INFLCENCE OF HEAT AND HYDROGEN I O S COKCESTRATIOS ON BIOLOGICAL TRhKSPORTATIO1\‘ SYSTEMS COXTAINING SULFUR’. B Y F. F. SORD?
Proceeding from considerations of Hottinger3 the suggestion was put forward recently that an intermediate systems with undetermined structure serves as nucleus of those highly reactive oxygen addition compounds formed by cysteine and reduced gluthatione, which mere supposed to have a decisive role in reversible physiological processes. The latter addition-compounds probably represent new members of that group of systems which-as we know also from certain processes concerning the cell-might be regarded as the “transportation form”$in contradistinction to the usual structural forms of biologically active substances. Since the work reported in this paper was completed it has been indicated6 that the chief characteristic of the transportation form of a compound or system may be regarded as its capability either to mediate in intermittent actions, or to enable irreversible reactions to proceed, especially in case3 where the use of a potentially higher energy content is involved. I t is not supposed to exist in a form which can be investigated successfully by means of our present tools as a chemical entity. Since its capacity to promote the aforementioned types of biological reactions is probably due in the main to an electron transfer caused by the ionic antagonism within the cell, it is probable that model experiments on analogous systems will not at present authorize US to do more than intimate their manner of functioning. Ionic antagonism, we know, also exerts a great influence upon enzyme action, and there are certain reasons for assuming that the same is also true for the influence of adsorption. However, lacking convenient terms for its demonstration, it seems to be difficult, even in biochemical relations, to abandon the structural conceptions at present and to adopt on a larger scale a much-needed and much more flexible visualization, such as that conveyed by the term “transportation form’ ’ . Besides the aforementioned sulfur systems which again might differ in the model experiment and the cell, only a few examples of transportation forms are supposed to exist (e.g. certain compounds of the bile promoting the Work carried out in 1926 in the Section of Biochemistry, the Mayo Foundation, Rochester, Minn. Present address: Division of Agricultural Biochemistry, rniversity of Minnesota, S t . Paul, Minnesota. A. Hottinger: Schweizerische medisin. Wochenschrift, 53, 430 (1923). Edward C. Rendall and F F. Sord: J. Biol. Chem., 69, 315 (1926). F. F. Sord: Chem. Rev., 3, 49 (1926). F. F. S o r d : Science, 65, (1927) (in press).
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hydro-diffusion of the cell, the enol forms of pyruvic acid, and y-glucose) which we already know differ in their thermostability or the conditions of the pH necessary for their existence. Since the unit of the protoplasma is the cell which is surrounded by a colloid cell membrane, it is capable of exert.ing a great influence through the latter in the regulation for instance, of the permeability', the pH and oxidation-reduction potentials. The ionic antagonism in the cell is doubtless influencing the arrangement of the electrons within the system and simultaneously that of the transportation form of a compound. Especially since heat dissociates the complex of adsorption within the living cell2,it was considered advisable to investigate by means of model experiments the influence of changes in energy condition upon the latter compounds. these changes in energy condition being effected by heat or different pH. It is recalled in this connection that' the existence of the enol forms of pyruvic acid is strongly dependent on the hydrogen ion concentration and tk. ese forms again are responsible for its fermentability. The conditions concerning the range of pH are in this case similar to the reversible reaction between tissue, glutathione and hydrogen acceptop, in which on the other hand the GSH is supposed to be a thermostable part of the system. 'I hese facts warranted especially the submission of the newly-described, reversible addition systems to the effect of changes in heat, all the more as the chemical nat,ure of these biologically important transportation systems is unknown, and their splitting, following slight changes in the solution, indicates t,hat they cannot be isolated and studied as chemical entities. Some of the early results with solutions containing this type of system showed that, simply on standing, the solution lost its ability to act as an oxidizing agent. This has to be interpreted as a splitting of the essential oxygen additionproduct between hydrogen peroxide and cysteine. This work has established the fact that the oxygen addition-product is unstable to heat. Although it can be formed readily a t pH 7.4 it is not essential to have as high a concentration of hydroxyl ion and it is possible to demonstrate the activity of the system at much higher hydrogen ion concentrations. After it had been shown that cystine and oxidized gluthatione are readily acted upon by suitable reducing agents in the presence of a type of an oxygen transportation system, and since the characteristic of living chemistry is its mutabiky, it seemed possible that also in the reciprocal effect of the fixed -SH and the -SS groups these or similar transportation forms may be regarded as essential also in promoting the irreversible phase of the reaction. I n the absence of the hydrogen peroxide the formation of this hypothetical system could have been represented by some kind of double-linkage type of combination. Since a molecule in the free state exists in a more condensed phase than the one suitable for a reaction, it is necessary to supply energy to IF. F. Nord: Protoplasma, 2, No. 2 (1927)(in press). * A . de Gregorio Rocasolano: Rev. de la Academia de Sciencas de Zaragoza, 2,92,(1917).
* Data concerning systems, where the trimethylamine oxide of Suwa (Arch. ges. Physiol., 129, 231 (1909)) has the role of the hydrogen acceptor, are not yet available.
BIOLOGICAL TRANSPORTATION SYSTEMS CONTAININQ SULFUR
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the molecule to produce the phase change. This form therefore could have been still more readily explained on the basis of intermediate changes due to the intermittent attachment of an electronegative element. It has been impossible to prepare muscle or boiled yeast suspensions in a manner which does now show a slight reduction of methylene blue in the presence of glutathione. On the other hand no noticeable reduction of indigo carmine could be observed. Since the presence or absence of air produced only very little effect, there is no conclusive indication in these experiments that the same transportation system was present in these cases in a concentration which could be measured by this method. The present paper contains evidence concerning the energy properties of the reversible system of cysteine and cystine and the destruction of the transportation form in the solution by heat.
Experimental Part 1
The apparatus used for this work was based on the principles of that designed by Clark and Cohenl and represented chiefly a multiplication of their single unit. However, the flow of the oxygen-free nitrogen was conducted through a copper tube and greatly facilitated the necessary mobility of the electrode chambers. Two movable burettes, made the addition of different solutions very convenient. The hydrogen peroxide was diluted to the desired strength from three per cent commercial hydrogen peroxide. Phosphate buffer solutions were used for a pH of 7.4, and the volume was always I O O cc. For the other hydrogen ion concentrations the buffers used are given under the individual experiments. The electrode chambers were heated to 100’ by placing in an oil bath which was raised to the desired temperature by an electric heating element. I t was found that when the electrode chamber was so heated it could be immediately replaced in the oil thermostat without danger of cracking. Each of the eight solutions which were used to determine the influence of heat was prepared as follows: I O O cc of phosphate buffer pH 7.4 and 6 cc. of 0.1?: sodium hydroxide were placed in each of the electrode chambers and deoxygenated for one hour with oxygen-free nitrogen. I . j36 g. of cysteine2 hydrochloride was dissolved in 96 cc. of water. This concentration was equivalent to a 0.1h’ solution if the cysteine were pure. As it was only ninety per cent cysteine and ten per cent cystine, the actual concentration of cysteine was 0.09 N . This solution was deoxygenated for one hour and was then added to the phosphate buffer in the electrode chambers. The 6 cc. of sodium hydroxide neutralized all but 6 cc. of the hydrochloride of cysteine and the resulting hydrogen ion concentration of the solution was very near 7 4. Hydrogen peroxide, 0.1h’,and indigo carmine, 0.02 N, were deoxygenated in separate
’ IT. 51. Clark and B. Cohen: Public Health Reports, S o . 834 (1923); see also Edward C. Kendall and F. F. Kord: 1. c. Compare: 8 . Sakuma: Biochem. 2 , 142, 68 (19231,or K. F Hoffman and R . A . Gortner: J. Am. Chem Soc., 44,350 (1922).
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burettes. Ten cc. of indigo carmine was added to each electrode chamber. This produced a drop in potential and the indigo carmine remained in the oxidized form; after about forty-five minutes the stream of nitrogen was stopped and the following amounts of 0.1N hydrogen peroxide were added to the electrode chambers: I , I cc.; 2 , 2 cc.; 3, 3 cc.; 4, 4 cc.; 5 , 6 , 7 and 8, 3 cc. each.
FIG.I
After a few minutes the indigo carmine in each solution was reduced to a light yellow color. Kitrogen was again passed through the solutions about one hour after the peroxide had been added. The potential of all the solutions was then very near -0.19 volts. One cc. of 0.02 X reduced indigo was then added to each of the solutions; this produced the sharp increase in potential shown in the chart. After a few minutes the equilibrium point was reached and another cubic centimeter of reduced indigo was added.
87 I
BIOLOGICAL TRANSPORTATION SYSTEMS CONTAINING SULFUR
The potential produced immediately after the addition was in the neighborhood of -0.24 volts. The solutions represented in Charts I , 2 , 3, 4, 5 and 6 were then boiled. The solutions represented in Charts 7 and 8 were not boiled. As soon as the solutions had again reached the temperature of the thermostat, 30°, reduced indigo was added. A slight oxidizing reaction was present in some of the solutions, requiring two and sometimes three additions of reduced indigo in order to produce a potential of -0.24. After a high 4 1 1
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potential, due to the reduced indigo, had been produced, no oxidation of reduced indigo occurred in those solutions which had been boiled and from which air was excluded. In Chart I , I O cc. of air was admitted to the electrode chamber after the potential of the solutions indicated that the reduced indigo had not been oxidized. The solution was then allowed to stand over night without nitrogen being passed through it. At the twenty-first hour the return to the equilibrium point showed that the reduced indigo had been oxidized,
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and the addition of reduced indigo showed that the solution had marked oxidizing power. Also it was determined after the addition of indigo carmine that this solution would reduce indigo carmine. In the experiments depicted in Chart 2 , all oxygen was excluded. After boiling, nitrogen was passed through the solution continually and the potential after the addition of reduced indigo showed that the solution had no oxidizing power. The added indigo carmine oxidized the reduced indigo and dropped the potential t o approximately -0.15. The solution, however, was unable to reduce the indigo carmine. Subsequently by addition of I cc. of reduced indigo, the indigo carmine was reduced, and the addition of I cc. more raised the voltage to -0.25, but nothing was present in the solution which could oxidize the reduced indigo. Charts 3 and 4 are similar to Chart 2. Chart 5 is a duplicate of Chart I . Air was added after the boiling a t six hours and forty minutes. The solution could both oxidize reduced indigo and reduce indigo carmine. Chart 6 is similar to Chart 2 ; all oxygen was excluded and oxygen-free nitrogen was passed through the electrode chamber continually after the solution had been boiled. Chart 7 shows the oxidizing and reducing power of a solution similar to the others except that it was not boiled and I O cc. of air was admitted to the electrode chamber a t the point indicated by six hours and thirty minutes on the chart. Chart 8 is similar to Chart 7, except that no oxygen was admitted and the nitrogen passed through the solution continually for the entire thirty-one hours. Sulfuric acid, in the amount indicated, was added to neutralize the sodium hydroxide contained in the reduced indigo a t the point indicated by “J”. This produced a slight decrease in the reducing potential. Influence of Hydrogen Ion Concentration In order to prepare the oxygen addition-product in the solutions for Chart 9 to 1 6 , 4 cc. 0.03j N indigo carmine and I cc. 0.1 N hydrogen peroxide were added to each. The solutions represented in Charts 9 and I O were prepared by adding I 76 mg. of reduced glutathione neutralized with sodium hydroxide to IOO cc. of a buffer at a pH of 6.4. The buffer contained 6 0 cc. of M:I~ KHlPO, and 40 cc. of M/I j NaaHPOa. For Charts I I and 1 2 , 176 mg. of reduced glutathione was added to IOO cc. of phosphate buffer pH 5.9: 90 cc. of M / I ~KHIPOl and I O cc. of M / I ~ h’a2HP0,. For Charts I j and 16, 176 mg. of reduced glutathione was added to I O O CC. of buffer pH 3.8. This was prepared by adding 100 cc. of M / I ~potassium acid phthlate to 5.3 cc. of 0.j K hydrochloric acid.
Discussion of Details The increases in potentials shown in the charts were produced by the addition of I cc. of 0.02 N reduced indigo, except where a number not followed by a letter above the curve indicates that a greater number of cubic centimeters was added. Indigo carmine was added in I cc. amounts, causing a decrease in potential.
BIOLOGICAL TRANSPORTATION SYSTEMS CONTAINING SULFUR
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The volume in cubic centimeters of 0.I N sulfuric acid which was added is indicated by a number followed by the letter “J”. The letters “E” and ‘IF” in the charts designate the starting and stopping respectively of the flow of nitrogen through the electrode chambers. The times when reduced indigo and indigo carmine were added to the solution, which are not clearly indicated on the charts, are recorded in the following: Chart I : One cc. of reduced indigo was added a t 2 hr. 1 2 rnin.; 2 hr. 4 2 min.; and 3 hr. 6 min. The solution was heated to boiling between 3 hr. 20 min. and 4 hr. 2 0 min. One cubic centimeter of reduced indigo was added a t 4 hr. 2 0 min., and again a t A hr. 42 min. The nitrogen was stopped and I O cc. of air was added a t 6 hr. 30 min. The nitrogen was started at 2 0 hr. 54 min. At 2 I hr. 3 min. and at each of the points where subsequent sudden increases in the potential begin reduced indigo was added in I cc. portions. Indigo carmine was added to the solution a t 2 3 hr. 2 4 rnin., a t 2 4 hr. 1 6 rnin., and at 2 7 hr. 3 2 min. Chart 2 : The solution was heated to boiling at 4 hr. and was replaced in the thermostat and cooled at 4 hr. 58 min. One of cc. of reduced indigo was added a t 4 hr. 58 min., a t 5 hr. I O min., and at j hr. 4 2 min. One cc. of reduced indigo was added a t 2 j hr. 50 rnin., but this was all oxidized by the indigo carmine which had not been reduced and a second cubic centimeter of reduced indigo was required a t 2 6 hr. I 5 min. in order t,o increase the potential to -0.2 j volts. One cc. of indigo carmine was added a t 2 7 hr. 44 min. and I cc. of reduced indigo a t 2 8 hr. 5 2 min. Chart 3 : The solution was heated to 100’ between 4 hr. 40 min. and 5 hr. I I min. One cc. of reduced indigo was added at 5 hr. I j min.! a t 5 hr. 3 1 min. and a t 5 hr. 4 2 min. At 2 1 hr. 9 min. I cc. of reduced indigo was added. The decrease in potential a t 2 4 hr. 1 8 min. was caused by I cc. of indigo carmine. One cc. of reduced indigo was added a t 2 5 hr. 50 min. and a t 2 6 hr. I j min. The remaining additions of reduced indigo and indigo carmine are clearly indicated. Chart 4: The solution was heated to 100’ at 5 hr. 6 min. One cc. of reduced indigo was added a t 5 hr. 45 rnin., at, j hr. 5 j rnin., at 6 hr. 2 4 min., and at 7 hr. 4 min. The increase in potential at 2 1 hr. 1 2 min. was produced by I cc. of reduced indigo. One cc. of indigo carmine was added a t 2 6 hr. 3 1 min. One cc. of reduced indigo was added at 27 hr. 22 min. and a t 2 7 hr. 4 2 min. Chart 5 : The solution represented in Chart 5 was boiled at j hr. 3 j min. One cc. of reduced indigo was added to the solution after it was cooled at 6 hr. 1 2 min. and another cc. of reduced indigo was added at, 6 hr. 2 4 min. Ten cc. of air was added and the solution was allowed to stand until the twenty-first hour. One cc. of indigo carmine was added at the twenty-eighth hour. The other additions of reduced indigo and indigo carmine in I cc. amounts are clearly indicated. Chart 6 : The solution represented in Chart 6 was boiled at 6 hr. 3 min. One cc. of reduced indigo was added a t 7 hr. 3 min. after the solution had been brought to the temperature of the thermostat. One cc. cf indigo carmine was
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added a t 2 3 hr. 38 min., at 26 hr. 2 min., and at 28 hr. I Z min. The increase in potential a t 27 hr. 14 min. was produced by reduced indigo. Chart 7 : The solution ,epresented in Chart 7 was not boiled. Ten cc. of air was admitted at 6 hr. 31 min. and the solution stood without nitrogen bubbling through i t until the twenty-first hour. Each of the sudden changes from the equilibrium potential is produced by the addition of 0.02 N reduced indigo or 0.02 indigo carmine in I cc. portions.
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Chart 8 : The solution represented in Chart 8 was not boiled and nitrogen was passed through the electrode chamber without interruption. The reduced indigo and indigo carmine were always added in I cc. portions and the additions are clearly indicated on the chart. The solution was very sluggish in both oxidizing and reducing power after the twenty-first hour. The addition of reduced indigo and indigo carmine to the solutions represented by Charts 9 to 16 is clearly indicated.
BIOLOGICAL TRANSPORTATION SYSTEMB CONTAINING SULFUR
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These results shown in Charts 9 to 16 are good evidence that the oxygen addition-product can be formed and will function a t all hydrogen ion concentrations between a pH of 7.4 and 3.8. The ve1oci:y of reaction represented is, in distinction to the conceptions of Conant and Cutter1, not an accurate criterion of the influence of hydrogen ion concentration alone, since the most important factor is the concentration of the oxygen addition-product, and by the method used there was no certainty that the same concentration of the essential transportation form was present in each of the solutions. Charts 1 3 and 14 show a more active oxidation of reduced indigo and reduction of indigo carmine than Charts I I and I 2 , but the results are qualitative evidence that, as the concentration of hydrogen ion increased, the speed of oxidation of reduced indigo and the velocity of reduct,ion of indigo ca
