Fabian J. Lionelti and Edward J. Pastore
Boston University School of Medic~ne Boston 18, Massachusetts
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Isolation and Characterization of Radioactive Adenosinetriphosphate from Mouse Muscle
Teaching of laboratory exercises in hiochemistry to medical students has become partially, if not wholly, a project or group type of collaborative effort. The rapid growth of the science has brought a spate of methodological techniques which can hardly be taught on the former simple and individualistic basis. Laboratory courses consisting mainly of procedures for the analysis of the constituents of blood and urine are slolvly, but surely, being modified to include those which teach intermediary metabolism and characterization of proteins. The demands of such experimentation for the relatively expensive instrumentation of ultracentrifugation, electrophoresis, spectrophotometry, and isotope counting have been met moderately successfully by the group type of project. The design of a group experiment must embody features which provide for more than the mere manipulation of instruments The ones found so far to be most successfulin our course have demonstrated or assessed a biological principle making use of as much modern instrumentation as budgetarily feasible. A good example of this type is the isolation of radioactive adenosinetriphosphate (ATP) from muscle, accomplished by means of the preparation of the barium insoluble phosphate ester fraction from a trichloroacetate filtrate, the absorption and elution of P32labeled ATP on Dowex-1-chloride, and the chemical characterization of the molar constituents of ATP. The data from one of the better experiments are given below. The procedure for ATP is part of a more elaborate p~ograminvolving the same group of students taught a short course on the biological use of isotopes within the same two-week interval. This consisted of three main parts: (1) the identification and characterization of Pa2; (2) its distribution in the organs of the mouse; and (3) the preparation of ATP from muscle. Subgroups of two students were assigned the first two parts, and the remaining four to six students undertook the preparation of ATP. This latter group was able to accomplish the injection of mice with Pa2,the dissection of muscle, and preparation of barium salts of phosphate esters in one laboratory period of five hours. Simultaneously, the resin column was prepared. A second period of five hours was necessary to effect the column neparation and analytical procedures. A final conference of two hours with the assembled group allowed for presentation of data, interpretations, and quizzes. Several short lectures, demonstrations of equipment, and dispersal of mimeographed directions were helpful. The last two hours of the final period were reserved for a detailed discussion and presentation of data. One week was allowed for the submission of a formal report, some representative data of which are given here. 612
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Journal of Chemicol Education
Radioactive
Phosphate Esters from Mouse Muscle
Six to eight adult mice were injected with approximately 100 @curiesof Pa2as NaH2Pa204in doses of 0.1 ml. The animals were immobilized by wrapping them in a cone of thin ~ubbergauze and the injection made subcutaneously in the midback region. This was done so that activity which remained a t or near the site of injection would not contaminate the tissues subsequently dissected. The animals were sacrificed a t 15-minute time intervals for an hour, and a t 2, 3, and 4 hours, for isotope turnover studies in organs. The last three of these a t 2-4 hours postinjection were reserved for the ATP isolation. Placing the mice for a few minutes in a desiccator, the bottom of which contained a small piece of dry ice, was found a rapid and simple means of dispatching them. As much muscle as conveniently obtainable in 10-15 minutes was dissected (avoiding contamination with bone) and immediately placed in about 20 ml of ice-cold trichloroacetic acid (10%). An ice cube was added and the mixture homogenized for about one to two minutes in a small blendor. The homogenate was centrifuged for about 15 minutes a t 4'C a t 2500 rpm, and the supernatant containing the nonprotein tissue components filtered through glass wool. The extract was neutralized with 2 N NaOH until pink to phenolphthalein (ca. 5 1 0 ml) and the barium ion insoluble fraction containing the phosphate esters of adenylates, hexoses, and trioses precipitated by the addition of 0.5 ml of 1 N BaCL. The entire solution was placed in a freezer for about an hour or in a refrigelator a t 4'C overnight to allow for precipitation and flocculation. The p r e cipitated esters were centrifuged for 10 minutes a t 4°C and 2500 rpm, the supernatant discarded, and the precipitate suspended in 5.0 ml of water. Barium ion was exchanged for hydrogen ion by the addition of a gram or two (wet weight, approximated by use of a porcelain spatula) of Dowex-50-resin (hydrogen cycle). The precipitate was observed to dis~olveas the exchange occurred. Completeness of exchange of hydrogen for barium ion was confirmed when a drop of the clear solution was found to give a negative test for barium ion with dilute HZSOI. The resin was separated by decantation and the pH of the solution adjusted to 8.3 with dilute NHIOH (few drops) until slightly pink to phenolphthalein. The solution was then diluted to exactly 35 ml in a large test tube, and an aliquot of 0.1 ml was reserved for a count. Chromatographic separation of the adenosinetriphosphate was undertaken on this extract on a subsequent lab day. The solution was frozen until a resin column (Dowex-1-chloride) was prepared. A discussion of the theory of exchange resins a t this point in the experiment mas timely.
ATP by Column Chromotogrophy Ten grams of resin (Dowex-l-chloride 10% crosslinked, 200400 mesh) were placed in a liter beaker and washed by decantation 8-10 times to remove dirt and fine particles. A 10 ml serological pipet was used for the column with a small plug of glass wool inserted and drawn to the tip with aspiration. A plastic funnel was fitted tightly over the mouthpiece and plastic tubing with a pinch clamp attached over the tip. Resin was suspended in water, poured by decantation into the funnel, and allowed to settle gently by gravity into a packed bed. After the resin was floated into the pipet, a separatory funnel was attached to the mouth end by means of polyethylene tubing. The whole apparatus was clamped to a stand, and the column, funnel, and connecting tube carefully freed of air bubbles. The 35 ml of solution containing the radioactive esters were thawed and poured slowly on the column. Ten-milliliter portions of the effluent were collected in 10-ml graduate cylinders alternately changed and 0.1 ml from each counted wet in nickel cup planchets in an end window Geiger counter. Comparison of the count rate with that of the original filtrate showed that virtually all of the radioactivity had adhered to the resin. See figure near the origin. Seporotion of Rodioodive
irolotion of ATPSzfrom
esters of mouse muscle
All of the esters less polar than ATP were eluted in one fraction from the column with approximately 100 ml of 0.2 N NH,Cl (larger peak in figure).' Volume aliquots (0.1 ml) were taken from each tube and counted. Ordinarily the first few tubes collected contained the greateet poltion of the counts. When a minimum in the count rate was reached and a persistent low value was observed in several successive tubes, the elution of ATP was begun. This was accomplished by switching eluting solvents to more concentrated (0.5 N ) NHKl and collecting 5.0-ml fractions. The count rate of phosphorous in the ATP fraction was seen to reach a maximum and then fall to a constant minimum a t which volume the elution was discontinued. The cumulative volume for the elutions of the two phosphate peaks (including original effluent) usually totaled between 17&200 ml. The apparent broad, low peak in the figure in the region just preceding the elution of ATP is
unexplained and is unique in that it did not occur in other separations. ATPaz The 5-ml fractions corresponding to the maximum concentration of P32 (tubes 16, 17, and 18), were assayed for the constituents of ATP. Adenine was measured by its absorption a t 260 mp using a value for the molar extinction coefficient of 13 X loa. For a solution containing 0.01 pmole per ml, this corresponds to an optical density of 0.130. A dilution of 1:5 of the elutate from the column was usually found necessary. The absorption a t 260 mp measurement was made by the instructor and provided a rapid estimate of the amount of ATP so that proper dilutions for the other analysis could be made. I n this way, student use of expensive silica cuvettes could be avoided. The diluted solution was reserved for assays of ribose, total phosphate, and inorganic phosphate. Ribose was assayed using the orcinol proced~re.~Phosphate in both a 1-ml aliquot digested with HzSOP and an undigested sample was assayed by the Fiske and Subbarow procedure.% Reagents and standards for both these assays were previously prepared and made available to the student groups. Data for the three tubes corresponding to the peak in eluted counts in Figure 1 are given in the table. Standard curves (not shown) were obtained by using dilutions of a ribosed-phosphate standard solution (1 pmole/ml). The tube corresponding to the peak (tube 17) gave molar constituents for A: R: P equal to 1.1 : 1.0 : 2.9. There wele usually negligible amounts of free inorganic phosphate in the preparation indicating no decomposition of the ATP during the chromatography and subsequent analysis. All of the groups achieved good separation of phosphorylated components of the mixture on the column, and nearly all obtained acceptable data for characterization of the ATP. I n one instance when the chromatography was accidentally interrupted for a week of spring recess, the A : R :P values were close to 1 : 1 : 2 giving a more to be expected yield of ADP rather than ATP. I t should be emphasized that the active supervision of an instructor is required, or problems of counting, sample preparation, or intricacies of resin column chromatography can easily lead to poor results with beginning students and greatly reduce the effectiveness of the experiment. Characterization of ATP3z in Column Fractions Choroderizotion of
Fraction No. 16 17 18
Absorption at 260 r n ~ Pentose,' 0.169 0190 0.150
0.155 0.175 0.179
Ratio: T ~ t a l P , " . ~ A:R:PPP 0.432 0.505 0.461
1.1 :1.0:2.8 1.1 : 1 . 0 : 2 . 9 0.85:1.0:2.6
" Corrected for free inorganic phosphate (found to be very low or negligible).
Based on pentose as 1.W. The values given are p molesiml in fractionsdiluted five times.
' Successive elutions were made with NH4C1as shown. The total count in the original extract (0.1 ml counted 924 cpm, total volume 37.5 ml) wag: 37.5 X 10 X 924 = 347,000. The total count recovered by summing the counts in all the tubes was 326,000. MEJBAUM, W . ,2.Physiol. Chem., 258, 117 (1939). a F I S ~C. , H., AND SUBBAROW, Y., J. B i d . Chem., 66, 375 (1925). Volume 36, Number 12, December 1959
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