A modification of Fiske and Subbarow's method for determination of phosphocreatine 1,

Transcription

1 A modification of Fiske and Subbarow's method for determination of phosphocreatine 1, By N.-O. Abdon (Lund) and Erik Jacobsen (Copenhagen). (From the Pharmacological Department, University of Lund, Sweden.) The separation of orthophosphate and phosphocreatine (Pkr). which is necessary in determining the latter, is performed in two essentially different ways. In the»direct colorimetric rnethod«by Eggleton and Eggleton (1927) and Fiske and Subbarow (1927) it is made by calculations on the colour development curve. Other methods separate the inorganic phosphate from the Pkr by precipitating the former. For this purpose Lohmann (1928, Lohmann and Je n d rassik 1926) uses Mathisen's reagent, i. e. magnesium citrate in diluted ammonia. This reagent does not precipitate other than orthophosphate, while the esters remain in solution. For the determination of the»preformed inorganic phosphate«this method is therefore the most adequate, but if further separation is not made, the rapidly hydrolysing phosphate from adenosintriphosphoric acid (Atp) will be included in the Pkr fraction in certain cases (See experiment I). Lohmann's method for Pkr determinations was developed before his discovery of Atp (1931), but even since that time the influence of Atp present at Pkr determinations has not been regarded by other investigators. Several examples from the literature show that with this method Pkr has been found in animals, in which other methods failed to detect even traces of it. Using Lohmann's method, Riefler and Hansen (193'3 a and b) found considerable amounts of Pkr in cephalopods, molluscs, holothurians, and gephyrians. In these animals it had not been possible to detect any Pkr by the methods of Eggleton and Eggleton or Fiske and Subbarow (Eggleton and Eggleton 1928, Needham and co-workers, 193'2). On the basis of these facts Riefler criticized the latter methods, though he had not isolated Pkr 1 Received for publication april 24, 193'7.

2 A MODIFICATIO:-l OF FISKE AND SUBBAROW'S METHOD ETC. 101 from the animals in question. His criticism was rejected by Need h am and coworkers (193'3), who pointed to the fact that with Walpole's reaction it was impossible to find any traces of chemically combined creatine ijn these animals. Experiment I. Atp was prepared as barium salt from rabbit's muscle according to the directions of Lo hm ann (193'1, 1932). Before be~ng used the barium salt was dissolved in dilute nitric acid, the barium precipitated with Na 2SO!, the precipitate removed, and the solution neutralized with NaOH. On samples consisting of 2 ml of Atp solution the following analyses were made. Inorganic phosphate, according to Lohmann and Jendrassik mg P was added to the test, refound»leicht abspaltbares Phosphat«= Atp according to Lohmann»phosphocreatine«according to L 0 h m ann phosphocreatine after the removal of Atp through precipitation with Ba(OH) mg P, mg P, 0.Ij'3 mg P, 0.00 mg P. For the precipitation of inorganic phosphate Fiske and Subbarow (1929) use Ca(OH)2 in saturated CaCI 2. Differently to M a thi sen ' s reagent the calcium mixture precipitates pyrophosphate and Atp as well as the orthophosphate and perhaps some of the hexosediphosphate. In the following manipulations of the Pkr fraction the excess of calcium is not at all or only partly removed from the solution. We have found, however, that the presence of calcium will to a considerable degree deepen the blue colour of the reduced phosphomolybdate on which the colorimetric measurements are made. Consequently the Pkr values obtained by this method are too high. The following experiment shows the influence of calcium on the colorimetric phosphate analyses, according to Fiske and Subbarow. Experiment 2. Into a series of 100 ml volumetric flasks a quantity of KH 2P04 solution corresponding to 0.2 or 0.4- mg P was transferred. To this was added I) 70 ml 5!7-N H 2SOj saturated with CaSO. j 2) 10 ml 2.5 P: c. aqeous solution of ammonium molybdate and 3') 5 ml 0.25 p. c. aminonaphtolsulphonic acid. The volume was made up to 100 m!. Exactly 4- minutes after the addition of the reducing agent the extinction values of the solutions were determined by the aid of a Pulfrich photometer (Z'eiss). Filter S 72 and a cuvette length of mm. were used. The values obtained in this way were compared with a similar series of determinations on solutions, which had the same constituents but with the exception that no calcium was added. The mean values recorded below are the average of 15 determinations. Table I shows that the presence of calcium increases the extinction coefficient (E) by about 7 per cent. On the other hand calcium does not change the time relation of the colour development (see table 2).

3 102 N.-0. Aanox AND ERIK JACOBSEN: Table I. Series I (0.4 mg Pin 100 ml) without Ca I with Ca E= E= Series 2 (0.2 mg Pin 100 ml)] without Ca I with Ca E= E= mean value. standard deviation maximum value.. minimum value ± ± ± ± Table 2. The extinction coefficients at different intervals after the addition of aminonaphtolsulphonic acid. Concentration of phosphate = 0.2 mg P in 100 ml. The colorimetric measurements were made by the aid of a Moll colorimeter (Kipp and Zonen); no filter was used. Time after the addition of without Ca with Ca aminonaphtolsulphonic acid E= E= 30 sec min (, Later on Eggleton and Eggleton (1929) suggested the determination of the Pkr by precipitation of the inorganic phosphate with Ba(OH)2 in substance. They used the colorimetric method of 'Briggs (1922), previously criticized by Fiske and Subbarow (1925). Furthermore the»ba precipitation method«of Eggleton and Eggleton also works with the following error: The Pkr solution is saturated with Ba(OH)2 which is precipitated by the addition of H 2S04 The BaSOI. precipitate is not removed until just before the

4 A MODIFICATION OF FISKE AND SUBBAROW'S METHOD ETC. 103 colorimetric reading, i. c. 30 minutes after the addition of the reducing agent, hydrochinon. As shown by experiment 3, the presence of the BaSO1 precipitate reduces the extinction by about 15 per cent. Also in the determination of phosphate, according to the method of Fiske and Subbarow, the presence of BaS0 1 after the addition of ammonium molybdate and aminonaphtolsulphonic causes a marked decrease of the extinction values. If the barium was removed before the addition of molybdate, we obtained the calculated value of extinction. - On the other hand, when molybdate and H 2 S0 1 are heated together with barium the addition of hydrochinon or aminonaphtolsulphonic acid is followed by a development of an. intense blue colour even without the pres~nce of phosphate. Experiment 3. Into each of 15 flasks were transferred 0.2 mg P as KH 2PO[, 12.5 ml 2.5 P: c. ammonium molybdate dissolved in 5-N H 2S01, 3' ml 0.5 P: c. hydrochinon in 15 P: c. sodium sulphite and enough water to make the volume 45 m!. 3'2 minutes after the addition of hydrochinon the extinction coefficients were determined by the aid of a Pulfrich photometer, filter S 72 and a cuvette length of 10 mm were used. In a corresponding series of flasks the same constituents were transferred together with 200 mg BaS0 1 in substance. After 30 minutes the BaS0 1 was filtered away and exactly 3'2 minutes after the addition of hydrochinon the photometer readings were made. Extinction coefficients of tests without Ba standard deviation Extinction coefficient of tests wit h Ba standard deviation ± , , ± , Method. The method proposed below is nothing but a modification of the»calcium precipitation method«by Fiske and Subbarow. Instead of Ca(OH)2 CaC12 we use a saturated solution of Ba(OH)2' The inorganic phosphate is more rapidly precipitated by baryta and there is no risk of incompleteness in the precipitation. Using Ba(OH)2 one avoids, as a rule, the neutralizing of the protein free extract with concentrated NaOH, prescribed by Fiske and Subbarow. The most important advantage is, however, that by addition of Na2S01 the barium can be easily and completely removed before the addition of molybdate and amoninaphtolsulphonic acid. If the barium is not removed before molybdate is added it will greatly disturb the determinations (See above). With regard to the preparation of a protein free extract with trichloracetic acid, we refer to the description of Fiske and Subbar o w.

5 104 N.-0. ABDON AND ERIK JACOBSEN: Reagents: I. 2.5 per cent ammonium molybdate in 5-X sulphuric acid per cent aminonaphtolsulphonic acid; 0.5 g. of specially purified 1,2,4,-aminonaphtolsulphonic acid is dissolved in 195 ml 15 per cent sodium bisulphite and 5 ml 20 per cent sodium sulphite (calculated without crystal water). If the sulphonic acid does not dissolve, sodium sulphite is added in small portions until the solutions become clear. If well stoppered and protected from light the solution will keep for a week. The use of an old reagent changes the extinction. 3. standard phosphate solution, containing mg P in 5 ml; g. of So r e n s e n ls KHPO j is dissolved in 1000 ml saturated solution of Ba(OHh 5. saturated solution of?\asoj' 1-5 ml protein free extract containing about o. I 5 mg P as Pkr are transferred to a centrifuge tube with a capacity of 12m!. To this is added 5 ml saturated Ba(OH) solution. The mixture should be alkaline to phenolphtalein; if this should not be the case, the necessary amount of concentrated NaOH is added. The volume is made up to 10m!. Immediately after the addition of Ba(OH)2 a precipitation appears, which consists of orthophosphate, pyrophosphate, Atp, some hexosediphosphate, and carbonate formed through decomposition of trichloracetic acid. The precipitate is removed by centrifugation. 5 ml supernatant liquid is transferred into another centrifuge tube and I ml Na2S0 4 solution is added. The mixture is centrifugated. 3 ml of the barium free solution is transferred into a 10 ml volumetric. flask, I ml ammonium molybdate is added and the flask is left for 30 minutes. (It is not recommended to accelerate the breakdown of Pkr by heating.) After this time 0.5 ml aminonaphtolsulphonic acid and water ad 10 ml are added. At least 4 minutes after the addition of the reducing agent the colorimetric reading is made by the aid of a photometer. A red glas filter is used. As shown in Table 2 the further deepening of the colour is very slow from 3 minutes after the addition of aminonaphtolsulphonic acid: it is, however, not finished even after 2 hours. Therefore the readings should always be made at about the same point of time. - Every person dealing with colorimetric measurements of phosphate ought to determine for himself the extinction coefficients of known amounts of phosphate. For accurate work this ought to be done every day. Therefore no extinction coefficients or a standard table are given here. If only an ordinary colorimeter is available the readings are

6 A MODIFICATION OF FISKE AND SUBBAROW'S METHOD ETC. 105 made against a standard solution which is prepared simultaneously with the test. The standard solution consists of 5 ml KH 2P04 solution, 10 ml ammonium molybdate in 5-N H 2SO j and 5 ml aminonaphtolsulphonic acid diluted with water ad 100 ml. Calculation: reading of standard X o. 16 reading of the unknown mg P in the used amount of protein free extract. The accuracy of the method. (Experiment 4). To examine the accuracy of the method there was made a series of determinations on Pkr prepared from rabbit's muscle, according to Lohmann (1928). To each test 160 y Pkr-phosphorus was added. A series of 23 determinations on Pkr without addition of inorganic phosphate gave the following results : Mean value: o ± mg as Pkr standard deviation A series of IS determinations on the same amount of Pkr with the addition of KH 2P04, corresponding to 0.25 mg P, gave as results: Mean value: mg P as Pkr standard deviation Summary. I. The calcium sulphate present in the determination of phosphocreatine according to Fiske and Subbarow's»calcium precipitation method«, deepens the colour, thus causing too high values. 2. The BaS0 4 present after the addition of the reducing agent in the determination by Eggleton and Egg let o n vs method reduces the extinction. 3. A modification of Fiske and Subbarow's method, so as to avoid these errors, has been worked out. References. Briggs, A. P., J. Bioi. Chem Eggleton, P., and G. P. Eggleton, Biochem, J Eggleton, P., and G. P. Egg Ie eon, J. Physiol Eggleton, P., and G. P. Eggleton, ibidem Fiske, C. H., and Y. Subbarow, J. Bioi. Chern,

7 106 N.-0. ABDON AND ERIK ]ACOBSE1\: A MODIFICATION OF FISKE ETC. Fiske, C. H., and Su b b a r o w, ibidem Lohmann, K., and L. J'e n d r a s si k, Biochem. Z li Lohmann, K., ibidem Lohmann, K., ibidem. 193' Lohmann, K., ibidem. 193' Needham, D. M., J. Needham, E. Baldwin, and J. Yudkin, Proc. Roy. Soc. 193' Needham, D. M., J. Needham, E. Baldwin, and J. Yu d k i n, Z. plz~isiol. Client, Ri e s s e r, 0., and A. Hansen, ibidem. 193' Riesser, 0., and A. Hansen, ibidem