BGDB/Pracs/Enzymes

< BGDB‎ | Pracs

There are 3 parts to this experiment where we look at the interaction between:

  • A. Casein, trypsin, pancreatic trypsin inhibitor
  • B. Casein, chymotrypsin, pancreatic trypsin inhibitor
  • C. Casein, chymotrypsin, trypsin, pancreatic trypsin inhibitor

Casein is a milk protein whose peptide bonds are hydrolysed when incubated with pancreatic proteases (trypsin & chymotrypsin). It will be broken down into smaller peptide fragments.

Trichloroacetic acid denatures and precipitates proteases including trypsin and chymotrypsin, as well as intact casein and large casein fragments. Smaller casein fragments remain soluble in solution. Bradford reagent is added to the supernatant (some consisting of soluble casein fragments) and absorption spectrophotometry at a wavelength of 620nm is utilised to gauge the amount of casein which is proportional to the rate of activity.

Contents

Experiment A

Experiment A

There are the following types of tubes:

  1. casein + trypsin + trichloroacetic acid (immediate addition) = time zero
  2. casein + trypsin + trichloroacetic acid (added at different time intervals)
  3. casein + trypsin + pancreatic trypsin inhibitor + trichloroacetic acid (immediate addition) = time zero
  4. casein + trypsin + pancreatic trypsin inhibitor + trichloroacetic acid (added at different time intervals)
  • For tube type 2, the greater the exposure of casein to trypsin, the greater cleavage of smaller casein fragments and thus high absorption value. However, there is a limit where active sites of trypsin are saturated and rate of activity cannot further increase.
  • Although PTI inhibits the action of trypsin in tube type 4, it is not permanent and rarely trypsin is allowed to function accounting for the slow and low rate of activity.
  • For tubes 1 & 3, the immediate addition of trichloroacetic acid (TA) leads to immediate denaturing and precipitation of trypsin (and PTI), thus no reaction occurs.

Experiment B

Experiment B

There are the following types of tubes:

  1. casein + chymotrypsin + TA (immediate) = time zero
  2. casein + chymotrypsin + TA (added at different time intervals)
  3. casein + PTI + chymotrypsin + TA (immediate) = time zero
  4. casein + PTI + chymotrypsin + TA (added at different time intervals)


  • For tubes 1 & 3, the immediate addition of TA leads to immediate denaturing and precipitation of proteases, casein, large casein fragments (no action)
  • Tube types 2 & 4 activity are the same as PTI is specific for trypsin and doesn’t inhibit chymotrypsin. Thus the greater exposure of casein to chymotrypsin, the greater the rate of activity until saturation is reached.

Experiment C

Experiment C

There are the following types of tubes and substances are in order of addition:

  1. chymotrypsinogen + trypsin + TA (immediate addition) + casein = time zero
  2. chymotrypsinogen + trypsin + PTI (at different time intervals) + casein + TA (at least 1 min after addition of casein)

Trypsin converts chymotrypsinogen to its active form chymotrypsin.

  • For tube 1, the immediate addition of PTI inactivates trypsin which means chymotrypsinogen is not activated. As both proteases are inactive, no cleavage occurs.
  • For tube type 2, the addition of PTI at different time intervals means that casein is exposed to active chymotrypsin for different amounts of time. The longer the exposure to active chymotrypsin, the greater the rate of activity and production of small soluble casein fragments. That is, the greater the time allowed for trypsin to react with chymotrypsinogen to turn it to active chymotrypsin before addition of PTI, the greater the proportion of chymotrypsin in the solution and thus rate of activity.

Questions

  • 1. At a protein structure level, what does a trypsin inhibitor do?
    • Trypsin inhibitor is a competitive antagonist that binds to the active site of trypsin, inhibiting it from binding with a substrate and carrying out its function.
  • 2. What is the normal function of trypsin in digestion?
    • Trypsin activates inactive digestive proteases such as chymotrypsin, elastase, and carboxypeptidase.
  • 3. What size molecules are they?
    • PTI is approximately 6kD (kD= kilodaltans). This is a small protein.
  • 4. What is the relationship between pancreatic trypsin inhibitors and pancreatitis?
    • Normally, digestive enzymes secreted by the pancreas do not become active until they reach the small intestine. However if the zymogens are activated while still within the pancreas, pancreatitis/autodigestion will occur. Trypsin inhibitor is a safety mechanism which prevents this from occurring.
  • 5. What foods contain trypsin inhibitor proteins, and what is the role of the inhibitors in those foods?
    • Some plant seeds eg. lentils etc contain a coating of trypsin inhibitor to prevent their digestion (the inhibitor tastes unpleasant to insects etc). This enables the plants to proliferate.

Discussion

  • 1. What can you deduce from the results from Experiments A & B?
    • The longer the exposure of casein to digestive proteases, the greater the rate of activity until the enzymes are saturated. Pancreatic trypsin inhibitor is only specific to trypsin.
  • 2. Why do the enzymes hydrolyse casein but not themselves?
    • There are recognition groups within the enzymes themselves and are not able to hydrolyse themselves. Also, the target peptide bonds of the enzymes are well hidden from their external surface. They are eventually hydrolysed but at a much slower rate than casein.
  • 3. Why are Experiments A & B important controls for Experiment C?
    • Exp A & B confirm the functionality of trypsin and chymotrypsin and PTI. This is important to establish that the validity of the results from Exp C regarding the interaction between trypsin, chymotrypsin and PTI.
  • 4. What can you deduce from the results for Exp C?
    • Trypsin activates chymotrypsinogen to chymotrypsin which can cleave the peptide bonds in casein. Thus the presence of PTI not only inhibits trypsin, but the other zymogens that trypsin is responsible for activating.