Changes Of Trypsin Research Articles

The Internal Friction of Proteins

The rates of protein conformational changes are usually not only limited by external but also internal friction, however, the origin and significance of this latter phenomenon is poorly understood. It is often found experimentally that a linear fit to the reciprocal of the reaction rate as a function of the viscosity of the external medium has a non-zero value at zero viscosity, signifying the presence of internal friction. Furthermore, for a few proteins (including trypsin) the temperature dependence of the internal friction was also determined, which shows an Arrhenius-like behavior, with an activation energy in the range of tens of kJ/mol. This indicates that the internal structural rearrangement of proteins occurs in a rugged energy landscape. To better understand this phenomenon, we performed molecular dynamics simulations of the conformational changes of trypsin. The simulations allowed us to control the external viscosity over a wide range, well exceeding the experimentally accessible values. The results confirm not only the existence of internal friction, but also its Arrhenius-like temperature dependence, and that the activation energy of the internal friction is indeed several tens of kJ/mol, in good agreement with the experimentally obtained values.

Structural and functional characterization of solution, gel, and aggregated forms of trypsin in organic solvent-assisted and pH-induced phase changes

Objective: In this study the effect of three different physicochemical parameters on pHtriggered gelation and aggregation of bovine pancreatic trypsin changes and structural and functional changes in these changes in alcohol-water mixtures were studied. Methods: Trypsin gelation times were studied using inverted tube method. Trypsin stability was studied using trypsin enzyme assay. Protein secondary structural changes were monitored using FTIR spectroscopy. Gel and aggregate macrostructures and morphologies were viewed using Scanning Electron Microscopy. Results: The solution phase was observed in the absence of both NaOH and CaCl 2 . The gel phase was observed in the absence of the either. The aggregate phase was observed in the presence of the both agents all depending on trypsin concentrations used. Trypsin stability studies showed that there were a nearly 53 and 32% specific activity losses after the gelation and aggregation processes. According to FTIR studies β-sheet structure in 1637 cm -1 band disappeared in trypsin gel and trypsin aggregates. Increases in α-helix structure in 1651 cm -1 in trypsin gel and aggregates were observed. Iodoacetamide delayed the gelation and prevented the aggregation indicating the importance of intermolecular disulfides in the both processes. Conclusion: Trypsin gelation was caused by the denaturation of the protein three dimensional structures. The gel and aggregate formation indicates a secondary structural change towards α-helix structure formation at the expense of β-sheet structure and formation of intermolecular disulfide bonds.

Photobehavior and docking simulations of drug within macromolecules: Binding of an antioxidative isoquinolindione to a serine protease and albumin proteins

The principal intent of the present contribution is to decipher the binding domain and structural changes of trypsin (TPS), a proteolytic globular enzyme and two serum proteins, namely, bovine serum albumin (BSA), human serum albumin (HSA) association with a newly synthesized bioactive isoquinolindione derivative (ANAP) by employing steady state, time resolved fluorescence and circular dichroism (CD) techniques. Intramolecular charge transfer emission (ICT) of ANAP is found to be responsible for the commendable sensitivity of the probe as an extrinsic fluorescent marker to the protein environments. A sharp distinctive feature of determined micropolarities in proteinous media clearly demarcates the differential extent of hydrophobicity around the encapsulated ANAP. A proficient efficiency tunable fluorescence (Förster type) resonance energy transfer (FRET) from the excited tryptophan to ANAP reveals that ANAP binds in the close vicinity of the tryptophan residue in protein. Molecular modeling simulation has been exploited for evaluating the probable interaction site of ANAP in proteinous assembly which shows subdomain IIA are earmarked to possess affinity for ANAP in serum albumins whereas S1 binding pocket in TPS has been found potential binding region for ANAP.

The Interactions of Glutathione-Capped CdTe Quantum Dots with Trypsin

Due to their unique fluorescent properties, quantum dots present a great potential for biolabelling applications; however, the toxic interactions of quantum dots with biopolymers are little known. The toxic interactions of glutathione-capped CdTe quantum dots with trypsin were studied in this paper using synchronous fluorescence spectroscopy, fluorescence emission spectra, and UV-vis absorption spectra. The interaction between CdTe quantum dots and trypsin resulted in structure changes of trypsin and inhibited trypsin's activity. Fluorescence emission spectra revealed that the quenching mechanism of trypsin by CdTe quantum dots was a static quenching process. The binding constant and the number of binding sites at 288 and 298 K were calculated to be 1.98 × 10(6) L mol(-1) and 1.37, and 6.43 × 10(4) L mol(-1) and 1.09, respectively. Hydrogen bonds and van der Waals' forces played major roles in this process.

The effect of dynamic high-pressure microfluidization on the activity, stability and conformation of trypsin

Dynamic high-pressure microfluidization (DHPM) treatment of trypsin showed no significant effects with relative activities of 98.5% (80 MPa), 98.3% (100 MPa), 97.8% (120 MPa) and 97% (160 MPa). However, DHPM treatment enhanced the pH and thermal stability of trypsin. After 100 min of incubation at 45 °C, the residual activity of trypsin treated at 80 MPa was still as high as 96% while the untreated trypsin retained only 86% of its original activity. The optimum pH of trypsin maintained surprising consistency (pH = 7.6); nevertheless, the relative activities were about 97%, 102% and 103% at 80, 100 and 120 MPa, respectively. In addition, DHPM-induced conformational changes of trypsin were observed. The unfolding of trypsin, induced by DHPM treatment, was reflected in the increase in maximum emission fluorescence intensity and exposed SH contents, as well as the decrease in total SH contents, UV absorbance and α-helix intensity.

Changes of trypsin in activity and secondary structure induced by complex with trypsin inhibitors and tea polyphenol

To reveal the relationships between the activity of trypsin and its structural change, changes of trypsin in biological activity induced by complex with Bowman-Birk trypsin inhibitor (BBTI), Kunitz soybean trypsin inhibitor (KSTI, type I-S) and tea polyphenol (TP) were detected and their relationship with the secondary structure changes were studied by far-UV circular dichroism (CD) spectra measurement. BBTI and KSTI were also irradiated by ultrasonic to compare the effects on trypsin. The rank was found as KSTI > BBTI > TP according to their inhibitory activities against trypsin. Yet BBTI exhibited much stronger resistance against ultrasonic irradiation than KSTI. BBTI, KSTI and TP were found inactivate trypsin by modifying the secondary structures and far-UV spectrum of trypsin. Complex of trypsin with ultrasonic-treated BBTI and native BBTI and KSTI exhibited the similar modified effects in secondary structures, decrease of α-helix and β-turn content, increase of β-sheet content and unchanged random coil content basically. But complex of trypsin with ultrasonic-treated KSTI exhibited less modified effects because of inactivation by ultrasonic irradiation. The changes of trypsin in secondary structure induced by complex with TP showed different from those induced by complex with BBTI and KSTI, increase of α-helix content, decrease of random coil content and unchanged β-sheet and β-turn content basically.

Seasonal Changes in Trypsin and Chymotrypsin Activity in Viscera from Cod Species

Trypsin and chymotrypsin activity in cod, saithe, haddock, tusk and ling viscera from the Barents Sea, Icelandic Sea and South coast of Ireland from three seasons have been compared. Significant differences in activity between seasons are found for cod from the Icelandic Sea. Cod caught in the Barents Sea show no significant seasonal changes in activity. Significant differences between the five species are found. Activity in cod viscera from the Icelandic Sea differ significantly from the two other fishing grounds in February-March. A significant difference was also found between cod from the Icelandic Sea and Barents Sea in April-June, and cod from the Icelandic Sea and South coast of Ireland in October-December.

Modulation of stability properties of bovine trypsin after in vitro structural changes with a variety of chemical modifiers.

Controlled chemical modification of enzymes, targeting groups not involved in the active site, can lead to modified catalysts that are intrinsically more efficient and resistant to heat and denaturing agents. Bovine pancreatic trypsin was covalently modified up to 75-85% with monomeric glutaraldehyde (MGA), polymeric glutaraldehyde (PGA), oxidized sucrose and oxidized sucrose polymers (OSP 70 and OSP 400). Virtually no loss in activity occurred upon modification. Temperature optima of trypsin shifts from 45-76 degrees C and T50 from 54-76 degrees C for the best modified sample made with OSP. The efficiency of the modifiers in stabilization was ranked in the order: OSP 400-T > OSP 70-T > PGA-T > MGA-T > Sucrose-T. Half-life of modified enzymes also followed the same trend. Both stabilization factor and t1/2 decreased with increasing temperatures. The free energy of activation for inactivation delta(deltaG*) varies from 12-20 kJ/mol and the activation enthalpy delta(deltaH*) of the modified trypsin by 80-120 kJ/mol indicating stabilization. Inactivation of modified trypsin by urea is less noticeable. The character of the two-step inactivation process of trypsin changes with the degree of stabilization in that the duration of phase I one increased noticeably as stabilization increases. Native trypsin fluoresces less intensely showing a red shift under the influence of denaturation. Such a fluorescence change is not so obvious for the modified enzymes indicating conformational stability acquired by modification.

Changes in trypsin and chymotrypsin inhibitory activity on soaking of redgram (Cajanus cajan L.).

Changes in trypsin and chymotrypsin inhibitory activity of redgram seeds on soaking in distilled water, different salt solutions and mixed salt solution were investigated. Reduction in trypsin and chymotrypsin inhibitory activity was observed on soaking in salt solutions in comparison to soaking in distilled water. Maximum loss of trypsin and chymotrypsin inhibitory activity was observed on soaking the seeds in mixed salt solution.

Ontogenetic change in digestive enzyme activity during larval development of Macrobrachium rosenbergii

This study was conducted to determine the ontogenetic changes in trypsin, non-specific esterase and amylase activities in Macrobrachium rosenbergii during larval development. Two batches of M. rosenbergii larvae were reared separately in 300-l tanks at 50 larvae l −1. Rearing temperature and salinity were maintained at 30±2°C and 12 ppt, respectively. The larvae were daily fed with newly hatched Artemia nauplii at 5 nauplii l −1. Trypsin, esterase and amylase activities in the larvae were estimated using TAME, butyrylthiocholine iodide and corn starch, respectively, as substrates. The results showed that maximum peaks of trypsin and esterase activities occurred at stage V–VI. Amylase activity remained low until the larvae reached stage VI–VIII, indicating that the early-stage M. rosenbergii larvae are exclusively carnivorous. Maximum amylase activity was observed at stage XdashXI. In general, the ontogenetic digestive enzyme activities in developing prawn larvae seemed to coincide with hepatopancreas development.

P-nitrophenylphosphatase activity of plasma membrane H(+)-ATPase from yeast. Implications for the regulation of the catalytic cycle by H+.

The H(+)-ATPase from Schizosaccharomyces pombe belongs to the group of transport ATPases which displays two main conformational states, E1 and E2 (P-type ATPase). In this report, we show that, as in the case of other P-type ATPase, the purified enzyme exhibits a p-nitrophenylphosphatase activity which can be completely inhibited by vanadate. In aqueous medium, p-nitrophenyl phosphate hydrolysis proceeds at only 0.5% of the rate of ATP hydrolysis, and both activities can be stimulated 3- to 4-fold by decreasing the pH from 7.5 to 6.5. Addition of the organic solvent dimethyl sulfoxide (10-40%), which has been shown to favor the E2 conformation, stimulates the p-nitrophenylphosphatase activity but inhibits the ATPase activity. At pH 7.5, the Km for p-nitrophenyl phosphate decreases when dimethyl sulfoxide is present. In the presence of 30% (v/v) dimethyl sulfoxide, the phosphatase activity can be inhibited by ATP (K(i) 300 microM) or by P(i) (K(i) 1 mM). The H(+)-ATPase incorporated into liposomes retains pNPPase activity, but it does not support H+ transport. Gel electrophoresis reveals that the pattern of H(+)-ATPase cleavage by trypsin changes when vanadate, Me2SO, or both compounds are present in the medium, regardless of the pH used during trypsinization. We propose that p-nitrophenyl phosphate is hydrolyzed by a H(+)-ATPase conformation distinct from that which hydrolyzes ATP, most probably an E2-like form. We also suggest that, in addition to the E1-E2 transition, the enzyme activity can be regulated by protons at another step of the catalytic cycle.

Pressure effects on the Raman spectrum of proteins: stability of the salt bridge in trypsin and elastase

Pressure induced changes in trypsin and elastase have been studied with Raman spectroscopy. The secondary structure of the enzymes has been calculated by a singular value analysis of the amide I band. The pressure titration of trypsin indicates a conformational transition. The salt bridge between Asp(194) and Ile(16) is disrupted and the enzyme becomes inactive. No such changes are observed for elastase. It is concluded that the presence of the bulky Val(216) and Thr(226) in the substrate binding site prevents the enzyme changing its conformation as a function of pH and pressure. Starting from a simple electrostatic model, an estimate is made of the dielectric constant of the environment of the salt bridge in trypsin.

Effect of secretin upon the levels of pancreatic enzymes in blood serum

We have evaluated the serum changes of trypsin, pancreatic isoamylase and lipase, after rapid infusion of secretin, in 45 patients with chronic pancreatitis compared with 35 healthy control subjects. On the basis of duodenal intubation results, chronic pancreatitis patients were divided into two subgroups at different levels of functional impairment. Using the peak activities of the enzymes we have been able to separate the two chronic pancreatitis subgroups by statistical difference; only trypsin distinguishes healthy control subjects from mild to moderate chronic pancreatitis. Therefore, we propose to put into clinical practice this serum provocative test to evaluate the functional damage of an established chronic pancreatitis.

Complexing of trypsin with soluble copolymers based on vinyl pyrrolidone and carboxylic acids

The authors have studied the interaction of trypsin with soluble copolymers of vinyl pyrrolidone with crotonic or methacrylic acids, containing from 4 to 30 mole % carboxylic acids. It is shown that in the neutral pH region trypsin forms with these copolymers soluble complexes of a type varying with the mode of distribution of the carboxyl groups in the polymer chain, the functional composition of the copolymers and the molar ratio of copolymer: trypsin in the initial complex system. The paper defines the optimal conditions for obtaining trypsin complexes with copolymers of vinyl pyrrolidone with crotonic acid in which trypsin completely retains enzymatic activity for protein substrates. The methods of dispersion of optical rotation and circular dichroism are used to make a detailed study of the conformational state of the trypsin macromolecules in the complexes. It is concluded that certain structural changes in trypsin lead to partial inhibition of the enzyme in the complexes obtained with a molar ratio of copolymer: trypsin < 1 in the initial complex system.

Potentiometric and spectroscopic studies of the reaction between trypsin and its inhibitors on chemically modified solid surfaces

The potential of a titanium metal electrode modified with trypsin changes as a result of the complex formation reaction between trypsin and its inhibitor, aprotinin, dissolved in the solution. A similar potential change in the opposite direction occurs by the reaction between aprotinin-modified electrode and trypsin in the solution. The induced changes in both cases depend on the pH of the solution, showing the maximum change at pH = 9.5. The potentiometric response of the trypsin-modified electrode for the consecutive addition of aprotinin and proflavine proves that trypsin bound on the solid surfaces reacts with aprotinin much more strongly than with proflavine. This result is fully consistent with the spectroscopically observed behavior of a trypsin-modified quartz plate against these inhibitors. The surface coverage of trypsin on the quartz plate is also determined by a near-ultraviolet absorption measurement.

Effet du pH sur la fixation d'inhibiteurs compétitifs synthétiques par la trypsine

Conformational changes in trypsin (EC 3.4.4.4) induced by the binding of competitive inhibitors (benzylamine, butylamine, N-α- benzoyl- l-arginine ) and, in some favourable cases, of substrates ( N-α- benzoyl- l-arginine ethyl ester, N-α- benzoyl- l-arginine amide) have been studied at 10°, in the pH range from 1.5 to 12.5. The formation of the trypsin-inhibitor (or trypsin-substrate) complex produces changes in the enzyme's rotatory power; this modification is small at neutral pH but substantial in acid and alkaline pH ranges where trypsin is reversibly denatured. It seems that a more compact structure of the protein results from the formation of the complex. It has been shown by spectrophotometric measurements in alkaline medium that this conformational change is accompanied by the burying of one tyrosine residue which ionizes normally in the free enzyme. The formation of the enzyme-substrate complex coincides with a proton release at pH values lower than 6. Results obtained both by proton titration at pH 4.1 and by polarimetric measurement of the affinity of benzylamine for trypsin at pH 3.5 and 2.5 indicate the existence of an interaction between the positive charge of the inhibitor and the negative charge of a carboxyl group of the enzyme having a p K of 4.7. The role of the substrate in maintaining the structural and catalytic properties of trypsin is discussed.

On the mechanism of enzyme action. LXXIV. Conformational changes in trypsin upon acetylation in aqueous buffers and dimethyl sulfoxide

The immediate effects of acetic anhydride upon the activity of trypsin have been investigated by means of optical rotation. The data indicate that the initial inactivation and subsequent reactivation of the enzyme in alkaline solution can be explained by reversible changes in enzyme conformation. In weakly acid solution reversible acetylation of the reactive serine of trypsin apparently takes place. Studies of the acetylation of succinyltrypsin reveal that this enzyme derivative is more stable to the anhydride, but that the mechanisms operative in acid and alkaline media are the same as those described for trypsin. Optical rotatory studies of trypsin dissolved in dimethyl sulfoxide afforded some insight into the behavior of the enzyme in this particular solvent. The results of these observations have been related to the over-all significance of the free amino groups of the enzyme in catalytic activity.

THE ESTIMATION OF ACTIVE NATIVE TRYPSIN IN THE PRESENCE OF INACTIVE DENATURED TRYPSIN

Inactive denatured trypsin changes into active native trypsin in the protein solutions which have been used to estimate tryptic activity. If the digestion mixture, however, is alkaline enough and contains enough urea this change does not take place. Such a digestion mixture can be used to estimate active native trypsin in the presence of inactive denatured trypsin.