Phosphocreatine

MICROCHEMICAL

JOURNAL

15,

88-9 1 ( 1970)

Phosphocreatine III.

A

Potential

Error

in

the

of

Determination

of

Phosphate

Moiety

Phosphocreatine

TAO WONG AND H. T. RUAN Department

of Chemistry,

Mississippi Mississippi

Received

August

State 39762 20,

University,

State

College,

1969

Using the “calcium precipitation” procedure similar to that by FiskeSubbaRow (I), Vorobev reported in 1960 (2) that egg contains a significant amount of phosphocreatine. Using several methods including the “calcium precipitation” procedure, we were unable to detect a trace of this compound in egg (3). Subsequent searching for an explanation for the discrepancy leads us to reveal a potential error in the determination of the phosphate moiety of phosphocreatine. When LePage’s slight modification (4) of the Fiske-SubbaRow procedure (5) was used for the determination of the phosphate moiety of phosphocreatine, an artifact was observed under the condition described in the discussion section of Ref. (6). In addition, we wish to report the following findings concerning the artifact in this communication. (A) Before the addition of reducing agent, 20-minute (or 30-minute) incubation (to hydrolyze phosphocreatine to orthophosphate and others) is required for the appearance of the artifact (the blue color). Omitting the incubation causes the nonappearance of the artifact. (B) After addition of reducing agent, 1 or 2 minutes of incubation is required for the incipient appearance of the artifact (the blue color). The blue color increases rapidly with time and the deepening of color is interrupted by the addition of water to make the final volume. (C) The artifact (the blue color) is located at the surface; and, therefore, it may be speculated that oxygen is involved in the color formation. (D) Any factors which facilitate complete mixing cause the nonappearance of the artifact. Thus, the delivery of reagents by irregular pipets (such as fastspeed pipets) yielded no blue color at the surface. (E) The volume of sample in the above experiments was 1 ml. When the volume was reduced to 0.5 ml, there was less color. In the experiments with OS-ml volume, the reagent blank also yields a blue color, whereas in the experiments with a 1 ml sample, the reagent blank did not yield blue color. 88

Exp

I 2 3 3 5

no.

(~moles

80 100 I 20 120 120

Orthophosphatei

i’RtCIPITATIOI*;

OF

8.8 X.8 8.8 8.25 8.25

PH

ORTIIOPHOSPHATE

I

Alcohol

AS CALcl~h4

Conditions

TABLE AT

IO 0 0 0 IO

(’ ; : v/v,

SALT

ALKAI

pH

79.1 99. I I I9 121 I I9

(pmolesi

l’hosphate

INE

recowred

9x.9 99.1 99.0 100.9 99.1

Kecovery

in precipitate i’;,

,P ? -e

E z z 2 m

2 5 g

2

-j

90

WONG

AND

RUAN

The experiments described below were designed for two purposes: (a) to confirm the blue color being an artifact; and (b) to find out whether orthophosphate is completely precipitated as calcium salt at pH 8.25 and 8.8. There is controversy in the literature regarding the problem (b). Fiske and SubbaRow reported that [Table VIII of Ref. (I)] phosphate can be completely precipitated as calcium salt at alkaline pH, but recently Ennor (7) stated that phosphate as Ca salt is appreciably soluble at pH 8.8. One volume each of 10% and 5% trichloroacetic acid were added to orthophosphate (the quantities of orthophosphate are recorded in Table 1) . The solution was then neutralized to pH 7-8 with 0.5 NaOH. One fifth of the volume of 10% CaCl, was added (in many experiments, 95% alcohol was also added to make it a 10% alcoholic solution). Finally the solution was brought to pH 8.8 or 8.25. After centrifugation, the precipitate (calcium phosphate) was dissolved in 0.1 N HCI and was assayed by the Fiske-SubbaRow procedure (5). The recovered phosphate, as shown in Table 1, was 98.9-100.9%. The recovery studies demonstrated that phosphate, as calcium salt, is not appreciably soluble in alkaline solution at pH 8.25-8.8 with, or without, 10% alcohol. These studies also confirmed that the blue color is only an artifact. The artifact may be incorrectly interpreted as evidence for the incomplete precipitation of calcium phosphate, or incorrectly interpreted as evidence for the presence of phosphocreatine. Finally, it may be mentioned that the requirement of a 20-minute incubation to obtain the blue color (the artifact) as was described in (A) is significant. In determination of orthophosphate the incubation period is omitted, therefore, the potential error almost certainly will be obviated. REFERENCES I. 2.

3. 4.

5. 6.

Fiske, C. H., and SubbaRow, Y., Phosphocreatine. J. Biol. Chem. 81, 629479 (1929). Vorobev, V. I., Phosphorus compounds of hen eggs and their transformations during incubation. I. Phosphorus compounds of nonincubated hen eggs. Chcm. Abstr. 55, 23746a (1961); Inst. Tsitol. Sb. Rab. 1960, 156-159. Wong, T., Phosphocreatine. II. Concerning the phosphocreatine in the egg. Poultry Sci. 48, 352-354 (1969). LePage, L. A., Methods for the analysis ot phosphorylated intermediates. In “Manometric Techniques” (W. W. Umbreit, R. H. Burris, and J. F. Stauffer, eds.), 3rd ed., p. 272. Burgess, Minneapolis, Minn., 1957. Fiske, C. H., and SubbaRow, Y., The calorimetric determination of phosphorus. J. Biol. Chem. 66, 375-400 (1925). Wong, T., Phosphocreatine. I. Isolation and determination of phosphocreatine by Dowex 2 column chromatography. Anal. Biochem. 27, 218-230 (1969).

ARTIFACT

IN

PHOSPHOCREATINE

ASSAY

91

Ennor, A. H., Determination and preparation of N-phosphates of biological origin. I. Phosphocreatine. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds). Vol 3, pp. 850-851. Academic Press, New York, 1957.