Isolation and purification of bovine testicular hyaluronidase

We developed a single-step method for chromatographic purification of the hyaluronidase extract on Sepharose blue, which produced high yield of the enzyme (95%) with high purity (40). Purification of hyaluronidase allowed us to obtain biological preparations standardized by the protein composition and enzyme activity and useful for basic and applied researches.     

A rapid purification of T4 polynucleotide kinase using Blue Dextran-Sepharose chromatography.

A rapid batch procedure is described for purification of T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to near homogeneity using Blue Dextran-Sepharose chromatography. The enzyme preparation is sufficiently free of contaminating endonuclease and alkaline phosphatase activities to be suitable for radioactively labeling nucleic acids in vitro. Kinetic measurements indicate that the chromophore of Blue Dextran, Cibacron Blue F3GA, inhibits the activity of T4 polynucleotide kinase competitively with respect to single stranded DNA substrate and non-competitively with respect to the rATP substrate.     

In vitro correction of Hurler fibroblasts with bovine testicular hyaluronidase.

Bovine testicular hyaluronidase (endo-beta-N-acetyl hexosaminidase) has a significant corrective effect on cultured Hurler fibroblasts. Nonspecificity of this effect is indicated by its equally strong corrective effect on Hunter fibroblasts. Although all specimens of hyaluronidase also possessed iduronidase activity, a separate corrective effect could be attributed to the endo-N-acetyl hexosaminidase activity of at least one hyaluronidase (Wyeth M-151) for four reasons: (i) its very low content of iduronidase activity; (ii) a decrease in intracellular macromolecular mucopolysaccharides (believed to be largely dermatan sulfate) with a corresponding increase in intracellular and extracellular oligosaccharides; (iii) no measurable increase in iduronidase activity of hyaluronidase-treated cells despite near maximal correction; (iv) direct correlation between Hurler cell correction and hyaluronidase activity when enzymes of different strength were used at less than maximal correction.     

Heparin-sepharose affinity chromatography for purification of bull seminal-plasma hyaluronidase.

Bull seminal-plasma hyaluronidase was purified 180-fold by chromatography on concanvalin A-Sepharose, heparin Sepharose, Sephadex G-200 and Sephacryl S-200. With hyaluronic acid as the substrate, the specific activity and turnover number of purified hyaluronidase were 3.63 mumol/min per mg (104000 National Formulary units/mg of protein) and 214 min-1 (mol of product formed/mol of enzyme per min) respectively. Polyacrylamide-gel electrophoresis indicated that the purified enzyme migrated as a single band on 7.5 and 10% (w/v) gels at pH 4.3 and 5.3. Bull seminal-plasma hyaluronidase was markedly inhibited by hydroxylamine, phenylhydrazine and semicarbazide. Purified hyaluronidase (1.25 munits; 1 unit = 1 mumol of N-acetylglucosamine liberated/min at 37 degrees C) dispersed the cumulus clot of rabbit ova in 1 h at 22 degrees C.     

Mechanism of action of bovine testicular hyaluronidase. Mapping of the active site.

The reactions of purified, homogeneous bovine testicular hyaluronidase have been studied with radioactively labeled oligomers of hyalobiuronic acid, (GlcUA-GlcNAc)n, as substrates and acceptors. Transglycosylation occurs by transfer of a glycosyl residue with retention of configuration from a leaving group to an acceptor. On the basis of detailed examination of cleavage and transglycosylation patterns for the trimer; comparison of trimer, tetramer, and polymer as substrates; comparison of acceptors; equilibrium binding; and other data, it is proposed that the enzyme's active site consists of five subsites for hyalobiuronate residues. In the terminology of Schechter, I., and Berger, A. ((1966) Biochemistry 5, 3371), these are s2-s1-s' 2-s3, where the reducing terminus is to the right, and cleavage occurs between s1 and s' 1. It is proposed that subsite s'2 has a high affinity for a substrate residue, while s1 and s'1 have low substrate affinity, and s2 and s' 3 are intermediate in affinity. This proposal is seen to have mechanistic implications. The reactions of several substrates show similar bell-shaped pH dependences, with optima in the region of pH 5 to 5.5.     

A new affinity method for purification of bovine testicular hyaluronidase enzyme and an investigation of the effects of some compounds on this enzyme

Abstract

In this study, a new affinity gel for the purification of bovine testicular hyaluronidase (BTH) was synthesized. l-Tyrosine was added as the extension arm to the Sepharose-4B activated with cyanogen bromide. m-Anisidine is a specific inhibitor of BTH enzyme. m-Anisidine was clamped to the newly formed Sepharose-4B-l-tyrosine as a ligand. As a result, an affinity gel having the chemical structure of Sepharose-4B-l-tyrosine-m-anisidine was obtained. BTH purified by ammonium sulfate precipitation and affinity chromatography was obtained with a 16.95% yield and 881.78 degree of purity. The kinetic constants K_M and V_Max for BTH were determined by using hyaluronic acid as a substrate. K_M and V_Max values obtained from the Lineweaver–Burk graph were found to be 2.23?mM and 19.85?U/mL, respectively. In vitro effects of some chemicals were determined on purified BTH enzyme. Some chemically active ingredients were 1,1-dimethyl piperidinium chloride, β-naphthoxyacetic acid and gibberellic acid. Gibberellic acid showed the best inhibition effect on BTH.     

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.     

A rapid purification procedure for tetrodotoxin derivatives by high-performance liquid chromatography

A simple procedure for purification of tetrodotoxin (TTX) derivatives by high-performance liquid chromatography is described. Chemically oxidized TTX, C₁₁-nortetrodotoxin (nor-TTX), was purified and collected by reverse-phase chromatography. The separation of nor-TTX from unreacted TTX was excellent and recovery of nor-TTX was more than 90%. The isolated nor-TTX was further coupled with lysine, and the coupled product was purified again by high-performance liquid chromatography on a cation-exchange column. The separation of all compounds required less than 15 min. The uv monitoring at 230 nm allowed the detection of TTX derivatives at the 2- to 3-ng level.     

Murine C4-binding protein: a rapid purification method by affinity chromatography

A simple, 3-step method was described for purification of murine C4 binding protein (C4-bp), a recently recognized serum protein that functions as one of the regulatory proteins of the complement system. The method consists of 1) affinity chromatography using TNBS-BGG-conjugated Sepharose beads, 2) gel filtration on a Sepharose 6B column, and 3) heparin-Sepharose chromatography. By this method, milligram quantities of C4-bp can be easily purified by more than 500-fold from EDTA-serum of various mouse strains, and the whole purification process can be completed within 1 wk. The overall yield of C4-bp is about 15%. The C4-bp thus prepared is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and immunelectrophoresis. The purified mouse C4-bp showed physicochemical properties very similar to those described for human C4-bp. Like human C4-bp, mouse C4-bp is composed of several apparently identical subunits of the m.w. of 80,000. However unlike the human counterpart, the subunits of mouse C4-bp are not linked by disulfide bonds but are connected by non-covalent forces that can be disrupted by SDS. The purified mouse C4-bp retained binding affinity for C4 and showed unaltered antigenicity. Immunization of rabbits with the purified mouse C4-bp resulted in the production of potent and monospecific antisera.