The Effect of Organic Solvents and Other Parameters on Trypsin-Catalyzed Hydrolysis of Nα[alpha]-Benzoyl-arginine-p-nitroanilide. A Project-Oriented Biochemical Experiment

Abstract

The study of enzymatic catalysis is a classical biochemistry experiment for undergraduate classes. We propose the utilization of the serine protease trypsin to discuss several parameters affecting enzyme catalysis. Hydrolysis of the chromogenic substrate N α -benzoyl-arginine-p-nitroanilide (BApNA) was followed by spectrophotometric monitoring. The optimal pH and temperature values were found to be 8.0 and 40 °C, respectively. Km and Vmax values were obtained by adjustment to Michaelis-Menten, Lineweaver-Burke, and Hanes equations. We then investigated the effect of organic solvents (a series of alcohols) on the hydrolysis of the chromogenic substrate. The reaction rate was reduced in the presence of methanol and further reduced by ethanol, 1-propanol, and 2-propanol, when compared to the data obtained with buffer. Finally the students were asked to measure the molar absorptivity of p-nitrophenol in the presence of the alcohols employed for the kinetic experiments. Thus they could learn that the value of this parameter varies with the solvent. These experiments were designed as a project-oriented approach to teach biochemistry methodologies and theoretical aspects of enzyme kinetics. They took about four months with four to six hours per week spent in the laboratory.

Immobilization of trypsin on chitosan gels: Use of different activation protocols and comparison with other supports

“…Hundred and fifty microliters of solution (soluble enzyme) or suspension (immobilized enzyme) of trypsin were added to 3 mL of the synthetic substrate solution (0.4 mM in sodium phosphate buffer 50 mM and pH 8.00, in the presence of ethanol). After determining the initial reaction velocity in Abs/min, the enzymatic activity in UBA p NA was determined using a molar extinction coefficient of 7.68 AU mM −1 cm −1 , determined by Correia and Bocewicz [15].…”

Partial characterization of a novel amphibian hemoglobin as a model for graduate student investigation on peptide chemistry, mass spectrometry, and atomic force microscopy

“…Especially for undergraduate students there are some problems related to the course time schedule but a well planed research project has been successfully conducted [27]. One of us had also a successful project‐oriented course for second‐year undergraduates in which the students had some instructor guidance to develop a project in enzymology allowing the students to introduce themselves to a relevant research problem in biotechnology, i.e., the effect of organic solvents on enzymatic catalysis [28, 29]. Thus, it is clear that project‐oriented approaches to graduate and even to undergraduate students must include an innovative research activity.…”

“…Peptidase enzymes may be immobilized in nanostructured materials such as silica [31]. The action of those immobilized enzymes upon natural or synthetic substrates can be evaluated by spectrophotometry, but also by chromatography followed by MS. Other aspects such as the effect of organic solvents over the activity of immobilized (or free) biocatalysts can also be investigated by the students [28, 32]. From the physiological chemistry viewpoint some aspects can be discussed with the students.…”

A Comprehensive Enzyme Kinetic Exercise for Biochemistry

“…Theoretical articles addressed new ways of considering the kinetic equations of enzymology where Ault introduced fractional representation of kinetic equations, Alberty considered rapid equilibrium forms for two substrate enzymes, Northrop considered the meaning of K m and V / K m from the Michaelis–Menten equation, Hofstee recommended equal distribution of data points about the K m value and against use of the double reciprocal plot, Chong demonstrated errors inherent in least-squares treatment of linearized equation forms, Martin addressed the preferred equation forms to extract accurate characteristic parameters, and Bruist focused on enzyme kinetic simulation using spreadsheets. A wide range of experimental articles covered the many aspects of enzymology including innovative visual demonstration of enzymatic activity, colorimeters interfaced to computers, coupled reactions in the visible wavelength range, the effect of alcohols on trypsin amidase activity, and specific activities of glucose oxidase–peroxidase coupled reactions …”