In most papers on phosphagen published to date, its presence has been considered to be connected with the function of motion. This point of view is no longer tenable, since phosphocreatine has been found not only in every muscular tissue of the Vertebrata and in spermatozoa, but also in nerves, brain and tumours; labile phosphorus has recently been found in the eggs of Amphibia (Zieliński, 1935).

Among the above-mentioned tissues, frog’s eggs are the least specialized and differ most in structure from muscle, where phosphocreatine was first found. For this reason, the present author considered it important to prove that the unstable phosphorus detected by him in the eggs and in different stages of the embryonic life of the frog (Rana temporaria), by Eggleton’s barium method, is identical with muscle phosphocreatine. With a view to throwing light on its possible significance in the egg and its relation to other substances, creatine, creatinine and certain phosphorus compounds which accompany phosphagen in other tissues were estimated.

The method for phosphorus determination used previously (Zieliński, 1935) was slightly modified. In the filtered trichloroacetic acid extract from the eggs from the oviduct, inorganic orthophosphate was precipitated by Eggleton’s barium method in one portion immediately and in another portion after the lapse of 24 hours’ hydrolysis in 0·1 N trichloroacetic acid at 22°. Phosphagen phosphorus was calculated as the difference between the inorganic phosphorus determinations in both portions. The true value for inorganic phosphorus was found in the first portion corrected by the amount of phosphorus which remained in the solution in the second portion, after barium phosphate precipitation. This correction was necessary because barium phosphate (which is highly insoluble in pure water) was partly retained in solution by colloids which remained in it after trichloroacetic acid deproteinization. Pyrophosphate was calculated from the hydrolysis curve of phosphorus compounds in 1N HC1 at 100° according to Lohmann’s (1928) method.

For the total creatine (i.e. creatine and creatinine) determination, 1 or 2 ml. of the trichloroacetic acid extract was taken, hydrochloric acid added to 2N and heated at 60° for 24 hours. After neutralization of the acid with NaOH solution in the presence of one drop of p-nitrophenol solution (according to Koplowitz, 1929), creatinine was estimated in 10-ml. flasks by the Folin micro-method as described in Hunter’s monograph (1928). The creatinine content of each flask was calculated from the extinction measured in a Pulfrich-Stufenphotometer with filters S 47, S 50, and S 53 against the pure reagent solution. In this way, the values measured gave the increase in extinction by the red colour development. According to Lieb&Zacherl (1934), the extinction, if measured by filter S 53 against water is proportional to the creatinine content. In order to obtain the exact values of the coefficients of proportionality, extinction was measured with the above-mentioned three filters for standard solutions of creatinine (pure creatinine made by the Pfanstiehl Chemical Co., Waukegan, Illinois). The following values are the means and the root mean square errors (σ) of n determinations of k = c/e, where e is the extinction coefficient measured as described and c is the creatinine content in mg. per 10 ml.

Attempts have been made to estimate the free creatine by Walpole’s (1911) diacetyl method. The application of colorimetric measurements, however, was quite impossible, since the creatine concentration was too small and many other substances were present in the solution, giving with the reagent a brownish colour, differing from that which was developed when the reagents were added to pure water.

All the results are calculated for 100 eggs in the same manner as in the previous publication, i.e. on the assumption that the total volume of the extract obtained from the eggs was equal to the sum of the amount of water contained in the eggs and the volume of trichloroacetic acid solution added. This mode of calculation is correct if the part of the water, bound by the colloids of the protoplasm and by the jelly membranes, is small (or if the extracted substances are evenly distributed in the whole volume of water) and if there is a negligible adsorption on the surfaces. It seems probable, however, that these sources of error were not the most important ones, as the variations in the figures obtained were independent of the proportion of the acid solution added to the eggs.

Velocity of phosphagen hydrolysis

The identification of such small amounts of phosphocreatine as those occurring in frog’s eggs could not be carried out by isolating the chemically pure compound and by determining its composition and properties. The only method possible was to compare the process of hydrolysis of the labile phosphorus extracted from the eggs with the hydrolysis of the muscle phosphagen. With this in view, to each centrifuge tube containing 1 ml. of extract neutralized with baryta, 0·1 ml. of approximately normal trichloroacetic acid was added. The hydrolysis was carried out in a thermostat at 22·0°. The results obtained in two experiments are given in Table I, where they are also compared with the calculated data. The value of the hydrolysis velocity constant was obtained at the same acid (about 0·1N) and barium salt concentration for phosphagen from frog’s musculus gastrocnemius.

Table I.

Percentage of phosphagen hydrolysis in 0·1N CC13COOH

Percentage of phosphagen hydrolysis in 0·1N CC13COOH
Percentage of phosphagen hydrolysis in 0·1N CC13COOH

It is in agreement with the value calculated from the results of Fiske&Subbarrow (1929) for 0·1N HC1.

One of the most characteristic properties of phosphocreatine is the acceleration of its hydrolysis by the presence of molybdate. Fig. 1 represents the results of the measurements of the colour development, when Kuttner&Cohen’s (1927) reagents were added (final concentrations 1N H2SO4, 0·7 per cent ammonium molybdate, and 0·01 per cent SnCl2) to the extract from the eggs (Curves I and II) ; the curves obtained are exactly similar to those for m. gastrocnemius extract (curve III).

Fig. 1.

Curves I and II, extracts from eggs; curve III, muscle extract.

Fig. 1.

Curves I and II, extracts from eggs; curve III, muscle extract.

Creatine and creatinine

Table II contains a summary of all figures obtained for total creatine extracted from the eggs and compared with those for inorganic phosphate and phosphocreatine. In the last three columns, the results are expressed in 10−6 mois per 100 eggs. In spite of the great variations in their content, it is evident that about one-third of the total creatine is bound as phosphagen. If all inorganic phosphate could be converted in some cases into phosphocreatine, a little more than half of the total creatine would be bound. These results, when compared with those of Gerard&Tupikow (1931) for muscle and nerve, indicate that the ratio of phosphagen to the free creatine in the egg is low.

Table II.

Inorganic phosphate, phosphagen and total creatine calculated per 100 eggs. Average, minimum and maximum values of thirteen analyses of frog’s eggs

Inorganic phosphate, phosphagen and total creatine calculated per 100 eggs. Average, minimum and maximum values of thirteen analyses of frog’s eggs
Inorganic phosphate, phosphagen and total creatine calculated per 100 eggs. Average, minimum and maximum values of thirteen analyses of frog’s eggs

As the Jaffé reaction is not specific for creatinine, it has been considered probable that a part of the red colour was caused by a substance other than creatinine. Hunter&Campbell (1917) showed by measurements of the rate of the red colour development that blood contains compounds, other than creatinine, which give the same reaction with alkaline picrate. A similar method was applied to the extract from the eggs. Here the reaction velocity was lower than that determined by the authors cited above, the picric acid concentration being five times lower. The extinction determinations were made by a Pulfrich-Stufenphotometer every 2 min. by means of filters S 47, S 50 and S 53. The curves obtained for pure creatinine were different for various wave-lengths of light and only rough approximations to a true exponential form. The values of K were calculated as if the reaction were monomolecular ; they were found not to be constant but to change with time. In view of the fact that between the tenth and the thirtieth minute their change did not exceed the variations evoked by experimental error, their average values for that time and root mean square errors were calculated and compared in Table III with those obtained in the same way for creatinine in the extract from the eggs. Here, the values for extinction should be corrected for the dispersion of light by turbidity ; the correction was taken to be equal to the extinction in zero time obtained by extrapolation, i.e. 0–14 per cent of total final extinction. If there were any compounds giving the red colour immediately, their content was lower than that correction. As the values quoted in Table III are in good agreement, creatinine is regarded as being solely responsible, or almost so, for the Jaffé reaction in the analysed samples.

Table III.

Velocity constant of the Jaffé reaction K=1tlnETEtEt Temperature, 15°. Time t, 10–30 min.

Velocity constant of the Jaffé reaction K=1tlnET−EtEt Temperature, 15°. Time t, 10–30 min.
Velocity constant of the Jaffé reaction K=1tlnET−EtEt Temperature, 15°. Time t, 10–30 min.

An attempt was made to determine the creatinine separately from creatine. The curves of extinction as a function of wave-length of light are given in Fig. 2. The colloids present in great amount in the extract are responsible for that part of the curve above λ = 5700 Å. When λ < 5700 Å., the curve is more inclined, indicating the presence of some creatinine. In order to make it possible to evaluate its content, the extinction caused by the colloids has been extrapolated graphically for that part of the graph on the assumption that log E is a linear function of log λ. The calculated results for creatinine are given in Table IV, and from these data it is evident that about 5 per cent of the total creatine in the egg is in the form of creatinine.

Table IV.

Preformed creatinine calculated from the curves of Fig.2

Preformed creatinine calculated from the curves of Fig.2
Preformed creatinine calculated from the curves of Fig.2
Fig. 2.

Extinction measured for the Jaffé reaction of the extract from eggs plotted against wave—length. 15 April,––24 April, …. extrapolated for colloids.

Fig. 2.

Extinction measured for the Jaffé reaction of the extract from eggs plotted against wave—length. 15 April,––24 April, …. extrapolated for colloids.

As the free creatine could not be estimated in the diacetyl reaction by colorimetric measurements, extinction coefficients have been determined for the coloured solutions obtained against blanks prepared simultaneously with them. As a result the values secured are equal to the difference between the absolute extinctions of both. When A < 4500Å. this difference is negative, but the absolute extinctions are positive. The curves show a maximum which is in all cases less prominent when phosphagen has not been hydrolysed. When these results were augmented by the calculated difference in the amount of free creatine before and after phosphagen decomposition, the curve was parallel to that obtained when phosphagen was hydrolysed. This would suggest that the amount of bound creatine is equal to that calculated from the phosphagen content.

Acid-soluble phosphorus

This was investigated by the Lohmann hydrolysis method. The results are given in Table V. When plotted on a graph, they show that there is about 0·012 mg. of pyrophosphate phosphorus per 100 eggs, probably bound with adenylic acid as adenosinetriphosphate, but no appreciable amount of fructosediphosphate or triose esters. After the pyrophosphate disappeared, further hydrolysis proceeded with an approximately constant speed corresponding to a velocity constant of the order of 0·5. 10−3.

Table V.

Phosphorus fractions in the acid extract expressed in mg. P per 100 eggs

Phosphorus fractions in the acid extract expressed in mg. P per 100 eggs
Phosphorus fractions in the acid extract expressed in mg. P per 100 eggs

After Lundsgaard’s discovery of muscle contraction without lactic acid production, phosphocreatine was looked upon as the first known source of energy of the working muscle. Now, in view of the results of Pamas and co-workers, Meyerhoff and Lohmann, phosphagen seems rather to be a reserve of phosphorus and energy for quick resynthesis of adenosinetriphosphate (Parnas&Ostem, 1935) and can be found in those organs of motion where such quick resynthesis is important for their function (Ostern et al. 1935). It can therefore be surmised that all tissues and organs having nothing to do with the function of movement and in which glycolysis takes place, should contain adenosinetriphosphate, but no phosphagen.

As frog’s eggs and embryos in the stages which cannot execute movements and where no organs are differentiated, contain both adenosinetriphosphate and phosphagen, another explanation of the biological significance of phosphagen is proposed herein. As in anaerobiosis, the glycolysis is not limited by the processes of oxidation, the constancy of the concentration of its coenzyme, the adenosinetriphosphate, seems to be much more important for the organ than in aerobiosis. If phosphagen is present, it can supply energy and phosphorus for the synthesis of adenosinetriphosphate if concentration of the latter has fallen too low, whilst phosphagen can be restituted if there is much adenosinetriphosphate. In such cases phosphagen is needed by and should be present in those organs or tissues which are adapted for anoxybiosis.

The above hypothesis is in good agreement with the facts stated for the early development of the frog. From fertilization until advanced neurula, the resistance to anoxybiotic conditions diminishes (Brachet, 1934; Brachet&Needham, 1935); the ability to increase glycolysis when oxygen is lacking can be regarded as a factor of this resistance. At the same time, there is a steady decline in phosphagen content in the embryo (Zieliński, 1935), and this parallelism does not seem to be without significance.

  1. Evidence is given that the labile phosphorus of the frog’s egg is identical with phosphocreatine.

  2. The sum of creatine and creatinine in the egg has been determined and identified by the measurements of the rate of colour development in Jaffé’s reaction. About one-third of this amount is bound as phosphagen and about 5 per cent is in the form of creatinine.

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