Journal of Biological Education Investigations with ...

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Journal of Biological Education

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Investigations with protease

Din Yan Yipa a Department of Curriculum and Instruction, Faculty of Education, The Chinese University of Hong Kong, Hong Kong Online publication date: 13 December 2010

To cite this Article Yip, Din Yan(1997) 'Investigations with protease', Journal of Biological Education, 31: 4, 249 — 252 To link to this Article: DOI: 10.1080/00219266.1997.9655572 URL: http://dx.doi.org/10.1080/00219266.1997.9655572

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Investigations with protease Din Yan Yip Two simple and reliable ways for measuring protease activity are suggested which can be used for a variety of investigations for both lower and upper secondary biology classes Introduction Practical investigations on enzyme properties and the effects of various factors on enzyme activity, such as substrate concentration, pH, and temperature, are required in most secondary biology curricula. An enzyme commonly used in such studies is protease. Protease is available in the form of trypsin or pepsin extracts of mammalian tissues from laboratory suppli ers, from commercial products such as meat tenderizer, biological washing powder or from the extracts of cer tain fruits. Because of the wide application of protease in everyday life, the study of protease activity is partic ularly relevant and popular in the school curriculum. Laboratory activities involving the use of this enzyme, however, often end up with failure and frustrations. Many students and teachers have the unpleasant experi ence that the measurement of protease activity, as based on methods recommended in textbooks or official syl labuses, often does not yield satisfactory results. The rate of protease activity is usually based on qualitative or semi-quantitative measurements such as the formation of clear spots on milk agar (CDC, 1992), digestion of milk or egg white suspension, or breakdown of gelatine in photographic film (Kirby and Clark, 1971; Mackean, 1983; Freeland, 1987). These methods usually fail to give clear, accurate results for enzyme activity and present considerable technical difficulties in the hands of secondary school students. This article suggests two simple and conve nient methods that give consistent and quantitative measurements of the activity of proteases obtained from a variety of sources.

Materials and methods 1. Using milk agar as the substrate The white colour of the milk agar is due to the pres ence of the milk protein, casein. In the presence of a protease, casein will be digested, giving a clear zone in the milk agar. To obtain clear and accurate results, the most important point is to use adequate quantities of constituents in preparing the milk agar plate. Too much milk may obscure the result and fresh milk coagulates readily when it is mixed with hot agar solu tion. A recipe for the preparation of milk agar plate that can yield good results is as follows: 1. Dissolve 1 g of milk powder in 100 cm3 of boiling water. 2. Add 1 g of agar powder with constant stirring. This quantity is sufficient for preparing four plates. 3. Pour a thin layer of the agar mixture into a clean sterilized Petri dish. 4. Replace the lid and allow the agar to cool and set. Below is the outline of an investigation using the milk agar plate to study protease activity from a vari ety of sources, based on a method suggested in the Hong Kong A-level Biology Teacher Assessment Scheme Handbook (HKEA, 1996). Outline of the procedure: 1. Prepare fruit juices from pineapple, kiwi fruit, and papaya as follows: Cut a 10 g fruit sample into small pieces and squash them in a plastic tube with a glass rod. Transfer the contents into a 25 cm' measuring cylinder and add distilled water until the final volume reaches 20 cm3. Filter the extract through a muslim cloth and collect the filtrate in a large test-tube. Prepare 2 per cent solutions of trypsin, meat tender izer, and protein removal tablet for contact lenses. [The meat tenderizer contains a protease called papain and the protein removal tablet for contact lenses con tains a protease called subtilisin.] 2. Prepare seven small wells on the agar plate by gen tly pressing a cork borer (6 mm diameter) into the agar and removing the bored agar core with fine forceps. 3. Carefully transfer a drop of each of the prepared solutions or water into one of the seven wells. 4. Incubate the milk agar at 40°C for about one to two hours. Journal of Biological Education (1997) 31 (4)

249


• Care should be taken when transferring the solu tions to the wells as any solution that is dribbled on to the surface of the agar may obscure the results.

Figure 1 Measurement of protease activity using a milk agar plate.

5. Examine the surface of the agar plate against a dark background. Compare the size of the clear zones developed around the wells. Figure 1 shows the results of such a study. The diameter of the clear zone around each well indicates the rate of protease activity. This method gives a more reliable and accurate result than a similar one in which paper discs soaked in enzyme solutions are placed on the surface of the milk agar. In the latter method, the amount of enzyme solution carried by the paper discs is variable, thus making it a less reliable instrument for comparing enzyme activity. The following precautions should be noted when conducting this investigation: • Cover the milk agar plate with a lid all the time dur ing the investigation. To reduce the chance of cont amination by microorganisms, expose the milk agar to air as little as possible and seal the Petri dish with adhesive tape during incubation and examination. • Do not incubate the milk agar plates for more than 12 hours to avoid formation of bacterial colonies which may be hazardous to health. If it is not con venient to examine the results immediately after incubation, the plates should be kept in a refrigera tor to reduce bacterial growth and examined in the next lesson. Plates kept under this condition can still yield good or even better results within a few days. • Any used or unused milk agar plates should be dis posed of as soon as possible in a safe way, such as by autoclaving or immersing in disinfectant solu tions for several hours before disposal. 250

Journal of Biological Education (1997) 31 (4)

2. Using photographic film as the substrate The emulsion layer of photographic film contains pig ment granules impregnated in a layer of gelatine, which is a protein. If a drop of protease solution is placed on the surface of the film, the gelatine is digested and the pigment granules will be released. This will produce a clear spot on the digested region. This method is commonly used in biology practicals because it is simple and easy to carry out. As it is dif ficult to determine the exact time for the film to become clear, this method suffers from the disadvan tage that the result gives only a crude estimation of enzyme activity. It has therefore limited application in the study of enzyme properties. The revised method suggested here yields results that can be quantified and can be used for a variety of investigations on pro tease activity. Below is the outline of an investigation using this method to study the effects of enzyme con centration on protease activity. Outline of the procedure: 1. Prepare a standard protease solution by dissolv ing a protein removal tablet (0.1 g) for contact lenses, which contains a protease, in 10 g distilled water. 2. Use this solution to prepare the following serial dilutions: xl, x \ x \ x \ x 4 a n d water. 3. Transfer 2 cm3 of each of these solutions to a testtube. Put these tubes into a water bath at 40°C for at least five minutes. 4. Use a paper punch to prepare a number of small discs from a piece of exposed and developed colour photographic film. Put three film discs into each of the tubes in the water bath. 5. Compare the colour of the solutions after 30 min utes. Figure 2 shows the results of this investigation. The gradation in the intensity of the brown colour of the solutions gives a direct and reliable comparison of protease activity. (Note: Black and white reproduction only is shown here.) This method has the following advantages: 1. The colour differentiation of the different solutions is much better than that obtained when using black and white film. The colour difference can be com pared by inspection with the naked eye, which can be quantified in the following way: Pour the solution with the deepest colour (i.e. xl solu tion) into a test-tube to a depth of about 1 cm and record the exact depth in mm. Pour the solution of x \ dilution into a second tube until the intensity of the colour appears the same when both tubes are held side by side over a white card and

M


\\

Figure 2

xl/2

xl/4

xl/X

\ l l(>

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protease in each tablet may vary widely with the sup plier. The enzyme tablet used in this study is distrib uted by Bausch and Lomb Inc., each weighing 0.1 g and claimed to contain 25 mg subtilisin. The other two brands available in Hong Kong claim to contain 0.06 and 0.4 mg per tablet respectively. Based on the milk agar method and photographic film method, they were found to show much lower protease activity than the Bausch and Lomb brand. Pure subtilisin is also avail able from commercial suppliers. Using this enzyme source as a standard, a meaningful and interesting investigation can be designed to study whether the amount of protease claimed to be contained in each brand of protein removal tablet is justified.

Effect of enzyme concentration on protease activity.

viewed from directly above. Record the height of the x £ solution. Using the height of the xl solution as a standard, express the colour intensity of the x \ solution relative to the standard as a percentage, bearing in mind that the height of the solution is inversely proportional to the colour intensity. Repeat the procedure with the remaining solutions in sequence. If the colour difference between the standard and a certain solution proves too great, dilute the standard solution appropriately and use the diluted standard for comparison with the latter solution. More accurate and quantitative results can be obtained readily if a colorimeter is available. One way to do this is to prepare a calibration graph for measur ing the colour intensity of the various treatments as follows: a) Transfer the solution with the deepest colour (i.e. xl solution) into a cuvette and take a colorimeter reading. b) Make a serial dilution of this solution and take col orimeter readings. c) Use these results to plot a calibration graph. d) Obtain colorimeter readings for the other solutions and use the graph to determine their colour inten sity. 2. Distinct results are produced in about 30 minutes, so investigations using this method can be completed well within a double laboratory period. 3. Exposed and developed film can be readily obtained from the beginning of a roll of used colour negatives. 4. This method can be used to study the effects of tem perature and pH on protease activity, whereas the milk agar method will not be adequate as the milk protein will coagulate at extreme conditions. A note on the protein removal tablet for contact lenses may be useful to the readers as the quantity of

Conclusion Using the milk agar plate to measure protease activity is easy and simple, and it gives clear and visible results. As it demands little manipulative skills, it is most suitable for lower secondary classes, especially for comparing the activity of proteases from differ ence sources. Using the photographic film as the sub strate for protease action yields quantitative results and it works in a variety of conditions. It is therefore suitable for the more sophisticated study of protease action in upper secondary level such as investigating the effects of temperature, pH, and enzyme concentra tion on enzyme activity. A variety of interesting and original investigations can be initiated by a combination of both methods. For instance, the milk agar method can be used to obtain some preliminary information on the relative protease activity of a variety of commercial products, such as meat tenderizer, biological washing powder, and protein removal tablets for contact lenses, at room temperature. On the basis of the results obtained, indi vidual students or groups can then identify more spe cific problems for further study and design their own investigatory strategies using the photographic film method which can yield more accurate and quantita tive results. These may include finding the optimal temperatures of the proteases from different sources and how the enzymatic activity is affected by various factors such as enzyme concentration, temperature, and time of treatment.

References Curriculum Development Council (1992) Syllabus for Biology (advanced level). Hong Kong: Government Printer. Freeland, P. W. (1987) Investigations for GCSE biology. London: Hodder and Stoughton. Hong Kong Examinations Authority (1996) 1998 Advanced Level Examination: Handbook on the A-level biology teacher assessment scheme. Hong Kong: Hong Kong Examinations Authority. Journal of Biological Education (1997) 31 (4)

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Kirby, T. W. and Clark, H. P. ( 1971 ) Experimental biology: a practical handbook. London: Oxford University Press. Mackean, D. G. (1983) Experimental work in biology. London: John Murray.

The author Mr Din Yon Yip is an Assistant Professor in the Department of Curriculum and Instruction, Faculty of Education, The Chinese University of Hong Kong, Shatin, NT, Hong Kong.

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