The following points highlight the top four types of assays of enzymes. The types are: 1. Amylase 2. Urease 3. Catalase 4. Peroxidase.
Assay of Enzyme: Type # 1. Amylase:
Amylases are hydrolyzing enzymes which catalyze the hydrolysis of starch through the addition of elements of water to α (1 → 4) glycosidic linkage. The product of enzymic hydrolysis of starch is maltose which is a disaccharide reducing sugar.
The maltose molecules may be further hydrolyzed in vivo by maltase, another hydrolyzing enzyme, into molecules of glucose. The assay depends on the estimation of residual starch in the reaction mixture as detected by colour reaction with iodine solution.
(a) Requirements:
1. Starch solution 1 % (w/v)
2. Iodine solution
3. Citrate buffer (citric acid + sodium citrate) – 0.05M, pH 5.0
4. Germinating seedlings of cereals like rice or wheat
5. NaH2PO4, 2H2O solution 0.2M
6. Na2HPO4, 2H2O solution 0.2M
(b) Procedure:
About 10-20 g fresh plant material is extracted with citrate buffer or simple water. The slurry thus prepared is strained through cloth and this crude extract serves as enzyme source.
Preparation of Buffer Solutions to Get the Desired pH Values:
Six conical flasks of 100 ml capacity are taken. To each flask, 10 ml buffered salt solution is prepared by mixing definite volumes of NaH2PO4 (the acid salt) and Na2HPO4 (the basic salt) solutions as described.
In this manner, 6 sets each with 10 ml buffer solution of different pH values ranging from acidic to alkaline reaction are maintained. To each flask, 2 ml of rice seedling extract is added as the enzyme source. The conical flasks containing the reaction mixtures thus prepared are left at room temperature for incubation.
Just after 1 minute incubation, 1 ml of reaction mixture containing starch is withdrawn from each flask and transferred separately to dilute iodine solution taken in 6 test tubes of uniform diameter. The contents of the tubes are shaken well and the intensities of starch-iodine colour are noted.
In a similar way, same amounts of reaction mixtures are transferred at an interval of 5 minutes to a separate set of 6 tubes containing dilute iodine as before to test the presence of residual starch. The various colour intensities are indicative of variable amounts of residual starch left after the stipulated periods of enzyme activity.
Such an assay method described here permits the students to make a time-course study on the rate of starch hydrolysis, while the enzyme reaction in the flask is allowed to proceed undisturbed for any desired length of time.
(c) Results:
The observations are recorded by putting + sign as degree of colour intensity.
(d) Conclusion:
The results indicate that solution with low pH. having higher H+ ion concentration, can promote amylase activity. On the contrary, solutions with progressively increasing pH having lower H+ ions inhibit amylase activity. The maximum and minimum activities are exhibited at 4 and 8.5 pH respectively, while intermediate pH favouring intermediate activities.
Assay of Enzyme: Type # 2. Urease:
Urease is a hydrolysing enzyme which catalyses the hydrolysis of urea leading to the formation of ammonia and carbon dioxide. NH3 gets dissolved in water and NH4OH is formed making the reaction mixture alkaline. Such alkaline solution is titrated with a standard HCL solution whereby the amount of acid consumed to neutralize the alkalinity can be taken as a measure of urease.
The enzyme urease is historically important, because this enzyme was first isolated in pure crystalline form by J. B. Sumner in 1926 from the extracts of jack bean.
(a) Requirements:
1. Urea 1 % (w/v)
2. HCL dilute as N/50
3. Phenolphthalein 0.5% (w/v)
4. Test material – soaked seeds of Cajanas cajan (Arhar)
(b) Procedure:
About 10-20 g of soaked seeds of Cajanas cajan (Arhar) are extracted with simple water. The slurry thus obtained is strained through cloth. This crude extract is diluted to a definite volume (100 ml) which serves as the source of enzyme.
Reaction mixtures are prepared in conical flasks by taking 10 ml water, 1 ml urea as substrate followed by the addition of 1 ml enzyme extract. The flasks containing the reaction mixtures are incubated at room temperature for 30 minutes.
At the end of the incubation period, urease activity in terms of alkali (ammonia) production is tested by adding a few drops of phenolphthalein as indicator to each conical flask. The entire content of pink coloured reaction mixture is titrated with HCL till the colour disappears. Urease activity is expressed in terms of the volume of HCL required for titration.
(c) Additional Lesson:
Determination of the effect of increasing substrate concentrations on enzyme activity:
This experiment may be done by including varying amounts like ranging from1 ml to 6 ml of urea in reaction mixtures taken in 6 conical flasks, while other components are kept constant. At low substrate concentrations, the velocity of the enzyme-catalyzed reaction is nearly proportional to the substrate concentration.
However, as the substrate concentration is increased, there is less increase in the reaction rate. When the substrate concentration is further increased, the reaction rate becomes independent of substrate concentration and approaches a constant value.
The results of this experiment may serve a useful purpose. The approximate value of Michaelis constant (Km) can be obtained by measuring the initial velocity at different concentrations of the substrate with a fixed concentration of enzyme.
Assay of Enzyme: Type # 3. Catalase:
Catalase is a heme enzyme which catalyses the decomposition of hydrogen peroxide into water and oxygen. The enzyme contains heme or iron-porphyrin as the prosthetic group.
2H2O2 → 2H2O + O2
The enzyme in fact catalyses a bimolecular oxidation-reduction reaction in which one molecule of H2O2 is oxidized to O2 while another molecule is reduced to H2O.
(a) Requirements:
1. Dilute H2O2 – 0.5 vol. H2O2 is generally available in 20 vol. strength. To prepare 0.5 vol. H2O2, 5 ml of H2O2 (20 vol.) is diluted with distilled water to 200 ml.
2. KMnO4 solution – 0.02N. It is known that 2 molecules of KMnO4 in acid solution give 5 atoms of available oxygen:
2KMnO4 + 3H2SO4 = K2SO4 + 2MnSO4 + 3H2O + 5O
i.e., 2KMnO4 = 5O = 10H,
... KMnO4/5 = H
... Equivalent weight of KMnO4 = molecular weight/5 = 158/5 = 31.6
Therefore, 0.02 N KMnO4 solution should contain 31.6/50 or 632 mg of KMnO4 per litre of the solution.
3. Phosphate buffer (NaH2PO4 + Na2HPO4) – 0.05 M, pH 6.8
4. 10% H2SO4
5. Any plant material like germinating seedlings, potato tuber or cabbage leaves.
(b) Procedure:
About 10 – 20 g fresh plant material is extracted with phosphate buffer or simple water and strained through cloth. The extract is centrifuged in cold and the supernatant suitably diluted is used as the source of enzyme. If refrigerated centrifuge is not available, the crude extract without centrifugation may also be used as enzyme source.
Reaction mixtures are prepared in conical flasks by mixing 10 ml buffer or simple water, 1 ml H2O2 and 1ml of plant extract serving as enzyme. A blank mixture is maintained by taking all ingredients like water and H2O2 except the enzyme extract. The flasks containing the reaction mixtures and the blank are incubated at room temperature for 30 minutes.
At the end of incubation period, the reaction is stopped by adding 5ml of 10% H2SO4. Since in mangano-metric titration, presence of H2SO4 is essential, the reaction mixtures as well as the blank should contain H2SO4. Then the contents of all the flasks are titrated with KMnO4 solution running from a burette.
Initially, KMnO4 is added at a fast rate and the pink colour is found to disappear rapidly. Then KMnO4 is added slowly until the end point is reached with the appearance of a faint and persistent pink colour.
The titer values of KMnO4 corresponding to the reaction mixtures indicate the residual amount of H2O2 at the end of the enzymic reaction, while that corresponding to the blank determines the un- reacted H2O2. The differences between these two titer values give the volume of KMnO4 the equivalent of which in terms of H2O2 is thought to have been decomposed by enzyme action.
The amount of H2O2 decomposed can be calculated by using the formula of equivalence:
1 ml of N KMnO4 = 17 mg of H2O2
... 1 ml of 0.02 N KMnO4 = 0.34 mg of H2O2
It is known that KMnO4 in acid solution is rapidly reduced by H2O2 to a colourless solution:
2KMnO4 + 3H2SO4 + 5H2O2 = K2SO4 + 2 MnSO4 + 8H2O + 5O2
(c) Calculations:
Let blank reading = a ml KMnO4 and
Reaction mixture reading = b ml KMnO4
The amount of H2O2 decomposed can be derived from (a – b) ml value:
(a – b) x 0.34 = x mg of H2O2
So, the result can be finally expressed as x mg of H2O2 decomposed per g plant material per hour.
Assay of Enzyme: Type # 4. Peroxidase:
Like catalase, peroxidase is also a heme enzyme that contains iron-porphyrin as the prosthetic group. It catalyses the oxidation of substrates with the help of oxygen derived from H2O2 decomposition.
where substrate AH2 may be a variety of substances like phenol, β-amino benzoic acid (PABA), ascorbic acid or leuco form of oxidation-reduction indicators. The assay depends on the conversion of colorless reduced substrate (AH2) into the dark-brown coloured oxidized product (A).
(a) Requirements:
1. Catechol 0.5%. It is o-dihydroxyphenol.
2. H2O2 – 0.5 vol.
3. Phosphate buffer (NaH2PO4 + Na2HPO4) – 0.05M, 6.8 pH.
4. Any plant material like germinating seedlings, potato tuber, cabbage, radish, etc.
5. Photoelectric colorimeter.
(b) Procedure:
About 10-20 g of fresh plant material is extracted with phosphate buffer or simple water and strained through cloth.
The extract is centrifuged in cold and the supernatant after suitable dilution is used as the source of enzyme. For a classroom demonstration work, the crude extract without centrifugation may also be used as the enzyme source. Reaction mixtures are prepared in test tubes by taking 5ml buffer or simple water, 1 ml catechol and 1ml H2O2.
The reaction is started by the addition of 1 ml enzyme extract. The reaction mixtures are shaken well, and a portion of which is quickly transferred to a sample tube of the colorimeter. The initial optical density (OD) of the brown coloured solution is read at 490 nm using blue filter, and the final OD measured after 1 or 2 minutes of incubation.
The difference in these two OD readings (ΔOD) caused during such short period of incubation is noted which indicates the enzyme activity.
The extracts of different plant materials may be employed as enzyme source in separate reaction mixtures, which will permit to make a comparative estimation of enzyme activity in different plant materials.
Data may be entered in tabular form with the following headings:
