effect of trypsin

Introduction

Trypsin is a proteolytic enzyme found in the small intestine, which is essential for digestion. A proteolytic enzyme is one that catalyses the catabolism of proteins, which in this case is casein (protein in milk). This occurs by trypsin catalysing the hydrolysis of proteins into smaller peptides. These peptide units are then hydrolysed into amino acids via other proteases, rendering them available for absorption into the bloodstream. Additionally, as trypsin functions in human cells, its optimal temperature is 37 degrees Celsius as expected, which parallels the average human body temperature. Contrastingly, the effect of concentration on enzyme activity will be tested via gelatine-coated photographic film. Trypsin digests gelatine into soluble amino acids. These acids then dissolve the silver halides in the film into metallic silver. A similarity between both these factors is that trypsin activity will be tested through either medium’s transparency.

Thus, this experiment was chosen in order to examine the effect of concentration and temperature of trypsin activity, and the extent of these factors in enabling it to function at its optimal level. Conditions that deviate from the optimal elicit severe adverse effects as evident in the condition; malabsorption. This is a result of the pancreas producing insufficient amounts of trypsin, and thus hinders its ability to absorb nutrients from food. In time, this leads to a deficiency in essential nutrients, and can lead to anaemia or malnutrition. Hence, this signifies the importance of trypsin functioning at its optimal level.

Consequently, this research investigation contributes to a broader understanding of the ideal conditions that optimise trypsin activity, as well as a broader knowledge of the general trends of varying conditions on enzyme activity.

Risk Assessment

Method

Method

Results

Results

Discussion

As Figure 1 and 2 suggest, the hypothesis was supported. This is because the time taken for trypsin to catalyse the hydrolysis of casein and gelatine, was lowest at 40 degrees Celsius and 2g/mL, respectively, indicating that at these conditions trypsin activity was most efficient.

Firstly, in respect to temperature, from 0-40 Degrees Celsius, the rate of trypsin activity progressively increased as indicated by the decrease in time of catalysis. This can be attributed to the increased kinetic energy of particles due to the temperature rise, which causes more collisions, thus increasing the frequency of successful collision. Additionally, the increased kinetic energy means that enzyme and substrate particles are more likely to have energies that are equal or greater than the activation energy. Due to this, along with the lowered activation energy due to the enzyme, the proportion of successful collisions increase, and hence the rate of reaction. However, at 80 degrees Celsius, there is an increase in time for hydrolysis, and thus a drop in the rate of trypsin activity. This is a result of denaturation, which distorts trypsin's tertiary structure, and thus the active site, so it cannot effectively bind to casein. For this reason, its catalytic activity and function is impaired, which is generally irreversible.

On the other hand, from 0.01% to 2%, the results demonstrate a consistent increase in the rate of trypsin activity. This is because increasing trypsin concentration will generally increase the rate of reaction, as more enzymes are available to collide with substrate molecules, thus the frequency of fruitful collisions increase. However, this will only have an effect up to a certain extent, until substrate concentration becomes a limiting factor, although this could not be determined with the concentrations in this experiment.

Although the results were consistent with the hypothesis, there are several limitations that may have inhibited the reliability and validity of the data. Inevitably, a systematic error is the range of error in lab equipment. For instance, in the measuring cylinder, test tube and thermometer have an uncertainty of +/- 0.1 and 0.2mL. Although this does not affect reliability, this falters validity especially as the experimental measurements are not reflective of true values. Furthermore, a random error is the use of improper photographic film. The film used was not gelatine-coated, and thus the transparency cannot be attributed to hydrolysis of protein (gelatine). Hence, this compromises the investigation's validity, and reliability. Moreover, an imperative aspect of this experiment is to maintain the required temperature in both Part A and Part B. However, although attempts were made to achieve this by constantly measuring temperature, it is likely that the temperature fluctuated from the requirement, and thus impedes reliability. For instance, the constant opening of the lids on water baths and door opening of the incubator may have resulted in invalid temperatures. Further attempts to reduce errors and biases are that the same person shook the test tubes to ensure the same amount of pressure was applied, and the same person measured the transparency with the indicator to negate observational errors.

To counteract these errors, future improvements that can be suggested include utilising calibrated equipment to ensure precision. The range of error is compounded due to its repeated use, and thus should be taken into consideration by taking a mean measurement to ensure reliability and precision. Furthermore, another suggested improvement is using the correct type of photographic film to ensure the results can be attributed to the dependent variable.

Furthermore, a broader improvement of this experiment is measuring a vaster range of concentrations, while maintaining controlled conditions. This should be conducted several times to increase its reliability, and in order for it to be conducive to real-world applications. As research on optimal temperature for trypsin is already well established, and conditions such as fever can lead to indigestion or constipation due to denaturation, there is a gap in terms of concentration. Nonetheless, if an effective concentration of trypsin can be determined, it could potentially be administered by medical professionals as an artificial source of trypsin to negate the effects of malabsorption as earlier mentioned. Further, this would alleviate the risks associated with insufficient trypsin levels, including nausea, kidney failure and even cancer. Consequently, this investigation has broader societal implications if further researched, as it can notably reduce the associated economic burden on the healthcare system.

Discussion

As Figure 1 and 2 indicate, the hypothesis was supported. This is due to the fact that the time taken for trypsin to perform hydrolysis of casein and gelatine was lowest at 370 C and 2g/mL, indicating that at these conditions trypsin activity is most efficient.

Firstly, in reference to temperature, at 0 to 400C, the rate of trypsin activity progressively increased, as demonstrated by the reduced time of hydrolysis. This is attributed to an increase in kinetic energy due to temperature rise. This results in reactant particles moving at a greater speed and increasing the amount of collisions, which in turn increases the frequency of fruitful collisions, and hence the rate of reaction. Along with this, an increase in kinetic energy also means that more reactant particles are likely to be equivalent or greater than the activation energy. For this reason, as well as the lowered activation energy due to the action of trypsin, significantly increases the proportion of successful collisions, and hence increases the rate of reaction. However, at 800C there was a drop in trypsin activity, illustrated by the increase in catalysis time. This is due to the denaturation of trypsin, which distorts the shape of the tertiary structure, and thus the active site. As a result, this impedes its ability to effectively bind with casein, rendering the function of trypsin impaired.

Furthermore, in terms of concentration, increasing concentration from 0.01% to 2% is proportionate to an increase in trypsin activity. This is because increasing trypsin concentration in turn raises the amount of enzyme that is able to bind with gelatine (protein). This, therefore, increases the frequency of successful collisions, and thus the rate of reaction. However, this only occurs to an extent until the substrate becomes ‘saturated’ and thus a limiting factor. However, due to the restricted range of the concentrations experimented, this could not be determined.

Conclusion

In conclusion, increasing temperature and concentration generally increases the rate of trypsin activity, with the optimal being 400C and 2g/mL, respectively. The hypothesis was supported, however, it may not entirely be reliable due to the limitations mentioned above. Further improvements include examining a diverse range of concentrations of trypsin as a more helpful determinant of its catalytic properties, so it is of more assistance in broader contexts.

Conclusion

References

References

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