Background
The study of the kinetics of biochemical reactions is significant for clinical, industrial, and laboratory applications, as it contributes to a better understanding of a substance’s behavior in solutions and mixtures, as well as its interactions with the body. Using empirical models helps identify patterns in the distribution of enzymatic reaction rates and thus estimate the optimal, threshold, and peak values required for full enzyme activation. Strictly speaking, enzymes are biologically active proteins that catalyze biochemical processes within an organism. Most enzymes function according to the principle of specificity, which states that each enzyme has a unique substrate (Copeland, 2023). The activity of an enzymatic reaction can be influenced by many factors, including the concentrations of the substrate and the medium (temperature, pH), as well as the characteristics of the medium (temperature, pH) in which the reaction occurs.
Papain is a natural protein-origin enzyme found in Carica papaya’s immature fruit. From a functional perspective, papain is a protease, meaning it cleaves peptide bonds within protein molecules (Tan, Chang, and Meng, 2019). The applicability of papain is quite broad: it has been used in the food, cosmetic, clinical, and pharmaceutical industries, indicating extensive practical applications.
In contrast, the substrate used in this work was azocoll, a collagen protein stained with azo (Tan et al., 2019). Due to its relatively simple structure, azocoll is readily cleaved by enzymes. In addition, azocoll has been widely used in laboratory, clinical, and biotechnology industries to study the kinetic properties of enzymes. In the present work, both substances, papain and azocoll, are used to evaluate the kinetic properties of their interaction. Thus, the present work aimed to construct a progress curve for the enzymatic hydrolysis of azocoll with papain and to study its kinetics.
Materials and Methods
The present laboratory work was based on a quantitative experimental design. One tablet of papain prepared in advance was dissolved in 10 mL of water to obtain the appropriate solution. 125 µl of azocoll substrate was introduced into the cuvette for spectrometric studies, after which 2.5 mL of the prepared papain solution was added. After vigorously stirring the cuvette contents for a few seconds, the sample was placed in a spectrophotometer at 520 nm.
The absorbance of the mixture was measured every minute for 30 minutes and recorded in a table; a total of 30 recordings were made. From the collected data, an absorbance vs. observation time curve was constructed, allowing evaluation of the progress of the investigated process and thus answering the question at which time point the enzyme activity appears to be maximal. In addition, a graphical analysis of the curve was performed, including determining the slope of the linear portion of the graph, which indicates the rate of the enzymatic reaction.
Results
The results of this work were the measured absorption rates of the mixture over time (see Appendix A): measurements were taken every minute for half an hour. Plotting the data as a scatter plot (Figure 1) shows that the relationship between absorbance and time is not linear but follows a sigmoidal shape (Han et al., 2019). The plot shown in Figure 1 was extended using graphical analysis techniques, as shown in Figure 2.
A deeper analysis of the plotted dependence yields several conclusions. First, at the beginning of the period (approximately the first 20 minutes), the dependence is linear: the absorption increases rapidly. Secondly, the absorption practically did not change over the last ten observations, reaching a horizontal plateau. Based on the data, the slope of the linear part of the diagram was 0.13, as shown in equation [1]. In addition, it was observed that the maximum absorption for papain was 2.22 — this corresponds to the pattern on the curve when the transition to the horizontal plateau.
slope=∆y/∆x=(1.42-0.50)/(13.03-5.90)=0.92/7.13=0.13 〖min〗^(-1) [1]
Discussion
The present work aimed to investigate the kinetic properties of the papain-catalyzed reaction with the azocoll substrate. The results of the constructed progress curve show that the absorbance increases with time: the maximum growth rate of this dependence was 0.13 min-1. The graph’s growth indicates that the synthesis (rather than decomposition) reaction is proceeding and that the reaction intensity is increasing. If the enzymatic reaction were based on decay (reverse reaction), this would decrease absorbance over time.
The results obtained are generally supported by published scientific and theoretical understanding. The sigmoidal shape of the curve is typical of an enzymatic process occurring in two stages (Benucci et al., 2020; Han et al., 2019). In addition, although the calculated slope for the linear part of the graph was not an indicator of the reaction rate in a direct sense, it did indirectly indicate an acceleration of the reaction over time. In other words, the increase in absorbance during the course of the reaction indicates that the product concentration of the enzymatic reaction increased over time while the substrate concentration decreased.
There are several other essential aspects to consider when discussing the results obtained. The data show that the papain enzyme reached its maximum activity about 20 minutes after mixing the enzyme and substrate. Since the absorbance after 20 minutes is almost unchanged and the graph reaches a horizontal plateau, this may indicate that azocoll is exhausted during the reaction and that the enzymatic reaction is suspended. In addition, the calculated maximum absorbance can be used to calculate the concentration of the resulting interaction product; however, this requires knowledge of the extinction coefficient.
Limitations and Potential Errors
In the present work, several limitations can lead to errors. Under the experimental conditions, the pH and temperature of the process were not controlled. Since papain is a protein, suboptimal pH and temperature values can lead to partial or complete denaturation and, consequently, a decrease in papain’s enzymatic activity (Ma et al., 2021). The experiment could be repeated under different pH and temperature conditions to verify this. In addition, the graphical analysis used approximate values, which could have reduced the data’s accuracy — the use of more advanced technologies would have improved the reliability of the results. Finally, the purity and shelf life of the reagents used were not controlled, which was also a limitation of the experiment.
References
Benucci, I., et al. (2020) ‘Papain covalently immobilized on chitosan – clay nanocomposite films: application in synthetic and real white wine,’ Nanomaterials, 10(9), pp. 1-11.
Copeland, R. A. (2023). Enzymes: a practical introduction to structure, mechanism, and data analysis. New York: John Wiley & Sons.
Han, Z., et al. (2019) 3DViewGraph: learning global features for 3D shapes from a graph of unordered views with attention.
Ma, X., et al. (2021) ‘Optimization of low-temperature lipase production conditions and study on enzymatic properties of Aspergillus Niger,’ Iranian Journal of Chemistry and Chemical Engineering, 40(4), pp. 1364-1374.
Tan, Y., Chang, S.K. and Meng, S. (2019) ‘Comparing the kinetics of the hydrolysis of by-product from channel catfish (Ictalurus punctatus) fillet processing by eight proteases,’ Lebensmittel-Wissenschaft & Technologie, 111, pp. 809-820.