Gerald S. Adams' a n d Craig V. Towers Whinier College Whittier, California 90608
Acid Phosphatase Characterization An undergraduate biochemistry laboratory experiment
This paper drscrihes an undergraduatr hiorhemical experiment which mvolves the isolation and chnrarteri~ation of acid phospharaie frnm readily availnl~lematerial. By i y i tematirally varying the ~ m r m assaving l prwedure, students can observe reaction velocity (rate of reaction) as a function of enzyme concentration, reaction velocity as a function of pH. enzyme activity as a function of temperature, and reaction velocity as a function of substrate concentration. Several years ago the authors were developing an undergraduate biochemistry laboratory course for chemistry and premedical students who had, as a orereauisite. one semester ~ ~ of general biochemistry. At that t i k e we onlyhad access to minimal equipment. An e x ~ e r i m e nthat t the authors wished to include in the laborator; course was one that involved the above stated principles and. a t the same time. could he nerformed in a reasonable amount of time, using material which is neither difficult to obtain nor to store. The authors surveyed hack issues (from 1966to the present) of this Journal and found that, of the few articles (1-6)on biochemical experimerits, all suffered from one or more shortcomings which, we believe, were overcome in this experiment on acid phosphatase. This experiment has the following advantages: (1) the starting materials (bovine aorta, beef liver, or beef heart) are readily available and inexpensive (the bovine aorta should he free, in most cases, from slauehtrrlng plants, while the othvr two materials can be purchased at a 11r.alsrrpermarkrt~,(2) the enzyme preparations are quite stable. (3) a minimum amount of eauiomint is involveri - - .-, (41 ~ - , most procedures can he performe: in a 3-4-hr~lahoratory period, and (5) the experiment is inexpensive to perform. Furthermore, we feel that this is an important experiment since: (1)it demonstrates the princi~lesand techniaues of enzyme isolation, (2) it illustrates thk theories on t h e properties of enzymes, (3) it reflects the type of experiment that is performed under actual research conditions, (4) it illustrates the proper use of controls in an ex~eriment.and ( 5 ) it can introduce the use of such equipmknt as a centrifuge and spectrophotometer. ~
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Theory The word phosphatase is used to describe those enzymes which catalyze the hvdrolvsis of comoounds of orthonhosphoric acid: ~ e p e n d i n gupon the specificity of individual phosphatases for the different t. w.e s of linkaees of nhosohorvl . . " group(s), they are classified as phospbomonoesterases, phosphodiesterases (ex., rihonuclease. deoxvribonuclease. or lipid phosphohydrofase), and phosphoric k h y d r i d e hy: drolases (ex., ATPases, inorganic ~ v r o ~ h o s o h a t a s e and s. nucleotide 6yrophosphatases).Phosphataieadivity wai first observed in 1907 ( 7 , 8 )in rice bran in which the enzyme split phosphoric acid from phytin (the Ca, Mg salt of inositol hexaphosphate). Between 1908 and 1912 the same enzyme was shown to be present in animal tissue such as hone, kidney, spleen, and pancreas (9,10). Today i t appears that phos. phatases are present i n practically all living materials-hone, hlimd plusa~a.*.r)ihroc)~es, leunrytes, liver, kidney, intestinal epithelium, milk, urine, rice bran, molds, and ieast. Phosphatases are involved in carbohydrate, nucleotide, and phospholipid metaholism, as well as hone formation. Prepa-
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' To whom communications should he addressed 780 1 Journal of Chemical Education
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3% EN-0-P=O I
+ HZO
+€NOH + 32H P 0 3-2
0Figure 1. Enzyme-phosphateformationand hydrolysis.
ration of phosphatases from liver, kidney, spleen, brain, in~ testinal epithelium, or mammary gland can he made from the simple water extraction of the ground tissue, from the autolyzed tissue, or from the powder of the specific tissue.
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This experiment deals with a nonspecific acid phosphomonoesterase (acid phosphatase). Phosphomonoesterases are classified as either nonspecific or specific (e.g., glucose-6-phosphomonoesterase, the action ofwhieh is an important step in the metabolic degradation of ... elvcoeen non- to elucose). Deoendine- uoon . their .DHootimum. . specific phosphomonoester&es are classified as either alkaline (EC 3.1.3.1)oraeid (EC3.1.3.2). The mechanism of action of phosphomonoesterase was reported in 1952 (11). The cleavage of phosphomonoesters by phosphomonoesterases in 180-enrichedwater is accompanied by the ineorporation of 180 into the liberated orthophosphate. Thus, one atom of oxygen is incorporated per molecule of liberated orthophosphate. Therefore, the hydrolysis of esters of phosphoric acid by phosphomonoesterases results in the cleavage of the 0-P bond rather than the C-0 bond. Some information on the nature of the active site has been reported (12).When alkaline phosphatase of Escherichia eoli is incubated with p-nitraphenyl phosphate (labeled with 32P), the enzyme becomes covalently attached to the phosphate group. Upon acid hydrolysis of the enzyme, phosphoserine and phosphaserine peptides are isolated; thus, it appears that the amino acid serine is involved in the active site (Fig. 1.). There are two traditional methods of assaying for enzymatic aetivity. The first involves the colorimetric determination of liberated orthophosphate by the method of Fiske and Suhbarow (13). The second assay technique is based on the cleavage of chromagenie aromatic phosphate esters as model substrates. A number of model substrates are used, but the most popular is disodium p-nitrophenyl phosphate. The liberated p-nitrophenol can be measured very easily spectrophotometrieally. Experimental Equipment The followingequipment is required: scissors, knife, cheese cloth, blender, constant temperature water bath, clinical centrifuge, and a spectraphotometer. Reagents The following reagents are required: Disadium p-nitrophenyl phosphate, 19mM, 4 mglml (Allied Chemical Co.)-the substrate is made up using the appropriate buffer;p-Nitrophenol,0.68 mM, 0.095 mglml (Eastman Organic Chemicals)-the molar extinction coefficient for p-nitrophenol is 17,500 at 410 nm (15);Citric acid-Sodium citrate buffer, 0.3 M, pH 5.6, approximately 44 ml of sodium citrate with 6 ml of citric acid (16);Trichloroacetic acid, 30%,300 gA (Mallinekrodt Chemical Works): Sodium hvdroxide. 1 N. 40 ell (Mal-
Preparation of Enzyme Bovine aortas, obtained immediatelyupon sacrifice,are placed in ice. First the fat and adventita are removed with scissors. Next. each
aorta (two of -6% in. in length per student) is cut in small pieces ('I&. squares) and immediately placed in a large beaker surrounded by ice. Approximately 200 g of tissue is ground with 200 ml of cold distilled water for 1 min a t high speed in a blender. The resulting mixture is kept cold. This homogenate is squeezed through two layers of cheese cloth (about 100 ml is recovered) and either used immediately or stored a t -20°C. This fraction is known as the crude homogenate.
Beef Liver For each student, approximately 100 g of frozen heef liver (from a local supermarket) is diced with a knife, ground with 100 ml of cold distilled water for approximately 5 s a t law speed in a blender, and immediately centrifuged at 2000 rpm for 3 min in a clinical centrifuge. The supernatant (-80 ml), known as the crude homogenate, is either used immediately or stored a t -20°C.
Beef Heart
proximately the same volume of crude homogenate is recovered.
Assay Procedure The assay procedure is the one described by Kirk (14). Into a tube are added: 1.0 ml of p-nitrophenylphosphate, 1.0 ml of magnesium chloride, 5.0 ml of citric aeid buffer, p H 5.6.2.5 ml of water, and 2.5 ml of crude homogenate. For each analysis an enzyme, reagent, and substrate blank should he included. The samples are incubated for 30 min a t 37-C. At the end of the incubation period, a 1.0-ml aliquot is removed end added t o 3.0 ml of water, plus 2.0 ml of 30%trichloroacetic acid. After standing in ice for 5 min, the samples are centrifuged for 5 min a t 20004000 rpm in a clinical centrifuge. To the supernatant 6.0 ml of 1.0 N NaOH are added. The samples are read immediately a t 410 nm.
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PH Figure 2 Activity profile of acid phosphatase in bovine aorta as a function of pH. Activity expressed as micromales hydrolyzed.
Standard Curve for the ~eterminationof Orthophosphate A stock solution (p-nitrophenol) is diluted to various concentrations. Then, to 1.0 ml of the sample solution 5.0 ml ofwater, plus6.0 ml of 1.0 N NaOH are added and the ahsorbanev a t 410 nm is measured. A standard curve can he oreoared hv olotiine the ahsorhance
Results Each student or pair of students is assigned the task of isolating and performing the general assay procedure. This experiment, using hovine aorta (with which we have the greatestexperience), will take -3-4 hr. We have found that crude bovine aorta homogenate can he stored at -20'C for over 18 mo with little loss of activity (crude beef heart and liver homogenate are stable for a t least 6 wk a t -20°C). Thus one variation to this experiment would be to prepare large quantities of crude homogenate to be stored for future student use. In addition, we have stored intact bovine aortas for 3 yr a t -20°C with little loss in activity. In either case the enzyme is extremely stable over long periods, which will allow flexibility in planning this experiment. Another exoeriment is to investizate the reaction velocitv as a function of
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TEMPERATURE
Figure 3. Activity profile of acid phosphatase in bovine aorla as a function of temperature. Activity expressed as micromoles hydrolyzed.
experiment can be performed using citric acid-sodium citrate buffer of p H 3.8,5.6, and 6 7. Acid phosphataw activity in bovine aorta shows maximum activity a t p H 5.5, although significant activity can he seen over a wide p H range (Fig. 2). Also a study of reaction velocity as a function of time can easily be done. We have found, using crude hovine aorta homogenate, that an excellent temperature profile can he ohserved if activity is measured a t 25O, 37', SO0, and 706C. The temperature optimum is -50PC for aeid phosphatase of bovine aorta (Fig. 3). By varying the initial substrate concentration from 19 mM to 75 mM, a Lineweaver-Burk plot for p-nitrophenyl phosphate as the substrate far acid phosphatase activity in crude bovine aorta homogenate reveals a Km of 3.6 X 1 0 - W (Fig. 4).
Additional Student Investigation
In addition to the investigation of acid phosphatase in a crude homogenate of hovine aorta, students can perform a number of other interesting experiments. They can investigate the properties of the enzyme in both heef heart and heef liver (both show excellent enzymatic activity) as wellas in other materialssueh as beef brain, yeast, etc. They can investigate substrate specificity using o-nitrophenyl phosphate, a-glycerol phosphate, AMP, glucose-&phosphate or py-
1I s Figure 4. Lineweaver-Burk plot of acid phosphatase in bovine aorta. Velocity expressed in A micromoleslmin. Substrate expressed in moiarity.
Volume 54, Number 12. December 1977 / 781
rophosphate. Students can study enzyme inhibition using phosphate, sulfate, or p-nitrophenyl sulfate and can investigate the effectof such divalent metal ions as MgZt, CaZ+,and Zn2+,etc. Finally, the effect of such SH group reagents as N-ethylmaleimideor p-chloromercuribenzoate can he studied.
Literature Cited I11 E%nder,M,L.,K€zdy.F, J..and Wed1er.F. C., J. CHEM. EDUC..44,85 (19671. (2) Hurlbut, J. A,, Bsl1.T. N., Pound, H. C., and Graves,J. L., J. CHEM. EDUC.,50,149
Conclusions
The authors have described a student experiment that can be performed with a minimum of equipment, expense, effort, and expenditure of time, and a maximum of satisfaction and edification. Another advantage is the experiment's relevance to contemporary research problems such as atherosclerosis. For example, i t has been reported (17 ) that in experimental or spontaneous atherosclerosis, a change in tissue metabolism reflects an earlv increase in ~ h o s ~ h a t aactivity. se Also, it has been reported 118)that phohphatase activity iaimportant in the discussion of the pathogenesis of arterial calcification. With this supportive information, we feel it willnot be difficult to generate a great deal of student enthusiasm and interest for this experiment.
782 / Journal of Chemical Education
244.266. J. E., J. Geronlol. 14,161 (19591. (161 Colowiek, S. P..and Kaphn, N. o.."MethcdsinEnrymologj." Academic Press. he., New York,Val. 1.1955.p. 110. (171 Zemplenyi. T., Ladavec, J. H.. and Mrhoui. 0 , J. Athwoaclero8is R e s , 5, 540 (19651. (181 Kirk, J. E., and Praetorius, E. Science. 111,334 (1950). (151 Kirk,
