Quantitationof Lipase Activity from a Bee An Introductory Enzyme Experiment Kathleen A. Farley and Marjorie A. Jones1 Illinois State University. Normal. IL 61761 Anvone who has ever exoerienced a bee stine or snake bite knows the pain and sweliing that are associated with this affliction. One of the reasons the skin reacts in this fashion is due to the material injected under the skin by the bee or snake. One such material is a lipase; an enzyme that catalyses the hydrolysis of a n ester linkage from a phospholipid, releasing a carboxylic acid group into the surrounding area. This enzyme can, therefore, destroy the phospholipids that are important for cell membrane integrity. I n order t o understand how a lipase can affect the tissues into which i t is injected, it is important to determine how active the enzyme is. Activity of a n enzyme is measured as the number of moles of a reactant (substrate) the enzyme can convert t o a oroduct in a -given amount of time (minutes if the enzyme is very active or hours if it is rather inactive). Thisactivity is then divided by the amount of protein in the sample toaccount for variations from one animal to another. The final value is called the specific activity of the enzyme. This 4-hour experiment utilizes a bee as a source of the enzyme and a radioactive substrate, ['4C]-phosphatidylcholine, to make the determination of the specific activity of the enzyme easy and very accurate. T h e reaction is as follows:
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['4~-phwophatidylcholine ['%-fatty acids +glycerol + phosphorylcholine
This experiment is also set up t o give the students experience in a wide variety of methods: visible spedrophotometry, liquid scintillation spectrometry, thin-layer chromatography (TLC), and the accurate dispensing of small volumes. For a school that does not have access t o a liquid scintillation soectrometer. a Geieer-Muller counter can be substituted if'the sensiti&y of ;he counter is a t least 150 keV (the e n e r w of carbon-14 beta radiation is 156 keV). his e x p e r s e n t could also be performed using a nonradioactive substrate. Hydrolysis of the standard phosphatidylcholine (PC) could be detected using TLC only. While the qualitative estimate of the activity of the enzyme using a nonradioactive substrate would not include the classroom use of liquid scintillation spectrometry, visible spectrophotometry and TLC would still be used.
Solutions Needed Sodium phosphate huffer: 0.01 M sodium phosphate 0.001 M CaC12, nH72 re-
Pbosphatidylcholine (PC) standard: 5 mg of PCImL CHCb (Sigma Chemical Co.). Hydrochloric acid: 10 M HCI. TLC solvent: chloroform/methanol/28% ammonia in the ratio of 65:25:52.
' Corresponding author.
Rouser, G.; Kritchevsky, G.; Yamamoto, A. Methods In Emymol* W; Academic: New York, 1969; Vol. 14, pp 272-317. Bligh, E. G.; Dyer. W . N. Can. J. Blochem. Physiol. 1959,37,911917.
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Chemicals Needed Bovine serum albumin (0.97 mg1mL Hz0). 1,2 di['4C]palmitoyl-L-3-phosphatidyleholine (Amemham, 80-120 mCiImmol). Liquiscint lrquid scintillation cocktail (National 1)iagnosticsJ. Reference standard, such as 14.,'HI toluene (Amemham) with an activity (dpm) calculated by the company. [Note: this is only needed if the liquid scintillation spectrophotometer you will be using reports the data in counts per minute (cpm) rather than disintegrationsper minute (dpm)]. Methanol. Chloroform. Solid iodine (in a suitable chamber with a tight lid). Other HPTLC plates (silica gel 60) (AUtech Associates). Pi~etman(Gilson) (used to disuense small volumes). ~icrocentrifueetubes ( ~ e k m & Co.). Bio-Rad protein assay dye reagent i~io- ad Laboratories).
Lipid Hydrolysls (Radioactive substrate) Sacrifice a live bee by exposure to fumes from diethyl ether, then separate the abdomen from the rest of the bee. Using a mortar and pestle, homogenize the abdomen in 500 pL of sodium phosphate huffer. The reaction can be run in microcentrifuge tubes. The fust tuhe contains 100 pL of bee homogenate and 10 pL of radiolabeled phwphatidylcholine,while the second tuhe contains 1M) pL of sodium phosphate huffer and 10 pL of radiolabeled phosphatidyleholine. Each tube is allowed to incubate far 1h at room temperature, after which the reaction is ptoooed with the addition of 70 uL of 10 M lioidsare then extracted bv the mkthod of . HCI to .. enchtuhe. - The -~~~~~ Hligh and Dyer.) In brief; tu each microcentrifuge tube, add 200 pl. of CHCI, and 200 uL of CH30H, mix well, and alluw phasr separation. Carefully remove the CHCIa layer (the denser Layer) to another tube, and dry under a stream of nitrogen gas or evaporate in a fume hood. Resuspend the lipid from each tuhe in 20 pL of CHC13, and spot 10 pL from each tube onto a separate lane of a high-performance (HP) TLC date. The standard ~hos~hatidvlcholine standard should also be spotted on the plate (use 5 p ~ h a n e )giving , a total of three spots on the HPTLC plate. After chromatography, visualize the lipids on the plate hy exposing the HPTLC plate to an atmosphereof Iz (e.g., iodine tank containing solid 41,and calculate the retention factor (Rt)for the PC standard. The Rlis calculated as the distance traveled by the sample divided by the distance traveled by the solvent ~~~~~~
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Lipid Hydrolysis (Nonradioactive substrate) For a qualitative estimate of the bee lipase activity,the nonradioactive PC may be used as the substrate in the reaction. In this case, the bee extract (100 pL) would be incubated with 10 pL of the PC solution (he sure the tubes are capped tightly so that evaporation does not occur). Several of these tubes should be prepared so that the reaction can be stopped at various times (10 min, 1h, 24 h) hy the addition of 70 pL of 10 M HCI to each tube. The control tube would contain 100 pL of huffer with 10pL of the PC solution. After the reaction has been stopped, the lipids are extracted as above by the method of Bligh and Dyers. Once the lipid from each tuhe has been dried down under nitrogen, it can he resuspended in 20 pL of CHCIa. Results will be determined by monitoring the number of spots on the TLC plate after exposure to the Iz vapors. Liquid Scintillation Counting (Radioactive Substrate) Using a pencil and a ruler, each of the HPTLC lanes can be divided into 10 equal segments for a total of 20 segments (the
standard PC is not counted),and each segment can be scraped into a scintillation vial. Each vial is marked for later identificationand 10 mL of scintillation cocktail is added to each. The vials can now he counted with a liauid scintillation soectrometer. While the snectrometer cannot actually detect the weak beta radiation emitt~ng frum the ['TI-PC, the beta radiation excites the scintillation cocktad and causes it to emit flashes of light (tluorescence) which the spectrometer can detect and report as disintegrations per minute (dpm) or counts per minute (cpm). If the spectrometer reports the radiation as cpm, the data can be converted into dpm by using the relationship, dpm X E = cpm (where E = counting efficiency). Efficiency is determined by counting a small volume of a radioactive standard with a known dpm. Once the sample has counted, a known volume of a radioactive standard is added (spike) into the original sample and counted again. The first count is then subtracted from the second count giving an adjusted cpm value. By dividing the adjusted epm value by the dpm spike, an efficiency can be calculated. Since mwt spectrometerscan detect many Rourcesof heta radiation, it is imponanr toset the machine to detect only ["(:I. If using the Geieer-Mullercounter. hold the ~robealmonttouchinc the TLC plate, i d scan each lane while reco;ding the readings. Protein Content of the Bee Homogenate Using the Bio-Rad protein assay dye reagent and the procedure of Bio-Rad laboratories, the protein content of several aliquots of bee homogenate was determined using bovine serum albumin (BSA) as a standard for comparison. Eight known amounts (where 1mL of water contains 0.97 mg of BSA) of BSA (10,20,30,40,50,60,80 and 100 rL) were prepared and mixed with 5 mL of Bio-Rad reagent each. At the same time, 100 fiL of the hee homogenate was also mixed with5 mL of Bia-Rad reagent. Aftera 30-min wait, the results are determined by putting each sample into the spectrophotometer and recording the ahsorbance of each sample at 595 nm. The micrograms of the standard BSA protein can then be plotted against the absorbance reading that was obtained for each standard sample (Fig. 1). Once the graph has been drawn, the protein concentration of the bee homogenate can be determined hy locating the absorbance obtained for the bee homogenate on the standard line (reading left from the y axis). By tracing this point down to the x axis, the micrograms of protein in the sample can be ohtained. Results
Radioactive Substrate Our results show that the crude bee homoeenate contains lipase activity. Figure 2 shows a graph of dishtegrations per minute (dpm) versus fractions scraped from the HPTLC
plate. T h e incubation with bee homogenate reduced the original [W]-PC by some 40% under these reaction condi(PC) tions. From dpm, the moles of ~hosphatidvlcholine . . present can becalculated hy using thecorrectjon factor2.6 X loa dpm = 1 rmol of PC (this factor is ohtained from the literature that comes with the radioactive substrate). The calculated Rl of the standard PC was found to he 0.38, while the R , of the I I T - P C was found to be 0.43. T h e R I of the radioactive P C is thbught to differ from the standard because of inaccurate spotting techniques on the HPTLC plate. Too large a spot (such a s the one we obtained) results in a greater margin of error. Once the moles of P C are obtained, the specific activity (SA) of the lipase can be calculated. T h e SA of the livase is eaual to the moles of substrate (PC) converted to product in i h divided by the amount of protein in 100 pL of bee homogenate. The specific activity for the bee homogenate was 11.8 nmol/h/mg protein. This is much higher than the specific activity we obtained using a yellow jacket homogenate with these s&e procedures (SA = 1.3 nmol/h/mg protein). This procedure is therefore very useful for comparisons of various lipase sources. Nonradioactive Substrate For the experiment using the nonradioactive PC, a qualitative estimation of enzvme activitv can be determined bv comparison of the P C spot size withvand without bee extract in the incubation. T h e control lane on the TLC plate, because of the absence of lipase activity, exhibited only one spot with a Rf of 0.38. T h e P C spot in the presence of the enzyme decreased in intensity as the lipase broke the bonds of the P C molecule. As the PC molecule continued to break down. a t least two other soots anneared on the TLC d a t e . In this experiment, a&lysis is especially useful to enable students to compare data from one species to another. A bee that contains a large amount of protein should also contain a n active lioase. Students. therefore. who have a rather inactive enzyme, can justify their results'if the protein content is also low. Cleanup This experiment contains several chemicals that require special handling. The organic chemicals (scintillation cocktail and chloroform) and radioactive samples should be
DISTANCE (crn)
Figure 1. This protein standard curve was obtained by using bovine serum albumin at various mcenbations.
Fiaure 2. Radimctivcitv, Il1'C1-ohasohatidvlcholiml associated with various . ,. . . tlactlons scraped trom me HPnC plate The reaction run with bee homage. nats is aymboilred with closed dots, wnie the reactom run w o t h a ~ tDee homogenste is symbollred with crosses Mtgallan at the a~thentlcPC standard is shown with an arrow. Volume 66
Number 6
June 1989
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pooled in a glass container and disposed of properlyl. The 10 M HCI and Iz vapors should also he handled carefully. Note In all procedures, safety glasses and hand protection are strongly encouraged. Students should also he informed of the potential hazards of working with these reagents.
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Journal of Chemical Education
Acknowledgment
Special thanks to Rick Hammond for collection of the bees for this experiment and for preparing solvents. Thanks also to Otis Rothenberger and Sol Shulman for help with this manumript. Armour, M. A.: Browne, L. M.; Weir, G. L. Hazardous Chemicals InformationandDlsposa1Guide: University of Alberta: Alberta, 1984.
