Effect of enzyme concentration on the initial rate of an enzyme-controlled reaction

Introduction

Enzymes are types of proteins necessary for catalyzing chemical reactions in the body. Enzymes are synthesis just as ribosomes do other proteins synthesis in the cell.  Enzymes are substrate specific and therefore react with specific substrates to enhance the rate of chemical reactions in cells (Karbach, Veit, & Ewe, 2009). Chemical reactions would otherwise be slower in the cells were it not for the action of enzymes and thus inhibiting basic human functions like breathing and digestion.

Trypsin is an enzyme that acts on proteins; it is secreted in the inactive form of trypsinogen from the pancreas into the small intestines. It acts on protein substrates resulting in the formation of amino acids and peptides (Hughes, Cohen, Arvan, & Seamonds, 2009).  Trypsin just as any other enzyme is pH specific and therefore requires specific levels of alkalinity and acidity for optimum action.

In the context of this experiment, the main objective was to determine how enzyme concentration changes the effectiveness of trypsin enzyme.

Health and safety

1% to 5% of Trypsin solution is an irritant and should be handled with care. If the solution splashes on the skin, it should be washed using plenty water as quick as possible. Eye protection should also be worn by all students in the laboratory to ensure their eyes are safe from the solution. In any case, the solution gets in any students eye; it should be reported immediately to the laboratory instructor (Poupon, Poupon, Grosdemouge, & Erlinger, 2009). Extra caution also needs to be taken when handling the enzyme’s solution; the enzyme may be kept in a refrigerator, but students should ensure that the experiment is completed within one session as the activity of protease enzymes decline with time.

Silver halides are low hazard materials and therefore can be used without considering extra caution, it should, however, be noted that old-fashioned photographic film is used for this experiment as the modern films do not use gelatin coatings (Zhang & Vardhanabhuti, 2014).

Background

Trypsin (an example of protease) acts on milk protein (casein), the action results in breaking down of the milk from the opaque white colour into a clear solution (Poupon, Poupon, Grosdemouge, & Erlinger, 2009). The reaction can, therefore, be monitored by a colorimeter or a spectrometer as the clear solution allows for easy monitoring. The reaction apparatus can be arranged as shown in fig 1.0.

Fig 1.0: Apparatus for monitoring the rate of reaction.

 

 

Procedure

1% trypsin solution was taken and diluted using distilled water into various test solutions of 0.2%, 0.4%, 0.6% and 0.8% respectively. The solutions were made up to 10 cm3 in boiling tubes and each of the boiling tubes containing solutions labeled using a lab maker. The resultant solutions were recorded in the laboratory notebook. 2 cm3 of trypsin, and 2 cm3 of water were added to the cuvette and used to set the absorbance of the calorimeter/spectrophotometer to zero. A suitable filter/wavelength for analysis of a blue solution was determined from the BIOL4A graph. 2 cm3 of milk powder solution was measured and added to a second cuvette. To the milk solution, 2 cm3 of trypsin solution was added and the solution quickly mixed and placed on the calorimeter/spectrophotometer and the stopwatch started.

The value of absorbance was measured immediately and subsequently at 15 seconds intervals until there was little change in the absorbance. The results were recorded in the table of results and used for further analysis. The procedure was repeated using new cuvettes with different concentration of trypsin from 1% to 0.2% and the results recorded in the table of results.

Results

Trypsin Concentration /% Absorbance
0 15 30 45 60 75 90 105
1 0 3.5 5.5 6 7.8 8.8 9.4 9.7
0.8 0 2.7 4.5 5.8 6.8 7.5 8.2 8.7
0.6 0 1.7 3 4 5 5.8 6.5 7
0.4 0 1 1.7 2.5 3 3.8 4 4.5
0.2 0 0.5 1 1 1.7 2 2 2.3
0 0 0 0 0 0 0 0 0

 

Fig 2.0: A plot of Absorbance Versus Time

Fig 3.0: A plot of initial rate of reaction versus enzyme concentration.

For a comparison of the rates of reaction of the five sets of reaction, the rates at the beginning of the reaction were determined. This is because once the reaction begins, there is a variation of substrate concentration as there is a conversion of the substrate to the product at different rates. The differences in reaction rates captured from the beginning are therefore important indicators of difference in enzyme reactions caused by the difference in the concentrations of the enzymes (Hughes, Cohen, Arvan, & Seamonds, 2009). The initial rate of enzyme reaction was calculated from the slope of the curves of the five different reactions at 30 seconds from the beginning of the reaction. An ideal case should, however, be as close to zero as possible which is not practical to implement. A second graph was then drawn with the initial rate of reaction plotted against the concentration of the enzyme in the reaction.

Fig 4.0: Absorbance Versus the wavelength

A plot of absorbance versus wavelength results in an absorption spectrum of a sample under investigation. It is useful in the identification of unknown samples because samples have unique characteristic absorption spectra.  The spectra shown in fig 4.0 was used for this experiment, the enzyme peaked at an approximate wavelgth of 600 nm.

Discussion

Trypsin concentration was the independent variable in the experiment while the rate of reaction (absorbance) was the dependent variable in this experiment. A rapid reaction occurs between the milk substrate and the enzyme resulting in the quick depletion of the milk and thus formation of a clear solution. At the beginning of the experiment, controlled variables such as substrate concentrations are normally the same at all levels for the independent variable thus making it easier for comparisons to be done. Absorbance values for the experiment may be higher than the actual values due to the presence of systematic errors. Other factors that may affect the rate of an enzyme-catalyzed reaction include the pH (Karbach, Veit, & Ewe, 2009). A change in pH of the reaction results in changes shape and size of the active site of the enzyme thus resulting in a variation in the rate of enzymatically catalyzed reactions. Enzymes thus work best at their optimum pH values.

From the graph, it is clear that a linear relationship exists between the initial rate of reaction and enzyme concentration that is the reaction rate is directly proportional to the concentration of the enzyme. This is because doubling or increasing the number of enzymes in a reaction increases the active sites available for slotting in of the substrate (Poupon, Poupon, Grosdemouge, & Erlinger, 2009). It should, however, be noted that the increase of enzyme concentration can be inhabited by the unavailability of the substrates.

Other factors that may inhibit the rate of enzyme catalyzed reactions include; the concentration of the substrate, the pH, temperature among other factors. An increase in substrate concentration also results in an increase in the rate of enzyme catalyzed reaction; this is, however, subject to the concentration of the enzyme and other factors. Temperature is also a vital component for optimum functioning of the enzymes (Zhang & Vardhanabhuti, 2014). Enzymes work best at specific temperatures, an increase in temperature beyond these temperatures denatures the enzymes and thus result in reduced rates of enzyme controlled reactions. A decrease in temperature below the specified temperature for enzyme operation results in inactivation of enzymes and thus slow enzyme reactions.

Enzymes work in specific pH values beyond which they are damaged. For example protease in the stomachs work under acidic environments and are damaged in alkaline conditions.

Conclusion

Enzymes are proteins and are useful in chemical reactions in the body as they lower the activation energies of reactions thus catalyzing the reactions. All chemical reactions in the body including anabolic and catabolic reactions are enzyme controlled. Enzymes are site specific and therefore are unique in structure and shape thus binds only to specific sites of the substrate resulting in the formation of an enzyme-substrate complex. Substrates reshape the size and shapes of binding thus resulting in perfect binding of the substrates and the enzymes.

Factors that affect the size and shape of the active site such as higher temperatures than the enzymes optimum temperature, high and low pH values, the presence of non-competitive inhibitor reduces the enzymes ability of binding with the enzyme and thus its activity (Poupon, Poupon, Grosdemouge, & Erlinger, 2009). The rate of reaction of en enzyme-catalyzed reaction may also be reduced by the presence of competitive inhibitors that reduces the likelihood of the substrates to bind thus reducing the rate of reaction.

Trypsin is an example of protease and thus acts on proteins to form peptides and polypeptides. Trypsin being a protein it works under specific conditions of temperature, pH, and concentrations of body fluids.  In this experiment, the effects of trypsin concentration on the rate of enzyme catalyzed reactions were examined, and the results analyzed as shown in the discussion section above (Karbach, Veit, & Ewe, 2009). Trypsin concentration affects the rate of digestion of proteins that is an increase in trypsin results in an increase in the initial rate of enzyme catalyzed reactions. It is, however, wise noting that the rate of reaction of an enzyme-catalyzed reaction is inhibited by other factors such as the concentration of the substrates.

The main objective of the experiment was achieved as the initial rate of reaction of the enzyme was compared to the concentration of the enzyme (Hughes, Cohen, Arvan, & Seamonds, 2009). A linear relationship was observed which is true for the initial.

 

 

References

Hughes, W., Cohen, S., Arvan, D., & Seamonds, B. (2009). The Effect of the Alkaline Tide on Serum-Ionized Calcium Concentration in Man. Digestion15(3), 175-181. http://dx.doi.org/10.1159/000198001

Karbach, U., Veit, J., & Ewe, K. (2009). Postprandial Cholylglycine Serum Concentration and Extent of Heal Inflammation or Resection in Crohn’s Disease. Digestion34(3), 202-206. http://dx.doi.org/10.1159/000199330

Poupon, R., Poupon, R., Grosdemouge, M., & Erlinger, S. (2009). Effect of Portacaval Shunt on Serum Bile Acid Concentration in Patients with Cirrhosis. Digestion16(1-2), 138-145. http://dx.doi.org/10.1159/000198064

Zhang, S. & Vardhanabhuti, B. (2014). Effect of initial protein concentration and pH on in vitro gastric digestion of heated whey proteins. Food Chemistry145, 473-480. http://dx.doi.org/10.1016/j.foodchem.2013.08.076

 

Advertisements

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s


%d bloggers like this: