Enzymes Specificity For Substrates And Their Assay

Enzymes and Specificity

Discuss about the Enzymatic Assay of Chymotrypsin.

There are hundreds of different enzymes in organisms. For a compound to be found into active site of an enzyme, a precise fit is required. Hence enzymes are said to be very specific. This specificity of enzymes is utilized in the determination of the presence of enzymes in a tissue even without isolating each enzyme. A proper selection of substrate and reaction condition is a must for such experimentation. For instance proteolysis enzymes hydrolyse certain of peptide bonds of proteins but not the glucosisdic bonds of starch. On the other hand, Amylase has activity in hydrolysing glucosisdic bonds of starch but not peptide bonds.(Garysmith)

Chymotrypsin and trypsin  areproteolytic  enzymes and can hydrolyse  a protein such as casein to small peptides.To be specific, Trypsin and chymotrypsin are two pancreatic endopeptidases. They have a number of similarities in properties and mechanism of action. The class of enzymes to which they belong is known as the serine proteas. It is so because they have a serine residue at the active site and this residue becomes involved in the formation of covalent acyl enzyme intermediatewith the part of the substrate molecule during course of enzyme’s reaction.

The overwhelming guideline is the unmistakable and simple method of perception of the chemical response. Basic to all chemical catalysed responses is the way that a substrate ends up plainly changed over into an item and accordingly the point of any examine is to watch the time-subordinate arrangement of the item. To accomplish this, a method must be found to distinguish the item. Since development of item is specifically associated with the vanishing of substrate, its decay is a sufficient measure of the response. In situations where at least two items are framed, or at least two substrate particles are included in the response, the assurance of just a single segment is sufficient.4 Obviously the most straightforward distinguishable response segment will be picked.

A straightforward yet imperative condition is that substrate and item should vary in the watched highlight. The item might be exceptionally well distinguishable by a particular strategy, however in the event that the substrate demonstrates a comparable flag with equivalent force; no turnover can be seen by any stretch of the imagination. Regularly both parts demonstrate a little contrast of generally comparable vast essential signs, particularly when just little atomic adjustments happen, as with numerous isomerase responses (Figure 2). Such changes might be mainly noticeable, however are typically hard to measure, since substantial signs are for the most part subject to solid scrambling, so that the little change created by the compound response ends up plainly lost inside this clamour. In such cases the flag to clamour proportion must be broke down (Figure 2, right). When in doubt the power of the flag showed by the response must surpass the clamour at any rate by a component of two. This is a general issue, since any strategy is to a pretty much degree subject to disseminate. Dissipating can have different starting points, a few, e.g. shakiness of the instruments or estimations in turbid arrangements like cell homogenates, can't be kept away from, while others, similar to pollutions, turbidity brought on by pitifully dissolvable substances, dirtying, clean or air pockets can at any rate be decreased via cautious dealing with. Dispersing is likewise most minimal if just the watched segment (substrate or item) creates the flag (e.g. ingestion), while alternate segments demonstrate no flag (no retention) in the watched go, so that the response begins really at zero and any adjustment in the flag shows the progressing response.(Bisswanger, 2014)

Trypsin and Chymotrypsin

Chymotrypsin and trypsin are generally engrossed in breaking a peptide bond in a protein molecule or putting it simply they break amide bonds in protein molecules. They have slight different preferences of their action. Chymotrypsin is oriented more towards cleaving bonds to C-terminal side of an aromatic amino acid (for example phenylalanine,tyrosine,or tryptophan).To a small extent Chymotrypsin also breaks other hydrophobic amino acids. Trypsi, however, have preferences in the cleavage to the C-terminal side of basic amino acids, lysine and arginine to site a few. Still proteins are not the best substrate to use for accurate kinetic analysis of these enzymes. A number of works have been carried out using artificial substrate. In  most of the cases the artificial substrate used were simple amide derivatives of amino acids, and many a times ester derivatives of amino acids have also been used. These enzymes can break such amide or ester derivatives of the amino acids quite easily. A careful and judicious selection of substrate makes the assaying of chymotrypsin and trypsin very easy since there occur a change in U.V absorbance on hydrolysis, which may be followed spectro-photometrically.

Despite having similarities in their action and mechanism,Chymotrypsin and Trypsin can be distinguished based on their characteristic properties. For instance Chymotrypsin and Trypsin can be distinguished on the basis of their substrate specificity. The specificity of Trypsin is for peptide bonds. Also noticeable is that the affinity of Trypsin in not for all peptide bonds. Rather they have specificity for peptide bonds in which lysine and arginine furnish the carbonyl group. Chymotrypsin, on the other hand, has specificity for peptide bonds where tyrosine, phenylaline and tryptophan participate in the formation of the carbonyl group of peptide bonds. Thus ,based on the above mentioned properties N-benzoyl-L-argininamide (or ester) can be classified as a good substrate for trypsin .Based on the similar arguments that is to say based on the above mentioned characteristics N-benzyol-L-tryosinamide (or ester) is exemplified as a suitable substrate for chymotrypsin.

The characteristic properties of chymotrypsin and trypsin which help in their assaying are briefly mentioned below.

Chymotrypsin: Chymotrypsin is a proteolysis enzyme. It belongs to the group “hydrolases”.(Hyrdrolase or hydrolytic enzyme is an enzyme that catalyses the hydrolysis of a chemical bond. ) Chymotrypsin is secreted in pancreas. Its pH is at 8.0.it is an endopeptidase (an enzyme which breaks peptide bonds other than terminal ones in a peptide chain) that catalyses the hydrolysis of peptide bonds in which the carbonyl group is contributed by the aromatic residues, phenylalanine, tyrosine, or tryptophan .Like many other proteolytic enzymes, chymotrypsin also has esterase activity. This esterase activity of chymotrypsin is what taken into account to detect its presence qualitatively.

Description

The following procedure is for products with a specification for chymotrypsin activity. Insoluble chymotrypsin cannot be assayed using this procedure. It is a continuous spectrophotometric rate determination based on the following reaction.(Sigma-Ardrich)

BTEE +H₂ O     Chymotrypsin    _>N-Benzoyl-L-tyrosine + ethanol

Where:

BTEE-N Benzoyl-L-tyrosine ethyl ester

Unit Definition- One unit of chymotrypsin will hydrolyse 1.0 micromole of BTEE per minute at pH 7.8 at 25^o C.

Assaying Enzymes

TrizmaBase :2-Amino-2-(hydroxymethyl)-1,3-propanediol, THAM, Tris base, Tris(hydroxymethyl)aminomethane, Trometamol:

N?Benzoyl-L?Tyrosine Ethyl Ester)

Methanol)

Calcium chloride, dihydrate

Hydrochloric acid solution

Use ultrapure water (≥18 MΩxcm resistivity at 25 °C) for the preparation of reagents.

Buffer (80 mMTrisHCl Buffer, pH 7.8 at 25 °C) – Prepare a 9.69 mg/ml solution in ultrapure water using Trizma Base . Adjust the pH of this solution to 7.8 at 25 °C.

BTEE Solution (1.18 mMN?Benzoyl-L?Tyrosine Ethyl Ester) – Weigh 37 mg of N?Benzoyl-L?Tyrosine Ethyl Ester  into a 100 ml Class A volumetric flask. Add 63.4 ml of Methanol and mix by swirling. Bring the final volume of the solution to 100 ml using ultrapure water. Invert the flask several times to ensure complete mixing.

CaCl₂ Solution (2 M Calcium Chloride) – Prepare a 294 mg/ml solution in ultrapure water using Calcium chloride, dihydrate .

HCl Solution (1 mM Hydrochloric Acid) – Prepare a solution by diluting 0.10 ml of 1.0 M Hydrochloric acid solution to 100 ml with ultrapure water in a 100 ml Class A volumetric flask. Mix by inversion and place on ice.

Enzyme Solution (Chymotrypsin) – Immediately before use, prepare a solution containing 2?5 chymotrypsin units per milliliter in cold (2?8 °C) HCl Solution.

First of all a reaction mix is made, say of 3 ml. This reaction mix contains the final concentration of 38 mMTris, 0.55 mMN-Benzoyl-L-Tyrosine Ethyl Ester, 30% (v/v) Methanol, 53 mM Calcium Chloride, 0.03 mM Hydrochloric Acid, and 0.2?0.5 units of Chymotrypsin.

1.Following regents are pipetted into suitable quartz cuvettes:

Reagent	Blank (ml)	Test (ml)
Buffer	1.42	1.42
BTEE Solution	1.40	1.40
CaCl2 Solution	0.08	0.08

2.Mix by inversion and equilibrate to 25 °C using a suitably thermos tatted spectrophotometer.

3.Add the following to the cuvettes:

Reagent	Blank (ml)	Test (ml)
HCl Solution	0.10	–
Enzyme Solution	–	0.10

4.Immediately mix by inversion and record the increase in A₂₅₆ for 3?5 minutes.

5.Obtain the ΔA₂₅₆/minute for both the blank and test reactions using the maximum linear rate over a one minute interval using at least 4 points.

Calculations:

1.

Units/ml enzyme =	(ΔA256/minute Test – ΔA256/minute Blank) x (3) x (df)
	(0.964) x (0.10)

Where:
3 = volume (ml) of reaction mix
df = dilution factor
0.964 = millimolar extinction coefficient of BTEE at 256 nm
0.10 = volume (ml) of test sample used in assay

2.

Units/mg solid =	units/ml enzyme
	mg solid/ml enzyme

Description :

This procedure can be used with products having specification for Trypsin. The substrate used is N_α-Benzoyl-L-arginine ethyl ester (BAEE). This procedure involves a continuous determination of spectrophotometric rate. It is based on the following reaction;

BAEE + H₂O     Trypsin    _> N_α-Benzoyl-L-arginine + ethanol

Where:

BAEE – N_α-Benzoyl-L-arginine ethyl ester

Unit Definition – One BAEE unit of trypsin activity will produce a ΔA₂₅₃ of 0.001 per minute with BAEE as substrate at pH 7.6 at 25 °C in a reaction volume of 3.20 ml.

Reagents needed:

Sodium phosphate, monobasic

N_α-Benzoyl-L-arginine ethyl ester)

Instructions for preparation:

Use of ultrapure water (>= 18 MΩxcm resistivity at 25 °C) is recommended for the preparation of good reagent.

Making of Buffer (67 mM Sodium Phosphate Buffer, pH 7.6 at 25 °C) - an 8.04 mg/ml solution is prepared using sodium phosphate, monobasic using ultrapure water. pH is adjusted to 7.6 at 25 ^oC with 1 M NaOH solution.

Preparing substrate solution(0.25 mM N_α-Benzoyl-L-arginine ethyl ester) – a 0.0866 mg/ml solution is prepared making use of N_α-Benzoyl-L-arginine ethyl ester (BAEE) in buffer.

Preparing HCl solution (1 mM Hydrochloric acid) - a 1000 fold dilution of 1 M Hydrochloric acid solution is prepared in ultrapure water.

Preparing Enzyme solution (Trypsin) - a solution which contain 425-575 units/ml of Trypsin in cold (2?8 °C) HCl Solution is prepared. Care should be taken to prepare this solution right before use.

Again as before, a 3.20 ml of reaction mix is prepared. This reaction mix contains the 62.8 mM concentration of sodium phosphate, 0.23 mM concentration of N_α-Benzoyl-L-arginine ethyl ester ,0.031-0.063 mM concentration  of hydrochloric acid, and 42.5-115.0 units of trypsin.

Step 1: Following reagents are pipetted into convenient quartz cuvettes:

Reagent	Blank (ml)	Test (ml)
Substrate Solution	3.00	3.00
HCl Solution	0.200	0.125

Step 2: Mixing is done by inversion and the solution is brought to equilibrium to 25 oC.A suitable thermostatted spectrophotometer is used for the purpose. Then following is added to the resulting solution. Reagent Blank (ml) Test (ml) Enzyme Solution – 0.075

Care should be taken to make final volume in each cuvette 3.2ml per unit definition.

Step 3: then immediately inversion is used to mix and the increase in A₂₅₃/minute is recorded for 5 minutes. Then using a 1 minute time period and a minimum of 4 data points, obtain the ΔA₂₅₃/minute using the maximum linear rate for both the Blank and Test.

Calculations

1

BAEE units/ml enzyme =	(ΔA253/minute Test – ΔA253/minute Blank) x (df)
	(0.001) x (0.075)

Where:
DF = dilution factor
0.001 = the change in A₂₅₃/minute based on unit definition
0.075 = volume (ml) of test sample used in assay
Note: The total volume in the cuvette is not used in the calculation since the unit definition is based on 3.2 ml.

2

Units/mg solid =	units/ml enzyme
	mg solid/ml enzyme

(n.d.). Retrieved from Sigma-Ardrich: https://www.sigmaaldrich.com/catalog/product/sigma/b6125?lang=en&region=IN

Bisswanger, H. (2014). Enzyme assays. Perspective in Sciences.

Garysmith. (n.d.). Retrieved from UCDavis: https://fst123.fst.ucdavis.edu/~garysmith/123LData.dir/Experiment%201.pdf