Kinetic study of thermal inactivation for native and methoxypolyethylene glycol modified trypsin

Bovine pancreatic trypsin (Ti) has been modified with four kinds of methoxypolyethylene glycol (MPEG, molecular masses 350, 750, 2000 and 5000 Da) to enhance thermostability. The MPEG-modified Ti was more stable against temperature than the native form, the larger molecular mass moiety of MPEG showing higher thermostabillty. To investigate the mechanism of thermal inactivation, a new kinetic model, which has the ability of taking the thermal denaturation and autolysis effects of the proteases into account, has been used to analyze the thermal inactivation process of the native and modified Ti in detail. The kinetic analysis showed that the stabilization effect caused by MPEG modification was the result of a decrease in autolysis rate and a decrease in the rate of thermal denaturation. In addition, the possible mechanism of reduced autolysis and lower thermal denaturation rate were also discussed.     

Kinetic characterization of phospholipase A2 modified by manoalogue

Manoalogue, a synthetic analogue of the sea sponge-derived manoalide, has been previously shown to partially inactivate the phospholipase A2 from cobra venom (Reynolds, L. J., Morgan, B. P., Hite, E. D., Mihelich, E. D., & Dennis, E. A. (1988) J. Am. Chem. Soc. 110, 5172) by reacting with enzyme lysine residues. In the present study, the inactivation of the phospholipases A2 from pig pancreas, bee venom, and cobra (Naja naja naja) venom by manoalogue was studied in detail. Manoalogue-treated enzymes were examined in the scooting mode on vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol. Here the native enzymes bound irreversibly to the vesicles and hydrolyzed all of the phospholipids in the outer monolayer without leaving the surface of the interface. All three manoalogue-treated enzymes showed reduced catalytic turnover for substrate hydrolysis in the scooting mode, and the modified enzymes did not hop from one vesicle to another. Thus, inactivation by manoalogue is not due to the decrease in the fraction of enzyme bound to the substrate interface. This result was also confirmed by fluorescence studies that directly monitored the binding of phospholipase A2 to vesicles. A chemically modified form of the pig pancreatic phospholipase A2 in which all of the lysine epsilon-amino groups have been amidinated was not inactivated by manoalogue, indicating that the modification of lysine residues and not the amino-terminus is required for the inactivation. Several studies indicated that the manoalogue-modified enzymes contain a functional active site. For example, studies that monitored the protection by ligands of the active site from attack by a alkylating agent showed that manoalogue-modified pig phospholipase A2 was capable of binding calcium, a substrate analogue, lipolysis products, and a competitive inhibitor. Furthermore, relative to native enzymes, manoalogue-modified enzymes retained significantly higher catalytic activities when acting on water-soluble substrates than when acting on vesicles in the scooting mode. Intact manoalogue had no affinity for the catalytic site on the enzyme as it did not inhibit the enzyme in the scooting mode and it did not protect the active site from alkylation. Pig pancreatic phospholipase A2 bound to micelles of 2-hexadecyl-sn-glycero-3-phosphocholine was resistant to inactivation by manoalogue, suggesting that the modification of lysine residues on the interfacial recognition surface of the enzyme was required for inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Some physicochemical properties of modified trypsin]

Physico-chemical properties of trypsin covalently bound with human serum albumin by glutaric aldehyde have been studied. The modification of the enzyme practically caused no changes in the pH optimum of trypsin. The inhibition of modified trypsin by inhibitors from soy beans and human blood serum has been also studied. The apparent inhibition constants have been calculated. The modification has been shown to result in a deceleration of autolytic degradation. The autolysis rate constants have been calculated at 50 degrees C.     

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.     

Kinetic characterization of human phosphopantothenoylcysteine synthetase

Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the formation of phosphopantothenoylcysteine from (R)-phosphopantothenate and l-cysteine with the concomitant consumption of a nucleotide triphosphate. Herein, the human coaB gene encoding PPCS is cloned into pET23a and overexpressed in E. coli BL21(DE3), to yield 10 mg of purified enzyme per liter of culture. Detailed kinetic studies found that this PPCS follows a similar Bi Uni Uni Bi Ping Pong mechanism as previously described for the E. faecalis PPCS, except that the human enzyme can use both ATP and CTP with similar affinity. One significant difference for human PPCS catalysis with respect to ATP and CTP is that the enzyme shows cooperative binding of ATP, measured as a Hill constant of 1.7. PPCS catalysis under CTP conditions displayed Michaelis constants of 265 μM, 57 μM, and 16 μM for CTP, PPA, and cysteine, respectively, with a k_cat of 0.53 ± 0.01 s^? 1 for the reaction. Taking into account the cooperativity under ATP condition, PPCS exhibited Michaelis constants of 269 μM, 13 μM, and 14 μM for ATP, PPA, and cysteine, respectively, with a k_cat of 0.56 s^? 1 for the reaction. Oxygen transfer studies found that ¹⁸O from [carboxyl-¹⁸O] phosphopantothenate is incorporated into the AMP or CMP produced during PPCS catalysis, consistent with the formation of a phosphopantothenoyl cytidylate or phosphopantothenoyl adenylate intermediate, supporting similar catalytic mechanisms under both CTP and ATP conditions. Inhibition studies with GTP and UTP as well as product inhibition studies with CMP and AMP suggest that human PPCS lacks strong nucleotide selectivity.     

DNA sequencing separations in capillary gels on a modified commercial DNA sequencing instrument

DNA sequencing separations of standard DNA fragments of known sequence have been achieved in small diameter capillary gels electrophoresed and analyzed in parallel in a modified commercial DNA sequencer instrument. DNA sequencing in terms of base-calling accuracy is comparable to conventional slab gels; however, the separations in the capillary were performed somewhat faster and required less sample than those in the slab gel. Advantages of this approach vs. separations on conventional slab gels are discussed.     

Turkey serum trypsin inhibitors: description and characterization

1. Turkey serum trypsin inhibitors were studied on whole and chromatographically fractionated normal turkey serum using both quantitative (trypsin inhibitory capacity measurement) and qualitative (antitryptic activity detection methods) determinations, coupled to electrophoretic and isoelectrophoretic studies. 2. Five proteins with trypsin inhibitory activity were described, the most important ones being alpha 2 and beta-globulins with a multibanded pattern revealed by isoelectric focusing. 3. Trypsin inhibitory capacity assays, performed on individual sera, as well as isoelectric focusing studies, failed to find any quantitative and/or qualitative deficiency of these antiproteases. 4. Evidence is given that round heart disease in turkeys is not related to serum trypsin inhibitor deficiency.     

MALDI mass sequencing and characterization of filarialglutathione-S-transferase

Glutathione-S-transferase has been detected in the somatic extract and excretory-secretory products of different life stages of Setaria cervi, a bovine filarial parasite. The enzyme was subjected to MALDI-TOF followed by mass spectrometry and the nearest match found was Pleuronectes platessa GST. Molecular mass of the purified enzyme was approximately 26 kDa as determined by SDS-PAGE and MALDI-TOF. Setaria cervi GST exhibited high activity towards 1-chloro-2,4-dinitrobenzene and ethacrynic acid. Kinetic analysis with respect to 1-chloro-2,4-dinitrobenzene and glutathione as substrate revealed a K(m) of 2.22 mM and 0.61 mM, respectively. The activity was inhibited significantly by Cibacron blue and alpha-tocopherol.     

Chemically modified, immobilized trypsin reactor with improved digestion efficiency

Tryptic digestion followed by identification using mass spectrometry is an important step in many proteomic studies. Here, we describe the preparation of immobilized, acetylated trypsin for enhanced digestion efficacy in integrated protein analysis platforms. Complete digestion of cytochrome c was obtained with two types of modified-trypsin beads with a contact time of only 4 s, while corresponding unmodified-trypsin beads gave only incomplete digestion. The digestion rate of myoglobin, a protein known to be rather resistant to proteolysis, was not altered by acetylating trypsin and required a buffer containing 35% acetonitrile to obtain complete digestion. The use of acetylated-trypsin beads led to fewer interfering tryptic autolysis products, indicating an increased stability of this modified enzyme. Importantly, the modification did not affect trypsin's substrate specificity, as the peptide map of myoglobin was not altered upon acetylation of immobilized trypsin. Kinetic digestion experiments in solution with low-molecular-weight substrates and cytochrome c confirmed the increased catalytic efficiency (lower K(M) and higher k(cat)) and increased resistance to autolysis of trypsin upon acetylation. Enhancement of catalytic efficiency was correlated with the number of acetylations per molecule. The favorable properties of the new chemically modified trypsin reactor should make it a valuable tool in automated protein analysis systems.