human fetuses. In the retina, numerous astrocytes, densely distributed in the nerve-fiber layer and ganglion-cell layer, were stained strongly with the S-100 antiserum. The first immunoreactive astrocytes occurred at the posterior pole of the retina and spread gradually outward and toward the ora serrata with increasing age. Müller cells were not immunoreactive for S-100 during development, except in the retina of the latest fetus examined. S-100 immunoreactivity was also found in the nonpigmented ciliary epithelium and posterior epithelium of the iris, both of which are developed from the inner wall of the optic cup. On the other hand, the pigmented epithelium extending from retina to iris, derived from the outer layer of the optic cup, was free of S-100 immunoreactivity.     

Determination of octopamine in human plasma by capillary electrophoresis with optical fiber light-emitting diode-induced fluorescence detection

A new analytical method based on capillary electrophoresis (CE) separation and optical fiber light-emitting diode (LED)-induced fluorescence detection has been developed for the determination of octopamine. Naphthalene-2,3-dicarboxaldehyde (NDA) was used for precolumn derivatization of octopamine. The separation and determination of the derivative was performed using a laboratory-built CE system with an optical fiber LED-induced fluorescence detector. Optimal separation was obtained at 20 kV using a background electrolyte solution consisting of 25 mM sodium borate (pH 9.2). High sensitivity detection was achieved by the optical fiber LED-induced fluorescence detection using a purple LED as the excitation source. The limit of detection (signal/noise=3) for octopamine was 5.0 x 10(-9)M. A calibration curve ranging from 1.0 x 10(-8) to 5.0 x 10(-7)M was shown to be linear. Using this method, the levels of octopamine in human plasma from healthy donors were determined.     

Implication of the differential roles of metallothionein 1 and 2 isoforms in the liver of rats as determined by polyacrylamide-coated capillary zone electrophoresis

Metallothioneins (MTs), determined by polyacrylamide-coated capillary zone electrophoresis (CZE), coincided well with those described by enzyme-linked immunosorbent assay. By using CZE, MT isoforms 1 (MT-1) and 2 (MT-2) were well separated and determined in the liver cytosol of LEC rats and Wistar rats administered CdCl(2). The total concentrations of MTs in the liver cytosol of LEC rats increased age-dependently as 1.0, 2.1, and 7.2mg/g wet weight of the liver at the age of 5, 10, and 15 weeks, respectively, and those of Wistar rats that had received daily CdCl(2) also increased with time of CdCl(2) as 0.5 and 1.2mg/g wet weight of the liver for 3 and 6 consecutive administration days, respectively. The MT-1/MT-2 ratio in the liver cytosol of LEC rats decreased age-dependently as 1.75, 1.49, and 0.76 at the age of 5, 10, and 15 weeks, respectively. In contrast, that of Wistar rats increased with time of exposure to the metal ion CdCl(2) as 1.1 and 1.6 for 3 and 6 administration days, respectively. Copper accumulation in the liver of LEC rats has already been reported. The present results indicated that the mechanism of the induction of MT synthesis differs between LEC rats, who lack ATP7B, and Wistar rats, who were given a toxic metal ion. On the basis of these results, we propose that MT-1 is related to the metabolism or detoxification of toxic metals such as Cd, and in contrast, MT-2 is responsible for the homeostasis of essential metals such as Cu.     

Optimization by factorial design of a capillary zone electrophoresis method for the simultaneous separation of antihistamines

A 3(2) full factorial design was used to optimize the experimental conditions of a capillary zone electrophoresis method aimed at achieving simultaneous separation and quantification of the antihistamines brompheniramine, chlorpheniramine, cyproheptadine, diphenhydramine, doxylamine, hydroxyzine, and loratadine according to their therapeutic group. A statistical program, SPSS, was used to calculate the mathematical model with which to obtain the response surface. Critical parameters such as pH and applied voltage were studied to evaluate their effect on resolution and on efficiency. Optimum separation conditions were phosphate buffer pH 2.0, 5kV, and 2psis(-1) at 214nm. The analysis time was below 9min and the theoretical plates were between 6000 and 63,000N. Calibration curves were prepared for the antihistamines. The limits of detection were 4-14ngmL(-1), which allow their quantification in pharmaceuticals. The RSD% of each antihistamine was fairly good. Up to seven antihistamines belonging to the antihistaminic H(1)-receptor group were separated in the same electropherogram. The proposed method was then applied to the determination of antihistamines in pharmaceutical, urine, and serum samples with recoveries in agreement with the stated contents.     

Identification of a hyaluronic acid (HA) binding domain in the PH-20 protein that may function in cell signaling.

The macaque sperm surface protein PH-20 is a hyaluronidase, but it also interacts with hyaluronic acid (HA) to increase internal calcium ( [Ca(2+)](i) ) in the sperm cell. A region of the PH-20 molecule, termed Peptide 2 (aa 205-235), has amino acid charge homology with other HA binding proteins. The Peptide 2 sequence was synthesized and two recombinant PH-20 proteins were developed, one containing the Peptide 2 region (G3, aa 143-510) and one without it (E12, aa 291-510). On Western blots, affinity-purified anti-Peptide 2 IgG recognized the 64 kDa band corresponding to PH-20 in acrosome intact sperm and, under reducing conditions, recognized the whole 67 kDa PH-20 and the endoproteolyzed N-terminal fragment of PH-20. HA conjugated to a photoaffinity substrate specifically bound to sperm surface PH-20. Indirect immunofluorescence demonstrated that Fab fragments of anti-Peptide 2 IgG bound to the head of live sperm. Biotinylated HA was bound by Peptide 2 and by sperm extracts in a microplate binding assay, and this binding was inhibited by Fab fragments of anti-Peptide 2 IgG. Biotinylated HA bound to the G3 protein and this binding was inhibited by anti-Peptide 2 Fab, but HA did not bind to the E12 protein. Fab fragments of anti-Peptide 2 IgG inhibited the increase in [Ca(2+)](i) induced in macaque sperm by HA. Our results suggest that the Peptide 2 region of PH-20 is involved in binding HA, which results in the cell signaling events related to the elevation of [Ca(2+)](i) during sperm penetration of the cumulus.     

The effect of synovial fluid proteins in the degradation of hyaluronic acid induced by ascorbic acid

The degradation of hyaluronic acid induced by ascorbic acid and the effect of synovial fluid proteins, such as ceruloplasmin, transferrin, and albumin, were investigated on the basis of the elution volume and the molecular weight of hyaluronic acid using high-performance gel permeation chromatography. Hyaluronic acid was degraded to less than one-third of the original molecular weight in the range of the physiological concentrations of ascorbic acid. Synovial fluid proteins protected against the ascorbate-dependent degradation of hyaluronic acid at their physiological concentrations. It is suggested that the inhibitory activity of ceruloplasmin mainly depends on the ferroxidase activity and that of transferrin is probably due to iron binding property.     

Determination of the degree of substitution and its distribution of carboxymethylcelluloses by capillary zone electrophoresis

A method based on capillary zone electrophoresis (CZE) has been developed to determine the degree of substitution (DS) of carboxymethylcellulose (CMC). Separations were performed with borate buffer (pH 9, ionic strength 20 mM) as background electrolyte in capillaries of 75 microm ID, with an applied voltage of 10 kV, and for detection UV absorption at 196 nm was measured. The use of an internal standard (phthalic acid) to correct for mobility variations resulted in a strong improvement of the precision of the DS determination. Experiments with indirect UV detection indicated that the peak widths obtained actually reflect the variation in mobility, and with that of the DS value, of CMC samples. With the proposed method not only the average DS value but also its dispersity could be established for technical CMC samples. A small but definite effect of the polymeric size on the mobilities was observed. Therefore, DS calibration curves will have to be determined for a specific MM range. Since the size effect is small, a classification of CMCs as low-, middle-, or high MM will be sufficient to obtain accurate data on the DS distribution.     

利用毛细管电泳法分析甜菊糖苷的含量

本文介绍了一种用毛细管区带电泳法筛选甜菊糖苷突变体的有效方法。根据实验结果,优化的电泳条件为60mmol/LTris-硼酸缓冲液(pH8.0),柱温30℃,工作电压25kV。优化条件下,甜菊苷(Stevioside)迁移时间的R.S.D为0.45%(15次),且在7.45×10     

Measurement of phosphorylated phospholamban levels in cardiomyocytes (HL-1) by immunoprecipitation

Phospholamban (PLN) is a key regulatory protein involved in cardiac calcium signaling through the pumping of cytoplasmic Ca²⁺ into the sarcoplasmic reticulum (SR). Recent systems-level studies have focused on integrating quantitative data (e.g. protein expression levels) for a better understanding of cardiac systems biology. In this view, we developed a capillary electrophoresis (CE) based immunoprecipitation method for the measurement of phospho-PLN (ser 16) in cardiomyocytes (HL-1 cell line). Dose-dependent isoproterenol (Iso) treated cells were analyzed using CE, and the phospho-PLN levels were quantified using specific polyclonal antibodies. The CE method employed was accurate, quick and easier compare to other techniques and the results are useful for the subsequent computational systems biology research.     

寡糖8-氨基萘基-1,3,6-三磺酸衍生物在毛细管区带电泳中迁移时间相互关系

确定了寡糖８－氨基萘基－１,３,６－三磺酸（ＡＮＴＳ）衍生物在毛细管区带电泳中迁移时间的相互关系．分别将葡聚糖、甘露聚糖、木聚糖和甲壳质部分酸水解产生的不同聚合度寡糖的混合物,用ＡＮＴＳ胺化还原衍生,然后用毛细管电泳,在５０ｍｍｏｌ／Ｌ,ｐＨ２．５磷酸缓冲液中分离衍生物,分别得到１至２１个聚合度的寡聚葡糖衍生物电泳梯度图,以及聚合度均为１至７的寡聚甘露糖、寡聚木糖和寡聚Ｎ－乙酰氨基葡糖衍生物电泳梯度图．同类寡糖衍生物相对迁移时间（ｔｍ）ｒ与聚合度ｎ均成线性关系．寡聚葡糖衍生物相对迁移时间与其他３类寡糖衍生物的相对迁移时间存在线性关系     

The identification of abnormal glycoforms of serum transferrin in carbohydrate deficient glycoprotein syndrome type i by capillary zone electrophoresis

One of the biochemical characteristics of carbohydrate deficient glycoprotein syndromes is the presence of abnormal glycoforms in serum transferrin. Both glycoform heterogeneity and variable site occupancy may, in principle, lead to the generation of a range of glycoforms which contain different numbers of sialic acid residues, and therefore variable amounts of negative charge. Capillary zone electrophoresis was used to resolve the glycoforms of normal human serum transferrin and also of a set of glycoforms which were prepared by digesting the sugars on the intact glycoprotein with sialidase. The sugars on the intact glycoprotein were also modified by a series of exoglycosidase enzymes to produce a series of neutral glycoforms which were also analysed by capillary zone electrophoresis. The oligosaccharide population of human serum transferrin was analysed by a series of mixed exoglycosidase digests on the released glycan pool and quantified using a novel HPLC strategy. Transferrin was isolated from carbohydrate deficient glycoprotein syndromes type I serum and both the intact glycoforms and released sugars were resolved and quantified. The data presented here confirm the presence of a hexa-, penta- and tetra-sialoforms of human serum transferrin in both normal and carbohydrate deficient glycoprotein syndrome type I serum samples. Consistent with previous reports carbohydrate deficient glycoprotein syndrome type I transferrin also contained a di-sialoform, representing a glycoform in which one of the two N-glycosylation sites is unoccupied, and a non-glycosylated form where both remain unoccupied. This study demonstrates that capillary zone electrophoresis can be used to resolve quantitatively both sialylated and neutral complex type glycoforms, suggesting a rapid diagnostic test for the carbohydrate deficient glycoprotein syndromes group of diseases.Abbreviations CDGS Carbohydrate Deficient Glycoprotein Syndrome - CZE Capillary Zone Electrophoresis - hTf human transferrin - gu HPLC glucose units - EOF electroosmotic flow. Nomenclature: for describing oligosaccharide structures: A(1,2,3,4) indicates the number of antennae linked to the t trimannosyl core - G(0–4) indicates the number of terminal galactose residues in the structure - F core fucose - B bisecting N-acetyl glucosamine (GlcNAc) - S sialic acid - Gal galactose; M - Man mannose     

The role of superoxide and hydroxyl radicals in the degradation of hyaluronic acid induced by metal ions and by ascorbic acid

Purified commercial hyaluronic acid contains significant amounts of iron. Addition of Fe2+ to solutions of it causes depolymerization, which is inhibited by catalase and scavengers of the hydroxyl radical (. OH) but not by superoxide dismutase. Fe3+ is ineffective. Ascorbic acid also depolymerizes hyaluronic acid, apparently because it can reduce Fe3+ in the reaction mixtures to Fe2+. Ascorbate-induced depolymerization is inhibited by the specific iron chelator desferrioxamine, by catalase, and by scavengers of the hydroxyl radical. The relevance of these observations to rheumatoid arthritis and inflammatory joint diseases is discussed.