Experimental Design and Analysis
Substrate Specificity of Proteases
Assignment
Introduction
Proteases are proteolytic enzymes that break the peptide bonds in other proteins and polypeptides to generate smaller peptide fragments and amino acids. Trypsin and chymotrypsin can act as esterases as well as proteases. The protease cleaves the bond between the amino acid and the p-nitroanilide moiety to release free p-nitroaniline, which is yellow and easily measured in a spectrophotometer. They have numerous functions, one of which is to generate aminoacids from protein in food for re-utilization.
Protease reactions do not produce colored products directly, so we must adapt them for assay. There are many ways of doing this, including these two:
1. One method is to treat the colorless product with a chemical that then produces a color, i.e. if compound AB breaks down to A + B (both colorless), but B reacts with C to produce D (colored), then the amount of D can be used to indicate the amount of B produced. Ninhydrin is a compound that reacts with amines to produce an intense blue-purple color. All proteinshave a single free amino group at their N-terminus, but when they are hydrolysed the total number of free amino groups increases as peptides and amino acids are produced.
2. Another method to assay proteases is to replace the natural substrate with a synthetic substrate that will generate a color when it undergoes the reaction. The synthetic substrates you will use are of the type xxx-amino acid-p-nitroanilide; simple colorless esters which are recognized by the protease as a substrate. For example, trypsin, chymotrypsin can act as esterases as well as proteases. The protease cleaves the bond between the amino acid and the p-nitroanilide moiety to release free p-nitroaniline. After a set amount of time the reaction will be stopped by the addition of 30% acetic acid. The specific activities will be calculated from amount of free yellow p-nitroaniline formed as determined by measuring its absorbance at 410 nm in a spectrophotometer.
The activity of proteases is usually expressed in ‘units of activity’ where 1 unit is the amount of enzyme that will catalyze the formation of 1 µmol product per minute. A more useful measure is the specific activity which relates the activity to the actual quantity [mg] of protease in the assay. Hence specific activity is usually quoted in ‘units of activity /mg protease’ or simply ‘units/mg’.
Substrate Specificity of Proteases: When hydrolyzing the peptide bond in a protein various proteolytic enzymes exhibit different specificities for the amino acids contributing to the peptide bond. Some proteases (exopeptidases) start at the end of a polypeptide chain and sequentially remove one amino acid at a time. Others (endopeptidases) break internal peptide bonds and initially produce peptide fragments. For example, trypsin and chymotrypsin are endopeptidases. Some endopeptidases are relatively non-specific and hydrolyze the peptide bond between any two of the 20 amino acids while others have a strong or absolute preference for certain amino acids. Thus, trypsin only cleaves the peptide bonds after (on the C-terminal side of) the basic amino acids lysine and arginine. Chymotrypsin is less specific, cleaving after large hydrophobic amino acids such as phenylalanine, tyrosine, tryptophan, leucine and methionine. This difference in specificity depends on the nature of the amino acid side chains present in the active site of the proteases that bind to their substrates. In digestion, a combination of exopeptidases and endopeptidases with different substrate specificities will reduce most dietary proteins to individual amino acids.
Note: Don’t confuse the terms “substrate specificity” and “specific activity”
Aims
In this experiment your group will design experiments to compare the substrate specificities of trypsin, chymotrypsin and pepsin using three synthetic substrates: BAN, SPN and HBN.
Complete the following questions and hand your group work along with individual contribution sheet signed by all members of the group Deadline 5 pm, 12th April
1. Show all your experimental groups (10 marks)
Illustrate your randomization plan for conducting experiments using
2. Latin square arrangement (10 marks)
3. Show a complete random number sequence and assign your treatment combinations. (10 marks)
4. Your group decides to repeat twice per experimental group. Due to limited time for conducting experiments, you are to collaborate with another group forming a team, consequently, there will be two different experimenters. Illustrate your randomization plan of treatment groups. (10 marks)
5. What is the total number of subjects to be measured? What is the ratio of your sample size per student group? (10 marks)
6. What is the outcome measure (response variable)? What is the unit of the outcome measure? (10 marks)
7. What is your experimental type? (10 marks)
8. List 5 factors (write in full sentences) that may improve the response variable precision? (30 marks)