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At least 595 records · Page 33Linked to original sources
PubMed2001

Convection patterns in end-heated inclined enclosures.

The natural convection in inclined side-heated rectangular boxes with adiabatic walls is theoretically and numerically investigated. The study is focused on the characterization of the convection patterns arising at the core of the basic steady unicellular flow and covers the whole range of Prandtl numbers (0< or = Pr < or = infinity) and inclinations (from alpha=0 degrees, heated-from-below vertical cavities, to alpha=180 degrees ). The onset of the flow instabilities depends on the core Rayleigh number R identical with K Ra, defined in terms of the local streamwise temperature gradient, K Delta T/L. The critical value of R for transversal and longitudinal modes is determined by the linear stability analysis of the basic plane-parallel flow, which also provides the stability diagram in the (Pr-alpha) chart. Anyhow, the effect of confinement can decisively change the stability properties of the core: if the steady unicell reaches the boundary layer regime (BLR) the local temperature gradient vanishes at the core leaving a completely stable core region. A theoretical determination of the frontier of the BLR in the space of parameters (alpha, R, and cavity size) yields an extra criterion of stability that has been displayed in the stability diagram. As confirmed by numerical calculations, the core-flow instabilities can only develop for Pr<O(1) whereas, at larger Pr the core region remains stable and the instabilities may only develop at the boundary layers. The analysis of the instability mechanisms reveals several couplings between the momentum and temperature fields that are not possible in the horizontal (alpha=90 degrees ) or vertical limits. For instance, by tilting the cavity with respect to alpha=0 degrees, the (Rayleigh-Bérnard) stationary thermal mode is suppressed in cavities whose depth is smaller than a theoretically predicted cutoff wavelength. The inclination also alters the properties of the oscillatory longitudinal instability, extensively investigated in the horizontal Hadley configuration at low Pr (liquid metals). An analytical relationship for its frequency in terms of alpha, Ra, and Pr is derived. Throughout the paper, numerical calculations in two- and three-dimensional enclosures illustrate each type of multicellular flow and examples of instability interactions near the codimension-2 lines predicted by the theory.

Journal Article↗
PubMed2000

New approach for separating Bacillus subtilis metalloprotease and alpha-amylase by affinity chromatography and for purifying neutral protease by hydrophobic chromatography.

Proteases are commonly used in the biscuit and cracker industry as processing aids. They cause moderate hydrolysis of gluten proteins and improve dough rheology to better control product texture and crunchiness. Commercial bacterial proteases are derived from Bacillus fermentation broth. As filtration and ultrafiltration are carried out as the only recovery steps, these preparations contain also alpha-amylase and beta-glucanase as the main side activities. The aim of this study is to purify and characterize the Bacillus subtilis metalloprotease from a commercial preparation, in order to study separately the impact of the protease activity with regards to its functionality on biscuit properties. Purification was achieved by means of affinity chromatography on Cibacron Blue and HIC as a polishing step. Affinity appeared to be the most appropriate matrix for large scale purification while ion exchange chromatography was inefficient in terms of recovery yields. The crude product was first loaded on a Hi Trap Blue column (34 microm, Pharmacia Biotech); elution was carried out with a gradient of NaCl in the presence of 1 mM ZnCl2. This step was only efficient in the presence of Zn cations, because this salt promoted both protease stabilization resulting in high recovery yields and also complexation of amylase units into dimers resulting in amylase retention on the column and a better separation of the 3 activities. Beta-glucanase was mostly non retained on the column and a part was coeluted with the protease. This protease fraction was then loaded on a Resource Phe column (15 microm, Pharmacia Biotech) in a last step of polishing. Elution was carried out with a linear gradient of 100-0% ammonium sulfate 1.3 M; protease was eluted at the beginning of the gradient and well separated from amylase and glucanase trace impurities. The homogeneity of the purified protease was confirmed by SDS-PAGE, which showed that its MW was about 38. pH and temperature optima were also determined on the fraction.

Bacillus subtilis↗
PubMed1996

Why fisheries collapse and what to do about it.

With the collapse of fisheries in many parts of the world causing widespread economic harm, attention is focused on a possible cause and remedy of fishery collapse. Economic theory for managing a renewable resource, such as a fishery, leads to an ecologically unstable equilibrium as difficult to maintain as balancing a marble on top of a dome. A fishery should be managed for ecological stability instead--in the analogy, as easy to maintain as keeping a marble near the base of a bowl. The goal of ecological stability is achieved if the target stock is above that producing maximum sustainable yield and harvested at less than the maximum sustainable yield. The cost of managing for ecological stability, termed "natural insurance," is low if the fishery is sufficiently productive. This cost is shown to pay for itself over the long term in a variable and uncertain environment. An ecologically stable target stock may be attained either with annually variable quotas following current practice or, preferably, through a market mechanism whereby fish are taxed at dockside if caught when the stock was below target and are untaxed otherwise. In this regulatory environment, the goal of maximizing short-term revenue coincides with the goal of ecological stability, thereby also maximizing long-term revenue. This new approach to fishery management is illustrated with the recently collapsed Newfoundland fishing industry. The Newfoundland cod fishery is expected to rebuild to an ecologically stable level in about 9 years and thereafter support an annual harvest of about 75% of the 1981-1990 average.

Journal Article↗
PubMed2005

Reproducible clusters from microarray research: whither?

MOTIVATION: In cluster analysis, the validity of specific solutions, algorithms, and procedures present significant challenges because there is no null hypothesis to test and no 'right answer'. It has been noted that a replicable classification is not necessarily a useful one, but a useful one that characterizes some aspect of the population must be replicable. By replicable we mean reproducible across multiple samplings from the same population. Methodologists have suggested that the validity of clustering methods should be based on classifications that yield reproducible findings beyond chance levels. We used this approach to determine the performance of commonly used clustering algorithms and the degree of replicability achieved using several microarray datasets. METHODS: We considered four commonly used iterative partitioning algorithms (Self Organizing Maps (SOM), K-means, Clutsering LARge Applications (CLARA), and Fuzzy C-means) and evaluated their performances on 37 microarray datasets, with sample sizes ranging from 12 to 172. We assessed reproducibility of the clustering algorithm by measuring the strength of relationship between clustering outputs of subsamples of 37 datasets. Cluster stability was quantified using Cramer's v2 from a kXk table. Cramer's v2 is equivalent to the squared canonical correlation coefficient between two sets of nominal variables. Potential scores range from 0 to 1, with 1 denoting perfect reproducibility. RESULTS: All four clustering routines show increased stability with larger sample sizes. K-means and SOM showed a gradual increase in stability with increasing sample size. CLARA and Fuzzy C-means, however, yielded low stability scores until sample sizes approached 30 and then gradually increased thereafter. Average stability never exceeded 0.55 for the four clustering routines, even at a sample size of 50. These findings suggest several plausible scenarios: (1) microarray datasets lack natural clustering structure thereby producing low stability scores on all four methods; (2) the algorithms studied do not produce reliable results and/or (3) sample sizes typically used in microarray research may be too small to support derivation of reliable clustering results. Further research should be directed towards evaluating stability performances of more clustering algorithms on more datasets specially having larger sample sizes with larger numbers of clusters considered.

Cluster Analysis↗
PubMed2006

Covalent photochemical functionalization of amorphous carbon thin films for integrated real-time biosensing.

Recent studies have demonstrated that carbon, in the form of diamond, can be functionalized with molecular and/or biomolecular species to yield interfaces exhibiting extremely high stability and selectivity in binding to target biomolecules in solution. However, diamond and most other crystalline forms of carbon involve high-temperature deposition or processing steps that restrict their ability to be integrated with other materials. Here, we demonstrate that photochemical functionalization of amorphous carbon films followed by covalent immobilization of DNA yields highly stable surfaces with excellent biomolecular recognition properties that can be used for real-time biological detection. Carbon films deposited onto substrates at 300 K were functionalized with organic alkenes bearing protected amine groups and characterized using X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The functionalized carbon surfaces were covalently linked to DNA oligonucleotides. Measurements show very high selectivity for binding to the complementary sequence, and a high density of hybridizing DNA molecules. Samples repeatedly hybridized and denatured 25 times showed no significant degradation. The ability to use amorphous carbon films as a basis for real-time biosensing is demonstrated by coating quartz crystal microbalance (QCM) crystals with a thin carbon film and using this for covalent modification with DNA. Measurements of the resonance frequency show the ability to detect DNA hybridization in real time with a detection limit of <3% of a monolayer, with a high degree of reversibility. These results demonstrate that functionalized films of amorphous carbon can be used as a chemically stable platform for integrated biosensing using only room-temperature processing steps.

Biosensing Techniques↗
PubMedDate not supplied

[Emoxipin correction of disorders of lipid peroxidation as affected by a slight excess of oxygen pressure].

The role of the emoxipin (Em.) (2-ethyl-6-methyl-3-oxipyridine) in the correction of the free radical oxidation and allied processes in lung tissues and blood plasma under high-pressure oxygen-prolonged action has been investigated. The studied oxygen exposure (0.3 MPa, 5h) causes the lung stage of oxygen intoxication. It is confirmed by exterior morphological assessment of the lung. The lipid peroxidation increase in lung tissue and blood plasma as well as erythrocyte membranes destabilization result from oxygen exposure. Lipid peroxidation intensity was estimated by determining of content of lipid peroxidation molecular products such as diene conjugates and Shiffs' bases. Erythrocyte membranes stability was evaluated with hemoglobin yield, total iron level and total peroxidase activity in blood plasma. Emoxipin was injected intraperitoneally in a dose 150 mg per 1 kg rats' weight just before the oxygen exposure. Emoxipin is found to improve physiological state of animals and to increase their survival; it normalizes morphology of the lungs and their state; stabilizes erythrocyte membranes injured under oxygen exposure; decreases intensity of lipid peroxidation processes in the lungs and in blood plasma which was previously increased under hyperoxia.

Animals↗
PubMed2002

Directed evolution of barnase stability using proteolytic selection.

We report the construction of a phage-displayed repertoire of mutants of the ribonuclease barnase from Bacillus amyloliquefaciens. The construction was guided by the natural variability between two closely related ribonucleases, barnase and binase from Bacillus intermedius. This repertoire was selected using a proteolytic selection method, allowing sorting of the library according to the resistance of the mutants toward proteolysis. Susceptibility toward proteolysis has been correlated with flexibility and unfolding, and is thus expected to yield mutants with increased thermal stability. Enrichment of barnase mutants with specific combinations of amino acid residues at four of the randomised positions was observed. Three of these enriched amino acid residues are present in neither barnase nor binase. For some of the mutations, the improvement in proteolytic stability does not lead to a pronounced improvement in thermodynamic stability, indicating that the factors governing the proteolytic stability in some cases may be different from those governing the thermodynamic stability, e.g. propensity to local unfolding.The results obtained add important knowledge to a novel use of phage display technology for selection of thermodynamically stable proteins. Only by carefully establishing the parameters that can be adjusted, and recognising the influence this will have on the outcome of selection, will it be possible to realise the powerful technique of proteolytic selection.

Amino Acid Sequence↗
PubMed2002

Diheteroarylethenes as thermally stable photoswitchable acceptors in photochromic fluorescence resonance energy transfer (pcFRET).

We have employed diheteroarylethenes as acceptors for photochromic FRET (pcFRET), a technique introduced for the quantitative determination of fluorescence resonance energy transfer (FRET). In pcFRET, the fluorescent emission of the donor is modulated by cyclical transformations of a photochromic acceptor. Light induces a reversible change in the structure and, concomitantly, in the absorption properties of the acceptor. Only the closed forms of the selected diheteroarylethenes 2a and 2b have an absorption band overlapping the emission band of the donor, 1. The corresponding variation in the overlap integral (and thus critical transfer distance R(o)) between the two states provides the means for reversibly switching the process of FRET on and off, allowing direct and repeated evaluation of the relative changes in the donor fluorescence quantum yield. The diheteroarylethenes demonstrate excellent stability in aqueous media, an absence of thermal back reactions, and negligible fatigue. The equilibration of these systems after exposure to near-UV or visible light follows simple monoexponential kinetics. We developed a general conceptual scheme for such coupled photochromic-FRET reactions, allowing quantitative interpretations of the photostationary and kinetic data, from which the quantum yields for the cyclization and cycloreversion reactions of the photochromic acceptor were calculated.

Journal Article↗
PubMed2000

The preparation of periapical lesions for flow cytometry.

AIM: To devise an optimal protocol and to analyse the leucocyte composition of periapical (PA) lesions by flow cytometry. METHODOLOGY: PA lesions were mechanically agitated, with and without proteolysis. This was with either 0.2% collagenase alone, or in combination with 0.02% DNA-ase in serial incubations until all tissue was digested. The efficacy of each method was assessed by counting total cell yield and cell viability. Phenotype stability was gauged by the percentage of peripheral blood leucocytes (PBL) which expressed CD45RB, CD3, CD20, CD4 and CD8 before and after mechanical and collagenase treatment. RESULTS: Disaggregation of PA lesions was superior if collagenase was present, but cell clumping was problematic unless the DNA-ase was also added, and serial digestion with this combination produced optimal cell yield and viability. Nevertheless, the total number of cells released rarely exceeded 105, though viability was in excess of 80%. Mechanical agitation and proteolysis adversely affected PBL phenotypes, but collagenase digestion limited to 10 min caused least damage. Flow cytometric analysis of disaggregated PA lesions failed to identify more than 7.9% (mean, range 6-10%) CD45RB + cells. CONCLUSIONS: Because of the necessity for single cell suspensions, flow cytometry is not easily applied to the analysis of leucocytes in PA lesions, and further refinements in tissue disaggregation and cell preparation are required.

Antigens, CD↗
PubMed2004

Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface.

The stability of tetrameric malate dehydrogenase from the green phototrophic bacterium Chloroflexus aurantiacus (CaMDH) is at least in part determined by electrostatic interactions at the dimer-dimer interface. Since previous studies had indicated that the thermal stability of CaMDH becomes lower with increasing pH, attempts were made to increase the stability by removal of (excess) negative charge at the dimer-dimer interface. Mutation of Glu165 to Gln or Lys yielded a dramatic increase in thermal stability at pH 7.5 (+23.6 -- + 23.9 degrees C increase in apparent t(m)) and a more moderate increase at pH 4.4 (+4.6 -- + 5.4 degrees C). The drastically increased stability at neutral pH was achieved without forfeiture of catalytic performance at low temperatures. The crystal structures of the two mutants showed only minor structural changes close to the mutated residues, and indicated that the observed stability effects are solely due to subtle changes in the complex network of electrostatic interactions in the dimer-dimer interface. Both mutations reduced the concentration dependency of thermal stability, suggesting that the oligomeric structure had been reinforced. Interestingly, the two mutations had similar effects on stability, despite the charge difference between the introduced side-chains. Together with the loss of concentration dependency, this may indicate that both E165Q and E165K stabilize CaMDH to such an extent that disruption of the inter-dimer electrostatic network around residue 165 no longer limits kinetic thermal stability.

Bacterial Proteins↗
PubMed1977

Fibrin and fibrinogen proteolysis products: comparison between gel filtration and SDS polyacrylamide electrophoresis analysis.

The proteolysis of purified human fibrinogen, stabilized and non-stabilized fibrin by plasmin were investigated by gel filtration analysis and SDS polyacrylamide electrophoresis of the reaction products. Plasmin proteolysis of fibrinogen followed the sequential steps previously reported and the two analytical methods yielded concordant results. Large molecular weight proteolysis products, of substantially greater molecular weight than native fibrinogen, were identified by gel filtration analysis following dissolution of stabilized and non-stabilized fibrin clots; with further incubation with plasmin, these proteolysis products gradually diminished in size. On the other hand, SDS polyacrylamide electrophoresis of these fibrin digests demonstrated that while non-stabilized fibrin yielded breakdown products similar in size to those obtained after proteolysis of fibrinogen, stabilized fibrin digests showed moieties of greater molecular size estimated to be of molecular weight 400,000 to 800,000. The final breakdown products of stabilized fibrin differed from those of fibrinogen and nonstabilized fibrin in that fragment D was present in the "double D" cross-linked form.

Antigens↗
PubMed2003

A single residual replacement improves the folding and stability of recombinant cassava hydroxynitrile lyase in E. coil.

Substitution of Ser113 for Gly113 in the cap domain of hydroxynitrile lyase from Manihot esculenta (MeHNL) was performed by site-directed mutagenesis to improve its self-generated folding and stability under denaturation conditions. The yield of the recombinant mutant HNL1 (mut-HNL1), which had higher specific activity than the wild type HNL0 (wt-HNL0), was increased by 2 to 3-fold. Thermostability of MeHNL was also enhanced, probably due to an increase in content of the beta-strand secondary structure according to CD analysis. Our data in this report suggest that Ser113 significantly contributes to the in vivo folding and stability of MeHNL and demonstrates an economic advantage of mut-HNL1 over the wt-HNL0.

Acetonitriles↗
PubMed2002

Anisole hydrogenation with well-characterized polyoxoanion- and tetrabutylammonium-stabilized Rh(0) nanoclusters: effects of added water and acid, plus enhanced catalytic rate, lifetime, and partial hydrogenation selectivity.

Following a comprehensive look at the arene hydrogenation literature by soluble nanocluster catalysts, six key, unfulfilled goals in nanocluster arene hydrogenation catalysis are identified. To begin to address those six goals, well-characterized polyoxoanion- and tetrabutylammonium-stabilized Rh(0) nanoclusters have been synthesized by the reduction of the precisely defined precatalyst [Bu(4)N](5)Na(3)[(1,5-COD)Rh small middle dotP(2)W(15)Nb(3)O(62)] with H(2) in propylene carbonate solvent. These Rh(0) nanoclusters are characterized by their stoichiometry of formation, transmission electron microscopy, and the two rate constants which characterize their mechanism of formation; previous studies in our laboratories have provided additional characterization of polyoxoanion-stabilized Rh(0) nanoclusters. Propylene carbonate solutions of the Rh(0) nanoclusters catalyze the hydrogenation of anisole (methoxybenzene) under mild conditions (22-78 degrees C, 30-40 psig H(2)). Proton donors such as water or HBF(4) small middle dotEt(2)O are discovered to affect both nanocluster formation and nanocluster arene hydrogenation catalysis. Under identical conditions, the Rh(0) nanoclusters are 10-fold more active than a commercially available, oxide-supported 5% Rh/Al(2)O(3) catalyst of the same average metal-particle size. A series of lifetime experiments shows that the Rh(0) nanoclusters are capable of at least 2600 total turnovers (TTO), a lifetime significantly longer than the approximately 100 TTO often seen for nanocluster arene hydrogenation catalysts, and a lifetime slightly better than the prior record of 2000 TTO for a literature nanocluster system. The present polyoxoanion-stabilized Rh(0) nanoclusters also display a record, albeit modest, 30% selectivity for the partial hydrogenation of anisole to 1-methoxycyclohexene with an overall yield of up to 8% at higher temperatures. In comparison to the 5% Rh/Al(2)O(3) catalyst, the polyoxoanion-stabilized nanoclusters yield a 4.7-fold higher maximum yield of 1-methoxycyclohexene. Finally, the seven main findings of the present work are summarized, including how they address five of the aforementioned six main goals in nanocluster arene hydrogenation.

Journal Article↗
PubMed1951

Mechanisms involved in fibrin formation.

That the role of thrombin in the conversion of fibrinogen to fibrin is essentially enzymatic, is established not only by the minute amounts of thrombin which are effective but also by the complete independence of fibrin yields and thrombin concentrations over a very wide range of thrombin dilutions and clotting times. The thrombin-fibrinogen reaction, in the phase beyond the "latent period" at least, seems fundamentally "first order." Technical requirements of the experiments leading to these conclusions include: (1) a highly purified (e.g. 97 per cent "clottable") fibrinogen, (2) absence of traces of thrombic impurities in the fibrinogen, (3) absence of fibrinolytic protease contaminant of the thrombin and the fibrinogen, and (4) sufficient stability of the thrombin even at very high dilutions. Four conditions affecting thrombin stability have been investigated. Fibrin yields are not significantly modified by numerous experimental circumstances that influence the clotting time, such as (1) temperature, (2) pH, (3) non-specific salt action due to electrical (ionic) charges, which alter the Coulomb forces involved in the fibrillar aggregation, (4) specific ion effects, whether clot-accelerating (e.g. Ca(++)) or clot-inhibitory (e.g. Fe(CN)(6)''''), (5) occluding (adsorptive) colloids, which have a "fibrinoplastic" action, e.g. (a) acacia and probably (b) fibrinogen which has been mildly "denatured" by salt-heating, acidification, etc. The data with which several European workers have attempted to substantiate the idea of a two-stage thrombin-fibrinogen reaction with an intermediary "profibrin" (allegedly partly "denatured") have been reanalyzed with controls which lead us to very different conclusions, viz. (1) denaturation and fibrin formation are independent; (2) partial denaturation is "fibrinoplastic" (see above); and (3) conditions of strong salinity and acid pH (5.1) usually do not completely prevent the thrombin-fibrinogen reaction but merely prolong the "latent" phase and lessen the time required for completion of essentially the same reaction (fibrin polymerization) when more favorable clotting conditions are restored. Thus, our experiments advance the modern concepts concerning the coagulation mechanisms along lines that, for the most part, agree with those of the Harvard physical chemists, and we oppose the European views concerning a two-stage reaction, "profibrin," and "the denaturase theory" of clotting.

Blood Coagulation↗
PubMed2002

Simultaneous analysis of sulfur-containing excitatory amino acids using micellar electrokinetic chromatography with diode array and laser-induced fluorescence detection.

The suitability of micellar electrokinetic chromatography (MEKC) coupled with diode array or laser-induced fluorescence (LIF) detection to analyze the four sulfur-containing excitatory amino acids (SEAA), homocysteine sulfinic acid (HCSA), homocysteic acid (HCA), cysteine sulfinic acid (CSA), and cysteic acid (CA) was investigated. 5-Carboxy-fluorescein succinimidyl ester was chosen as fluorescent reagent to derivatize HCSA, HCA, CSA, and CA. During method development, the yield of reaction dependent on pH and incubation time as well as the stability of the products were analyzed. The maximum yield was obtained after 30 min using a 0.1 M borate buffer (pH 8.9) as derivatization buffer. Each labeled amino acid exhibited high stability at room temperature over a period of 5 days. Baseline separation of labeled HCSA, HCA, CSA, and CA was obtained using a buffer consisting of 0.1 M borate, 50 mM sodium dodecyl sulfate (SDS), and 5% v/v methanol (pH 9.0). By applying LIF detection, limits of detection ranged from 0.9 x 10(-10) M for HCSA to 6.0 x 10(-10) M for CA, respectively. Slightly modified separation conditions enabled the analysis of SEAA in cerebrospinal fluid in the presence of the neurotransmitters glutamate and aspartate. In conclusion, MEKC coupled with LIF detection is a suitable technique for the simultaneous and sensitive analysis of SEAA. Further work will focus on the validation of the method with cerebrospinal fluid as sample matrix.

Amino Acids, Sulfur↗
PubMed2006

Luminescent properties of water-soluble denatured bovine serum albumin-coated CdTe quantum dots.

Chemically reduced bovine serum albumin (BSA) has been used to modify the surface of water-soluble CdTe quantum dots (QDs). It is demonstrated that the denatured BSA (dBSA) can be conjugated to the surface of CdTe QDs and thereby efficiently improve the chemical stability and the photoluminescence quantum yield (PL QY) of the QDs. It is inferred that a shell-like complex structure CdTe(x)(dBSA)(1-x) will form on the surface of the CdTe "core", resulting in the enhancement of PL intensity and the blue shift of the PL peak. This study of the effects of pH and dBSA concentration on optical properties of dBSA-coated QDs suggests that, at pH 6-9, the solution of dBSA-coated CdTe QDs can keep substantial stability and fluorescent brightness, whereas further increase of pH value leads to a dramatic decrease in PL QY and chemical stability. On the other hand, too high or too low initial dBSA concentration in the QD solution results in a decrease of PL QY for dBSA-coated CdTe QDs. This study provides a new approach of preparing stable water-soluble QDs with high PL QY and controllable luminescent colors for biological labeling applications.

Journal Article↗
PubMed1988

Hyaluronidase in ram semen. Quantitative determination, and isolation of multiple forms.

A study was made of hyaluronidase in ram semen. The end-group assay conditions used to determine activity quantitatively were chosen to ensure reliability as well as sensitivity [Gacesa, Savitsky, Dodgson & Olavesen (1981) Anal. Biochem. 118, 76-84]; they led to 1 W.H.O. Standard International Hyaluronidase Unit displaying 0.1263 EC munit (1 EC unit of activity releases 1 mumol equivalent of N-acetylglucosamine end groups/min at 37 degrees C). All the activity in the semen was shown to be sperm-derived, and intact spermatozoa were estimated to contain 1.23 EC units per 10(9) cells. In a low-ionic-strength medium, only some 20% of the hyaluronidase was extractable, although up to 80% of the activity could be extracted as the ionic strength was increased; further addition of detergent extracted the remainder. During purification of the enzyme, it was found that inclusion of poly(vinyl alcohol) in the media stabilized the activity; detergent inclusion also improved the yield, especially during early stages. As a consequence both of reliable quantitative determination and of stabilization, a number of forms of hyaluronidase could be isolated in high yield, by using anion-exchange chromatography, cation-exchange chromatography, affinity chromatography and gel filtration. The existence of all these forms was confirmed by electrophoresis and immunoblotting with the use of a monoclonal anti-(ram hyaluronidase) antibody, and their presence in very freshly prepared sperm extracts was demonstrated. The specific activity of the isolated major hyaluronidase form was 15.0 EC units/mg; this was equivalent to 119,000 W.H.O. units/mg, higher than any other previously reported values.

Animals↗