Effervescent Enzymes!

BackgroundEnzymes, by virtue of the myriad offunctions with which they are involved,are necessary for life. Consequently,studying enzymes and the roles whichthey play continues to be an integralpart of many science curricula. In thatregard, Curriculum for Excellence (CfE) isno exception and one of the SciencesExperiences and Outcomes at Level 4 [1]relates to practical work involvingmeasurement of enzyme properties:

I have taken part in practical activitieswhich involve the use of enzymes andmicroorganisms to develop myunderstanding of their properties and theiruse in industries. [SCN 4-13b]

In this short article, we present anexperimental protocol that we believe tobe both robust and accessible andwhich, therefore, should prove useful insupporting the delivery of outcome SCN 4-13b. Further experimental detailswill be made available on the Science 3-18website [2]. The protocol which followsis adapted from one produced by RogerDelpech of the Haberdashers' Aske'sBoys’ School - the original article can bedownloaded from the SAPS website [3].

The enzyme catalase is found in nearly allaerobic cells (animals, plants andmicrobes). The function of catalase is toprotect the cell from the harmful effectsof hydrogen peroxide which isgenerated as a by-product of cellmetabolism. Catalase speeds up thebreakdown of hydrogen peroxide intomolecular oxygen and water.

2H2O2 2H2O + O2

The following protocols use a verysimple yet elegant method todemonstrate the above reaction. Whenfilter paper discs are added to hydrogenperoxide they initially sink to the bottomof the container. If, prior to theirimmersion in the hydrogen peroxidesolution, the discs have been in contactwith a source of catalase (for example, ifthey have been soaked with suspensionsof living cells or in contact with livingtissue) oxygen will be produced. Some

of this oxygen becomes trapped in thefibres of the filter paper making the discsbuoyant and they eventually float to thesurface of the solution. The time takenfor the discs to rise to the surface can beused as a measure of reaction rate andhence the amount of catalase present.

ExperimentalWe suggest here two ways of using thistechnique:

■ investigating whether living and non-living materials contain catalase

■ investigating the effect of catalaseconcentration on the rate of reaction

Catalase activity in living and non-living materialsThe aim of this activity is to provide asimple method by which students caninvestigate catalase activity from anumber of sources including living (plantand animal) and non-living materials. Bypooling class results, students should beable to conclude that (i) catalase is foundonly in materials which contain live cells,and (ii) the activity of catalase variesdepending on the source selected.

■ We recommend a control experimentin which a piece of filter paper, whichhas not been in contact with any of thesamples to be tested, is added to aUniversal bottle containing 20 cm3 of1% hydrogen peroxide solution(IRRITANT). The disc should sink to thebottom and remain there.

■ Using a scalpel or a knife make a freshcut on the surface of plant or animalmaterial to be tested. Using tweezers holda filter paper disc against the test surfacefor 10 seconds and then add the disc to

the Universal bottle containing 1%hydrogen peroxide solution. The disc willsink to the bottom. However in this caseif oxygen is produced, indicating catalaseactivity, the disc will float to the surface.Students need to be ready to time howlong it takes the disc to float to thesurface. Alternatively the time taken for adisc to rise a given distance could be usedfor measurement (this would beparticularly useful if catalase levels in agiven sample were low).

■ Further trials using a variety of plantor animal or non-living surfaces can thenbe undertaken.

■ Students can design a table for resultsand make comparisons of catalaseactivity in living / non-living materialsand plant / animal tissues. Table 1 showspreviously published results for a varietyof plant materials [3].

F

Catalase activity present Catalase activity absent

apricot parsnips applebanana potato citrus fruitscherry radish peachesbroccoli red cabbage rhubarbcarrot turnip tomatocucumber fresh flowers senescent flowersonion leek

Table 1 - Catalase activity in some fruit and vegetables (taken from [3]).

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Liver contains a lot of catalase and thiscan be related to the role of the liver inthe removal of toxins from the body. Itwould be interesting to use a variety ofdifferent animal tissues (e.g. kidney,muscle) and make comparisons ofcatalase levels. However, it is worthnoting that the biological significance ofcatalase is quite complex; for example itappears that some organisms deficient incatalase can survive with little or noharmful consequences [4]. Someadditional information on the role ofcatalase in plants is available [5].

The effect of catalase concentrationon the rate of reactionThe aim of this activity is to provide amethod by which students can investigatethe effect of increasing catalaseconcentration on the rate of breakdownof hydrogen peroxide. Yeast or algalsuspensions are convenient sources ofcatalase.

Figure 1 shows a set of images when adrop of a yeast suspension has beenadded to a filter paper disc and placed inhydrogen peroxide solution.

The concentrations of yeasts and algaesuspensions can be easily varied. It isreasonable to assume that the greaterthe concentration of the yeast or algae

suspension the more catalase would bepresent in a fixed volume and so theconcentration of yeast or algae can beused as a measure of enzyme concen-tration. In the protocol that follows wewill only refer to suspensions of the algaScenedesmus quadricauda.

For this protocol we recommend thatyour students carry out a serial dilutionfrom a starting stock of algae. Depend-ing on the year group with which youdecide to use this activity the extent ofexplanation you give will be important;for example with ‘Higher’ studentscarrying out the serial dilutions could bedone as part of an investigation.

Bearing in mind that you will need onlysmall volumes of material for the assayyou will not need to prepare largevolumes of each dilution.

We recommend making dilutions to 5cm3 although smaller volumes would, inmost cases, be entirely adequate. So 0.5cm3 of a stock solution is diluted to 5.0cm3 with water and 0.5 cm3 of thatsolution is further diluted to 5.0 cm3 etc.(shown schematically in Figure 2)

■ A pipette is used to place one drop ofthe algal suspension on to a filter paperdisc - clearly more accurate measure-ments of the volume added would addrigour to the experiment.

■ Using tweezers add the disc to aUniversal bottle containing 1% hydrogenperoxide solution. The disc will sink tothe bottom. If oxygen is produced,indicating catalase activity, the disc willfloat to the surface. Students need to beready to time how long it takes the discto float to the surface.

■ The process is repeated with the rangeof serial dilutions.

■ As a control, the process should alsobe repeated with a drop of water on afilter paper disc.

■ Pupils can record their data in tablesand graphs and draw conclusions aboutenzyme activity against concentration.

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Effervescent Enzymes!

A B CFigure 1 - A single drop of a 1% suspension of yeast has been added to a filter discand then placed in a 1% solution of hydrogen peroxide. Photographs were taken at(approximately) 3 s (A), 8 s (B) and 13 s (C).

Figure 2 - Preparation of serial dilutions from a stock solution of Scenedesmus quadricauda

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Resources to support new Higher Sciences

The data in Table 2 show the time takenfor filter paper discs to float to thesurface after the addition of 1 drop of agiven algal suspension. We contend thatthis is a nice experiment and one whichoffers enormous scope for further invest-igation (some suggestions are includedin [3]).

Summary and ConclusionsThe method for the assay of catalase asdescribed in this article offers scope forpractical work at various levels of thesecondary sector. A full range ofcurriculum links is available atwww.science3-18.org. The resourcesrequired to undertake the practical areminimal and should be readily available /accessible to schools and colleges.

Technical Details1. Hydrogen peroxide is readily availablefrom chemical suppliers. 2. The filter papers used were WhatmanAA (13 mm) and purchased from FisherScientific. With an educational discounta pack of 1000 discs costs approximately£30. However, other filter papers willalso work and can be conveniently cut tosize with a cork borer or similar.Concentration of

algal stock (%) Time (s)(undiluted stock = 100%)

100 *10 211 85

0.1 1600.01 270

Table 2 - 1 drop of a given dilution ofScenedesmus quadricauda was added to thesurface of a filter paper and placed into aUniversal containing 1% hydrogen peroxidesolution. The time taken for the filter paperto float to the surface was recorded. *Thecatalase activity in the undiluted stock wassuch that the filter paper did not sink to thebottom of the Universal.

References

1. Curriculum for Excellence: Sciences - Experiences and outcomes – available at http://www.ltscotland.org.uk/learningteachingandassessment/curriculumareas/sciences/eandos/index.asp (accessed 1st December 2010).

2. Science education 3-18 website - http://tinyurl.com/Effervescent-Enzymes(accessed 13th December 2010); please note that to access the full suite ofresources on this website a log-in ID is required.

3. Delpech, R. (2003) Microscale Investigations with Catalase – available athttp://www-saps.plantsci.cam.ac.uk/worksheets/ssheets/SS24.pdf (accessed 4th December 2010).

4. Catalase - available at http://en.wikipedia.org/wiki/Catalase(accessed 5th December 2010).

5. Dann, L. Why do plants produce the enzyme catalase? Available at http://www-saps.plantsci.cam.ac.uk/records/rec580.htm (accessed 4th December 2010).

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Learning and Teaching Scotland hasinformed us that they are makingresources available on line to support thenew Higher science qualifications. Someof the materials have been developedwith substantial input from SSERC. Welook forward to having a good look ateverything on offer when we haveappropriate access.

The new CfE-inspired Highers in Biology,Chemistry, Human Biology and Physicswill be available for early adoption inScottish schools from June 2011.Learning and Teaching Scotland (LTS)have commissioned resource writers toproduce a wide range of supportmaterials for these new qualifications.These customised support materials willbe presented in a variety of engaging,modern formats including scientificanimations, short films, Powerpoints,interviews and web-links, as well as arange of more traditional paper-basedresources. The materials will be accessedfrom the National Qualifications website[1]. Latest news of developments may befound on the National QualificationsSciences blog [2] and resources on the

National Qualifications Sciences GlowGroups [3]. Theoretically, Glow users willbe able to access all the Sciences GlowGroups but we, at present, cannot.

Higher Biology/ Higher Human BiologyThe following resources have beenproduced for both the Biology andHuman Biology courses:■ Extensive teacher support notescovering the whole course.■ A wide variety of active learningactivities. ■ Nine ‘case studies’ on topical issues.These case-studies are either practical-based or research-based or involvestudying first-hand experimental data.An example of a practical case-study onPCR, which was developed with supportfrom SSERC, is already available in theBiology Glow group. Full details may beseen in SSERC Bulletin 233, Autumn2010 [4] ■ An ‘Evolution’ support pack adaptedfor the new Higher from an EdinburghUniversity resource.■ A skills development resource oneffective web research in Biology.

Higher Chemistry■ A teacher support pack on the newstyle ‘Open-ended questions’. ■ Three topical ‘Researching Chemistry’support packs looking at areas such asbiodiesel, antioxidants and alcohols.Each support pack consists of a teacher’sguide, a technician’s guide and a pupil’sguide containing a selection of possibleinvestigative activities. The support ofSSERC in developing these resources isgratefully acknowledged.■ Five skills development resources on‘carrying out web-based research’,‘planning and carrying out an exper-iment’, ‘processing and analysing data’,?

Figure 1 - National QualificationsSupport at LT Scotland

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