Potato Peroxidase for the Study of Enzyme Properties

Potato Peroxidase or the Study of Enzyme Propertes

BrianR. Shmaefsky

Many enzyme studies conducted in thington 1988, Cowen 1990). Hydro- 1 small container of silver nitrate general biology laboratorysessions in- gen peroxide serves as a substrate for solution volve using human saliva as a source peroxidasewhich degrades the hydro- 1 small container of 95 percent eth- Downloaded from http://online.ucpress.edu/abt/article-pdf/55/5/300/46800/4449664.pdf by guest on 01 October 2021 of amylase for catalyzing the hydroly- gen peroxide into water and gaseous anol sis of starch into maltose. While this oxygen. This reactionis detectableand practice is simple and inexpensive it quantifiablewithout instrumentation. exposes students to infectious agents It simply can be analyzed by observing that may be present in the saliva sam- the number of oxygen bubbles being Methods ple (Shmaefsky1990). Other easily ob- generated by the degradation of a Instruct the students to cut three tainable enzymes are available from known quantity of hydrogen perox- 5 mm-thick slices from the center of nonhuman sources. However, analyz- ide. In addition, special preparationis the potato. Have them place two slices ing the functions of these enzymes not requiredto extractthe enzyme. A in the boiling water, removing one may require elaborateprotocols or in- variety of enzyme action experiments slice after 60 seconds and the other struments that are not appropriatefor can be effectively conducted on the after three minutes of boiling. The high school students and freshman freshly sliced face of a large potato. slices should be placed on a paper college classes. Some simple-to-performenzyme stud- towel to drain and cool. Next the stu- One under-exploited source of en- ies include the influence of pH on dents should place the three potato zymes for classroom use is the potato enzyme action, the enzyme denatur- slices on a piece of wax paper serving (Solanumtuberosum), which tradition- ation potential of boiling water, the as the work space. The wax paper ally serves as the substrate for class- inhibitionof enzymes by heavy metals should be labeled to identify the dif- room amylase studies. Potatoes are a and the effects of salt concentrationon ferent treatments conducted on the ubiquitous specimen in the science enzyme effectiveness. potato slices. For example, one slice is classroom, serving as a model for labeled "unboiled" while the others plant growth cycles (Furman 1990), a are labeled "boiled for X minutes" as source of electrolytes for electrochem- Materials shown in Figure 1. ical demonstrations (Roffiaet al. 1988) Eachgroup of students will need the Next instruct the students to scratch and even as a crude osmometer for the surface of the unboiled potato into calculating the effects of salt concen- following materialsto conduct a series of enzyme properties exercises: six sections (Figure 2). Each section trations on living systems (Barrett will represent a different treatment 1984). They are also excellent for used to alter the abilities of the perox- demonstrating the action of the iron- Enzyme Preparation idase. The students should draw a bearing enzyme peroxidase found in diagramof the section lines to prevent plants and animals (Albertset al. 1989, 1 large potato (preferone with a 6 to 7 cm diameter) them from confusing the treatments. Worthington 1988). One section should be designated as Peroxidase and the related enzyme 1 sharp kitchen knife 1 setup for boiling water in a 250 ml the control. The remaining sections catalase (found in almost all animals should be treated as follows: and aerobic microbes) are responsible beaker 1 set of kitchen tongs or large for- for removing destructive peroxides Basic pH conditions section-Add that accumulate from normal cellular ceps 1 sheet of wax paper one drop of pH 9 sodium hydrox- metabolism. The metabolic peroxides ide. are a result of superoxide dismutase Substrate Preparation Acidic pH conditions section-Add activity which converts destructive one drop of pH 2 hydrochloric free radical oxygen to less cytotoxic 1 small beaker of household grade acid. peroxides (Alberts et al. 1989, Wor- hydrogen peroxide Concentrated salt conditions sec- 1 small containerof pH 2 hydrochlo- tion-Add one drop of 10 percent ric acid solution sodium chloride. BrianR. Shmaefsky, Ph.D., is professorof 1 small container of pH 9 sodium Heavy metal conditions section- biotechnology at Kingwood College, hydroxide solution Add one drop of silver nitrate. Kingwood,TX 77339. 1 small container of 10 percent so- Alcoholic conditions section-Add dium chloride one drop of ethanol.

300 THEAMERICAN BIOLOGY TEACHER, VOLUME 55, NO. 5, MAY1993 Concentrated Salt: 75 percent decrease in activity (mild denaturation) Heavy Metal:50 percent decrease in activity (inhibitionby chelation to the active site) Alcohol: 50 percent decrease in activity (inhibition by competition for active site) -un boiled sl-hort boil 60-Second Boil: No measurable decrease. 3-Minute Boil: 75 percent decrease in activity (extremedenaturation). The optimum conditions for potato long boil peroxidase using a hydrogen peroxide substrateare pH 7.0, 25?Cand approximately a 1 percent solution of sodium Figure 1. Potato slice boiled for X minutes. chloride (Worthington1988). Extension Activities Downloaded from http://online.ucpress.edu/abt/article-pdf/55/5/300/46800/4449664.pdf by guest on 01 October 2021 Potato peroxidase activity may be compared for different varieties or sizes of potatoes. Also, potatoes can be compared to other good sources of peroxidase: horseradish root, mango fruit and turnip radical (Worthington 1988). The peroxidase activity of fresh potatoes (or other sources) can be compared to that found in prepared foods (canned, frozen or freeze-dried). Students can be instructed to design experiments that determine the optimum ranges of conditions for peroxidase activity by subjecting the potatoes to a gradient of pHs, salt unboiled concentrationsand temperatures. The tests can be repeated on other peroxi- Figure 2. Unboiled potato. dase sources to determine species-spe- cific differences of the various peroxi- dases.

Let the solutions sit on the potato suming no experimental error, any References surface for 60 seconds to allow time for variation from the control is probably Shmaefsky, B.R. (1990). Artificialurine for the reagents to absorb into the tissue due to the treatments. the science laboratory. The American Biol- and interact with the peroxidase. ogy Teacher,52(3), 170-172. Once the last treatment application Expected Results Furman, E. (1990). Plant a potato-Learn reacts for 60 seconds the potatoes are about life (and death). Young Children, ready for peroxidase activity analysis. The rate of peroxidase activity for 46(1), 15-20. To do this assay, the students should the control section is dependent on Roffia, S., Conclalini, V. & Paradisi, C. the variety of potato, its degree of (1988). Tested demonstrations.Journal of first add one drop of hydrogen perox- Chemical Education, 65(8), 725-727. ide to the control section. They should maturityat time of harvest, the conditions at which the harvested potatoes Barrett,D.R.B. (1984).Osmosis and surface wait approximately five seconds and area to volume ratio. Journal of Biological then tally the number of oxygen bub- were stored and the temperature of Education, 18(4), 273-274. bles produced in a 30-second interval the room at the time of the experi- Alberts, B., Bray, D., Lewis, J., Raff, M., immediately following the five-second ment. After many classroom trials of Roberts,K. & Watson,J. (1989).Molecular incubation.This step must be repeated this experiment, the following results biology of the cell. New York: Garland for each treatment on the unboiled were noted for the different treat- Publishing Inc. ments: Worthington,C.C. (Ed.). (1988). Worthing- potato and once on each of the boiled ton manual. Freehold, NJ: Worthington potatoes. The number of bubbles pro- Acidic:100 percentdecrease in activ- BiochemicalCorporation. duced by the control section (unboiled ity (extreme denaturation) Cowen, R. (1990). Droopy plants drop potato) is the baseline peroxidase ac- Basic:80 percent decrease in activity hints of enzyme's role. Science News, 137, tivity for that particular potato. As- (extreme denaturation) 87.

POTATOPEROXIDASE 301