Stability and catalytic properties of chemically modified pig trypsin

Pig trypsin was chemically modified with the bifunctional compound ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester) to yield EG-trypsin. EG-trypsin showed greater thermal stability (100% active beyond 100 min at 55°C; native only 53% active at 100 min) together with slightly increased tolerance toward some organic solvents. Arg/Lys hydrolysis ratio changed little. Esterase/amidase activity ratio of EG-trypsin in buffer was 11-fold greater than that of native pig trypsin, but 5-fold less in 30% v/v acetonitrile. In buffer, EG-trypsin synthesized the dipeptide benzoyl-Arg-Leu-NH₂ at a 3-fold higher rate than native trypsin, but native trypsin outperformed EG-trypsin in 30% v/v acetonitrile.     

Activation of a cyclic nucleotide phosphodiesterase and of a protein kinase by chemically modified calmodulin

1. Several calmodulin derivatives prepared by chemical modification of lysine residues were tested using bovine heart cyclic nucleotide phosphodiesterase and wheat germ calmodulin-dependent protein kinase. 2. The effect of chemical modification on the activation capacity of calmodulin for the two studied enzymes was different. 3. This was particularly noticeable in the case of alkylated derivatives which exhibited a higher affinity than native calmodulin towards phosphodiesterase but a lower affinity towards protein kinase. 4. The efficiency of these derivatives (maximal activation) was higher than that of native calmodulin in relation with the protein kinase.     

Preparation and properties of trypsin chemically attached to EEDQ activated styrene-methacrylic acid copolymers

Styrene-methacrylic acid copolymers of varying combinations crosslinked with p-DVB (1-2%) and porous structure were synthesized to be used as carriers in trypsin immobilization. The styrene-methacrylic acid copolymers containing free carboxy groups were activated by conversion into the mixed carbonic anhydride with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) at pH 4.0. The degree of activation of copolymers were determined from the amount of p-aminobenzoic acid each could bind. The activated copolymers were incubated with trypsin in phosphate buffer (pH 8.0) at 4 degrees C for 24 h. The optimum conditions for enzymatic activity measurements determined and the activity tests were carried out in 1.5 x 10(-2)M CaCl(2) solution (pH 8.0) at 0.05 ionic strength with a pH-stat instrument. The dependence of the activity of styrene-methacrylic acid (SMA)/trypsin derivatives to pH was investigated and it was observed that the optimum pH of the immobilized trypsin derivatives moved to the basic region compared to the native trypsin. It was found that as the ionic strength increased, the shift in the optimum pH decreased and the activity increased. The Michaelis constants for the SMA-trypsin derivatives were determined with aid of Lineweaver-Burk diagrams. The thermal, storage, and operational stabilities of SMA-trypsin derivatives were assessed. It was found that the above stabilities for all the immobilized trypsin derivatives were better than that for the native trypsin.     

Use of chemically modified activated carbon as a support for immobilized enzymes

Loading and activity assays of the enzymes alpha-chymotrypsin, alpha-chymotrypsinogen, and glucose oxidase covalently bound to an activated carbon support are presented. The activated carbon support material was pretreated using either a radio-frequency oxygen plasma or an electrochemical oxidation to maximize the enzyme attachment. Cyanuric chloride or water-soluble carbodiimide linking reactions were used to covalently attach the enzymes to the carbon support. Discussion of the relative merits of each reaction scheme is presented.     

Synthesis, characterization and biological activity of chemically modified insulin derivative with alpha lipoic acid

A novel chemically-modified insulin, epsilon-N(B29)-lipoyl insulin, was selectively prepared by the covalent linkage of alpha-lipoic acid (LA) to the epsilon-amino group of Lys(B29) of insulin without any protecting agent and analyzed by PAGE, HPLC, MALDI-TOF-MS. Monolipoyl- insulin maintained the glucose-lowering effect as well as native insulin and showed a longer duration of action than native insulin and an inhibitory effect towards trypsin degradation.     

Chicken ovomucoid: determination of its amino acid sequence, determination of the trypsin reactive site, and preparation of all three of its domains

The complete amino acid sequence of chicken ovomucoid (OMCHI) is presented. OMCHI consists of three tandem domains, each homologous to pancreatic secretory trypsin inhibitor (Kazal) and each with an actual or putative reactive site for inhibition of serine proteinases. The major reactive site for bovine beta-trypsin is the Arg89-Ala peptide bond in the second domain. The equilibrium constant for hydrolysis of this peptide bond, K0hyd, is 1.85. The first and third domains of OMCHI are relatively ineffective inhibitors of several serine proteinases against which they were tested. OMCHI is a mixture of two forms: the major form with all of the amino acid residues and a minor form with Val134-Ser135 deleted. This polymorphism is present in all chicken eggs and is the result of ambiguous excision at the 5' end of the F intron. Procedures are given for preparation of modified chicken ovomucoid, OMCHI (in which the Arg89-Ala bond is hydrolyzed), of the first domain, OMCHI1 (residues 1-68), of the second domain, OMCHI2 (residues 65-130), and of the third domain, OMCHI3 (residues 131-186). In the case of the third domain, both the Asn175 glycosylated form, OMCHI3(+), and the carbohydrate-free form, OMCHI3(-), were obtained. These isolated native domains are useful in many studies of ovomucoid behavior.     

Affinity selection of chemically modified proteins: role of lysyl residues in the binding of calmodulin to calcineurin

In affinity selection, calcineurin selects from a population of randomly modified calmodulins those species with which it prefers to interact. The method shows that acetylation of lysines affects calmodulin so as to interfere with its ability to interact with calcineurin. Monoacetylation of any lysine of calmodulin reduces its affinity for calcineurin by 5-10-fold. Multiple acetylations amplify the loss of affinity; none of the modifications are imcompatible with activity. The lack of selectivity of calcineurin against any particular modified lysine indicates that the loss of affinity reflects changes induced by the removal of the charged groups and suggests an important role for electrostatic interactions in the cooperative structural transitions which calmodulin undergoes upon binding its target proteins or calcium. In the presence of calcineurin, a large and specific decrease in the rate of acetylation of Lys-75 and -148 of calmodulin is observed. The reactivity of the same residues is greatly increased in the presence of calcium alone [Giedroc, D. P., Sinha, S. K., Brew, K., & Puett, D. (1985) J. Biol. Chem. 260, 13406-13413]. Lys-75, located in the central helix, and the C-terminal Lys-148 [Babu, Y. S., Sacks, J. S., Greenhouse, T. J., Bugg, C. E., Means, A. R., & Cook, W. J. (1985) Nature (London) 315, 37-40] may act as sensors of the calmodulin allosteric transitions. Their reactivity changes in opposite directions in response to calcium-induced or calcineurin-induced structural changes. The reactivity of other residues such as Lys-21, decreased in the presence of calcineurin but not calcium, is also affected by a conformational change which is induced specifically by calcineurin.     

The amino acid sequence and reactive site of a single-headed trypsin inhibitor from wheat endosperm

The sequence of a trypsin inhibitor, isolated from wheat endosperm, is reported. The primary structure was obtained by automatic sequence analysis of the S-alkylated protein and of purified peptides derived from chemical cleavage by cyanogen bromide and digestion withStaphylococcus aureus V8 protease. This protein, named wheat trypsin inhibitor (WTI), which is comprised of a total of 71 amino acid residues, has 12 cysteines, all involved in disulfide bridges. The primary site of interaction (reactive site) with bovine trypsin has been identified as the dipeptide arginyl-methionyl at positions 19 and 20. WTI has a high degree of sequence identity with a number of serine proteinase inhibitors isolated from both cereal and leguminous plants. On the basis of the findings presented, this protein has been classified as a single-headed trypsin inhibitor of Bowman-Birk type.     

Cancer therapy with chemically modified enzymes. I. Antitumor properties of polyethylene glycol-asparaginase conjugates

The covalent attachment of monomethoxypolyethylene glycol (PEG) to asparaginases from Escherichia coli and Vibrio succinogenes by new coupling methodology produced conjugates that are active, stable, without significant immune response, and with greatly extended plasma half-lives in mice. Therapeutic efficacies were greater for the PEG-asparaginases than for the unmodified asparaginases in mice infected with the L5178Y lymphosarcoma or the 6C3HED tumor. Large single doses of native or modified enzymes were more effective against tumors than the same amount of enzyme given in smaller doses over several days.     

Stimulation of the erythrocyte Ca2+-ATPase and of bovine brain cyclic nucleotide phosphodiesterase by chemically modified calmodulin

Chemically modified calmodulins have been used to investigate structural features which are important for the interaction of the activator with targets. Carbamoylation of lysine residues had no influence on the ability of calmodulin to stimulate the plasma membrane Ca2+-ATPase whereas the stimulation of the bovine brain cyclic-nucleotide phosphodiesterase was reduced up to 50%. Different species of carbamoylated calmodulin have been isolated but no differences were detected in their interaction with the cyclic-nucleotide phosphodiesterase. Modification of arginine residues by 1,2-cyclohexanedione had no effect of the stimulation of the phosphodiesterase but reduced by 40% the stimulation of the erythrocyte Ca2+ ATPase. Mild oxidation of methionines by N-chlorosuccinimide produced a number of differently modified calmodulins. The different species have been purified and the modified residues have been identified. They affected the two different test enzymes to different extents indicating that methionines in the central helix of calmodulin are of greater importance for the interaction with the phosphodiesterase, whereas methionines located in the C-terminal half of calmodulin are more important for the interaction with the Ca2+-ATPase.     

Preparation and characterization of a chemically modified plastocyanin.

The reaction of plastocyanin with tetranitromethane results in the nitration of only one of the three tyrosyl residues present in the protein. The modification does not affect the blue copper chromophore as both the characteristic visible spectrum of the chromophore and the redox potential of the protein are unchanged. Photochemical assays show that the modified plastocyanin is fully active in the reduction of photooxidized P700 and in the photooxidation of cytochrome f. The pK of the nitro-tyrosyl residue is about 7.3 indicating that the modified residue may be located in a negatively charged environment. Examination of the recently published X-ray structure of poplar plastocyanin suggests that Tyr-80 would be a likely candidate for the site of modification.     

Dissociation of lepidopterous tissues with proteolytic enzymes: Comparison of collagenase,trypsin, and pronase

The fat body and certain other tissues of lepidopterous larvae and diapausing pupae can be completely dissociated by incubation in crude or purified collagenase. Damage to the cells is minimal. Dissociation with trypsin is incomplete, and pronase causes extensive damage. All three enzymes act principally by digesting the extracellular connective sheath that envelops the individual lobes of fat body, since the cells at this stage are not intrinsically cohesive. The experimental findings support the notion that collagen is an important structural component of insect connective membranes.     

Synthesis and characterization of a novel super-absorbent based on chemically modified pulverized wheat straw and acrylic acid

In order to develop an eco-friendly polymer, the material was prepared from pulverized wheat straw by chemical methods. And a super-absorbent hydrogel has been synthesized with chemically modified pulverized wheat straw (CMPWS) and acrylic acid (AA) in aqueous solution. Factors, such as weight ratio of AA to CMPWS, the amount of initiator and cross-linker, temperature and neutralization degree of AA that influence absorbencies of super-absorbent were investigated. Moreover, the super-absorbent had been proved with expectant polymerization structure and good thermo-stability via IR spectrum and TGA analysis. The morphological features were evidenced by SEM images. The excellent product was obtained with the absorbencies of 417 g/g in distilled water and 45 g/g in 0.9 wt% NaCl solution.