Yehuda Levin

Covalent coupling of nucleotides to agarose for affinity chromatography

Abstract An improved method for the preparation of agarose hydrazide for coupling of nucleotides and nucleosides is described. Adipic acid dihydrazide is coupled to the agarose by the cyanogen bromide procedure, giving in one step the required agarose hydrazide. Aldehyde of IO 4 − oxidized nucleotides di- and triphosphates are coupled to the resin by hydrazone formation. The application of NADP bound to Sepharose hydrazide for the adsorption of glucose-6-phosphate dehydrogenase is described.

Enzymatic mechanochemistry. I. The interaction of heavy meromyosin with “immobilized adenosine triphosphate”

Abstract ATP was covalently bound to an agarose gel. The insolubilized ATP was found to be capable of specifically binding heavy meromyosin. The adsorbed heavy meromyosin could be eluted by ATP in solution. Both binding and elution by ATP of heavy meromyosin were not much effected by Ca 2+ , Mg 2+ or EDTA. While the water-soluble polyalanine-myosin was also found to be adsorbed, myosin in 0.5 M KCl did not seem to be adsorbed by agarose-ATP. Both Mg 2+ and Ca 2+ appear to activate the splitting of bound ATP by heavy meromyosin to practically the same extent. We prepared water-soluble derivatives of ATP in which ATP underwent the same chemical modification required for its coupling to agarose but in which the agarose component was absent. Their splitting by heavy meromyosin was also activated by Mg 2+ though to a lesser extent but actin did not influence this reaction. Possible relations between our findings and the various stages of the reaction between myosin and ATP, as well as the potential use of columns filled with insolubilized NTPs for the separation and purification of myosin and of its subfragments, are discussed.

Purification and characterization of Aspergillus niger exo-1,4-glucosidase

A specific exo-1,4-glucosidase (1,4-alpha-D-glucan glucohydrooase, EC 3.2.1.3) from Aspergillus niger has been partially purified and subsequently characterized by biochemical, physico-chemical and optical methods. Molecular sieve chromatography yields an enzyme with maximal activity at pH 4.2-4.5 close to its isoelectric point. Reduction and carboxymethylation leads to complete loss of activity and O-acetylation of 3 of the 13 tyrosine residues results in loss of 20 % of the activity. Sodium dodecylsulfate-polyacrylamide gel electrophoresis indicates that the native enzyme consists of two major components of molecular weights 63 000 and 57 500, respectively. Small amounts of dissociated material of molecular weight 28 000 and 16 000 as well as aggregates of the order of 100 000 are also present to the extent of 2-5% of the total potein. Following reduction and carboxymethylation under forcing conditions, the bands around 60 000 diminish and the 28 000-30 000, 16 000 and aggregate bands are dominant...

Water soluble polycationic derivatives of trypsin and chymotrypsin - preparation and properties

Abstract Water-soluble polycationic derivatives of trypsin and chymotrypsin were prepared by the enzyme initiated polymerization of the a-N-carboxyanhydride of σ-triflouroacetyl- L -ornithine.Poly- L -ornithyl trypsin (POT) and poly- L -ornithyl chymotrypsin (POCH), containing 30–50 bound ornithine residues per mole of enzyme, were obtained upon removal of the protecting group. Branched poly- L -ornithyl poly- L -ornithyl trypsin (POOT) was obtained by further polymerization of the a, N-carboxyanhydride via initiation with POT and subsequent hydrolysis of the trifluoroacetyl groups. The POOT derivatives contained about 100–150 bound ornithine residues per enzyme molecule. The pH-esteratic activity profiles of the polycationic enzymes, determined at different ionic strengths, reflected the influence of the electrostatic field induced by the attached polyelectrolyte chains. Similarly, the different activity pattern towards a basic protein, compared to the native enzyme, was also attributed to electrostatic interactions.

Studies on amino acid and peptide derivatives of daunorubicine.

Daunorubicine and adriamycin are antracycline antibiotics that inhibit DNA synthesis and have been shown to possess potent antitumor activity [l-4]. It was demonstrated that such binding is due to two types of drug-DNA complex, i.e., an intercalation involving a base-pair and chromophore interaction and an electrostatic interaction between the amino group of the sugar residue of the antibiotic and the phosphate group of DNA [5]. The therapeutic application of daunorubi~ine and adriamycin is, however, restricted due to their toxic side effects, such as those manifested in the heart muscle and the proliferating bone-marrow cells [6]. It was demonstrated that some chemical modifications of the drugs could bring about a significant decrease in their toxicity [7], and chemically-modified adriarnycin has been reported to retain its anti-tumor activity [8]. We have taken the approach of derivatizing daunorubicine by binding peptides of various lengths to its amino group and analyzing the modified properties of the derivatized drug. The results summarized in this report have shown that the extent of binding of daunorubicine derivatives to DNA and their biological activity decreased with peptide chain length as measured by quenching of fluorescent emission and by inhibition of thymidine incorporation into drug-treated cells, respectively. Reversing the electric charge of daunorubicine or its peptidyl derivatives by succinylation strongly reduced their

Twentyfold increase in alkaline phosphatase activity by sequential reversible activation of the enzyme followed by coupling with a copolyme of ethylene and maleic anhydride

Alkaline phosphatase, APase, (EC 3.1.31) from calf intestine, after shifting the equilibrium by effector molecules towards the dimeric form of the enzyme, was coupled (ratio 1:2, protein: copolymer) to a copolymer of ethylene and maleic anhydride, EMA. The water-soluble APase-EMA was separated from APase and the unbound EMA by DEAE-cellulose ion exchange chromatography. The specific activity of the APase-EMA, compared to APase, increased 26-fold at pH 7.1 and 10-fold at pH 8.6. The pH optimum of APase-EMA was shifted down from pH 9.5 (native APase) to 8.6. This change could be interpreted in terms of polyelectrolyte theory. APase-EMA retained 50–70% of its optimum activity in the pH range 7–8, while APase retained only 5–15% of its optimum activity within the same pH range. Its isoelectric point, pI, was 4.2 (APase 6.0) and it migrated on polyacrylamide gel electrophoresis in a single band, anodic movement twice as fast as APase. Parallel with the kinetic measurements, the reactive-enzyme sedimentation method was used to measure S20,w values. S20,w values obtained for APase-EMA, activated APase, and APase dialyzed against wafer were 6.56S, 6.46S, and 5.17S, respectively. Molecular weights, Mr, were determined by equilibrium sedimentation: the values obtained were 180,000, 160,000, and 84,500. Mr values of APase-EMA and APase (native) estimated by Sepharose-4B gel filtrations were essentially the same. The above-mentioned values remained unchanged for APase-EMA after intensive dialysis against water, whereas for the activated APase, separation from the effector molecules caused the equilibrium to shift back to the monomeric, very slightly active enzyme with concomitant changes of S20,w to 5.15 and Mr to 82,000.

Flavin-protein interaction in bound glucose oxidase

Glucose oxidase was bound to Sepharose, Sephadex, gelatin, and dextran, yielding immobilized soluble and insoluble derivatives of the enzyme. The soluble preparations possessed higher enzymic activity than the analogous insoluble ones. The reversible dissociation process of the bound enzyme into apoenzyme and flavin adenine dinucleotide (FAD) was studied with the soluble and insoluble glucose oxidase in relation to enzymic activity and conformational changes as measured by circular dichroism and fluorescence methods. Bound apoenzyme was found to be more stable than the apoenzyme obtained from the unmodified glucose oxidase. The binding constant of FAD in bound glucose oxidase (Kdiss≈10-8M) calculated from fluorescent studies was lower than that of FAD in the native enzyme (Kdiss10-10M). The circular dichroism measurements indicated that dextran-bound glucose oxidase has a conformation similar to that of the native enzyme.

Oxidation of 2-mercaptoacetyl-phenylalanyl-leucine, a specific inhibitor ofPseudomonas aeruginosa elastase, in the presence of the bacteria and its prevention

The compound 2-mercaptoacetyl-l-phenylalanyl-l-leucine (HSAc-Phe-Leu) is a specific and potent inhibitor ofPseudomonas aeruginosa elastase with demonstrated potential as a drug for the treatment ofPseudomonas eye infections. The thiol moiety of this compound is lost in the presence of the bacterial at a rate greater than that seen in the absence of the organisms. Tritium-labeled inhibitor was prepared and used to examine whether this accelerated loss is due to the uptake of the inhibitor by the bacteria or due to its modification by some bacterial products. Our results excluded by possibility of uptake and indicated that the loss of the thiol resulted from its oxidation to the inactive disulfide form. The oxidation reaction is probably catalyzed by a low molecular weight extracellular bacterial product, and is effectively prevented in the presence of sodium bisulfite. It is suggested that HSAc-Phe-Leu preparations used for further investigation of the inhibitors therapeutic potential should include antoxidants such as sodium bisulfite to provide maximal inhibitory capacity.