belonging to the pseudoglobulins, forming a heat-stable, dialysable vasoconstrictor (Received 2 April 1942)

Transcription

1 284 J. Physiol. (I942) IOI, I :6I2.I46 PREPARATION AND SOME PROPERTIES OF HYPERTENSIN (ANGIOTONIN) BY P. EDMAN, U. S. VON EULER, E. JORPES AND 0. T. SJOSTRAND From the Physiology Department and the Chemistry Department of Karolinska Institutet, Stockholm, Sweden (Received 2 April 1942) The blood-pressure raising effect of fresh kidney extract was'discovered by Tigerstedt & Bergman [1898], who ascribed this action to a heat-unstable undialysable protein substance which they named renin. Later investigations [Kohlstaedt, Helmer & Page, 1938] showed that this substance required the presence of some other substance occurring in the blood for the development of its vasoconstrictor action. When this 'activator' and renin were allowed to interact a pressor substance was formed, which was heat stable and alcohol soluble. The new active substance was called angiotonin [Page & Helmer, 1939, 1940]. Braun-Menendez, Fasciolo, Leloir & Mu-noz [1939] in Houssay's laboratory found that venous blood from the ischaemic kidney contained a heat-stable, dialysable pressor substance which they named hypertensin, and showed also that incubation of a certain fraction of blood plasma or serum with renin led to the formation of a substance with similar properties [1940a]. It is now generally accepted that renin acts as an enzyme on a substrate in blood belonging to the pseudoglobulins, forming a heat-stable, dialysable vasoconstrictor substance (hypertensin, angiotonin). As to the chemical nature of this substance, Page & Helmer [1940], as well as Braun-Menendez et al. [1940b], have made certaln suggestions. The former authors [1940] claimed that they had isolated a crystalline picrate and oxalate of the active agent supposed to be a base. Page, Helmer & Kohlstaedt [1940] had previously shown that the pressor agent was not identical with tyramine. Braun-Menendez et al. only stated that the active substance behaves like a polypeptide, being dialysable, precipitable with ammonium sulphate and destroyed by proteolytic enzymes like pepsin and trypsin. In the present paper a report is given of some experiments undertaken in order to study the formation and some properties of this substance.

2 PREPARATION AND PROPERTIES OF HYPERTENSIN 285 PREPARATION OF HYPERTENSIN The crude hypertensin (angiotonin) was prepared as described by Page & Helmer [1940]. The renin was prepared from fresh hog-kidney cortex. The material was ground, extracted with 2 vol. of acetone and left to dry at room temperature. After drying, the kidney substance was finely powdered and extracted twice with 5 and 3 vol. respectively of a 2 % sodium chloride solution. To the extract, glacial acetic acid was added to 2 %, the precipitate filtered off and the clear filtrate dialysed against running water for 24 hr. The salt-free renin solution was then concentrated in vacuo to 1/10 of its volume and filtered again if necessary. 500 g. dry powder gave about 250 ml. renin solution. When tested on the atropinized cat under chloralose anaesthesia, 0-25 ml. of this solution produced a rise in blood pressure of some 50 mm. Hg. Since this crude solution showed no untoward side reactions it was used for the preparation of hypertensin without further purification. The globulins were obtained most easily from defibrinated horse blood, where the rapid sedimentation rate made centrifuging unnecessary. An equal volume of a saturated ammonium sulphate solution was added to the serum and the precipitate collected on filter paper. It was placed in cellophane sacs and dialysed under toluene at room temperature against running tap water for hr. The temperature of the tap water was fairly low even in the summer and kept the system cool. The contents of the cellophane sacs were emptied and mixed twice during the dialysis, and the sacs were kept in movement mechanically the whole time. The volume of the final globulin solution after dialysis was somewhat smaller than that of the serum. No adjustment of the ph of the globulin solution seemed to be necessary before adding the renim globulin solution obtained from 20 to horse serum were heated in an enamelled jar to 400 C. and 750 ml. to 1 1. renin solution (from 1-5 to 2 kg. dried pig-kidney cortex) were added. After 10 min. the globulin-renin solution was quickly denatured by heating in a steam bath. The temperature of the solution rose to 700 C. within 5-6 min. The heating was continued for 15 mn. After cooling, the globulins were filtered off and the precipitate washed with water. The filtrate was concentrated in vacuo to 500 ml. and 4 vol. of alcohol were added. The precipitate was removed and the filtrate concentrated to 50 ml. The solution was made faintly alkaline to litmus with 1 N NaOH, and 500 ml. of dry MeOH were added. After storage in the ice-box for hr. the precipitate was removed. This precipitate was inactive. Then 2 1. dry peroxide-free ether were added. The precipitate was collected in centrifuge tubes, washed with ether and dried in vacuo. The yield per litre horse serum was 100 mg. in one preparation and 200 and 250 mg. in two following batches.

3 286 P. EDMAN AND OTHERS All of them showed the same pressor activity, equivalent to mg. tyramine phosphate (T.P.) per mg. dry substance. A second precipitate of the same order of magnitude or somewhat smaller could be obtained on adding more ether to the filtrate. It showed a 7-8 times weaker activity, 1 mg. corresponding to 0.1 mg. T.P. Somewhat stronger preparations were obtained if the concentrate, after the removal of impurities with 4 vol. of alcohol, was fractionated with ammonium sulphate. To the concentrate, 400 ml., obtained from 101. of globulin solution ammonium sulphate was added to half and full saturation. The precipitates were collected, sucked dry and each was dissolved in 50 ml. of water. Dry methyl alcohol was added to 90% and the precipitate removed. To the mother liquor 3 vol. of peroxide-free ether were added. The precipitates A u J, 1. Fig. 1. Cat, chloralose, 1 mg. atropine sulphate per kg. intravenously. Blood pfsure from the femoral artery. A, 0-25 mg. tyramine phosphate; B, 02 ml hypertensin solution. removed with methyl alcohol weighed 3-6 and 0-9 g. respectively, the former being inactive. The active fractions precipitated with ether weighed 500 and 270 mg. and showed a pressor activity corresponding to 1 and 2 mg. T.P. respectively per mg. dry substance. All preparations were tested on cats under chloralose anaesthesia after the intravenous injection of 1 mg. atropine per kg. body weight. In order to obtain a quantitative measure of the effects tyramine was used as a reference substance. Though there was a certain difference in the type of action (Fig. 1) the relative activities of tyramine and hypertensin could easily be compared. THE PURIFICATION AND PROPERTIES OF HYPERTENSIN As to the chemical nature of the pressor substance, the findings of Braun- Menendez et al. that the hypertensin is destroyed through the influence of pepsin and other proteolytic enzymes were confirmed. It seems very probable, therefore, that the hypertensin is of protein nature.

4 PREPARATION AND PROPERTIES OF HYPERTENSIN 287 To a 1 % solution of pepsin (Parke, Davis and Co.) 1: 3000 in N/100 HCI an equal volume of a hypertensin solution was added. The activity was completely destroyed in 2 hr. at 370 C. and in 6 hr. at room temperature. In both cases, no loss of activity was experienced in using boiled pepsin and trypsin solutions under similar conditions. Nor was there any inactivation of hypertensin if it was stored at room temperature in N/2 hydrochloric acid or in N/2 sodium hydroxide for 10 min. or, in ten times weaker solutions, for 1 hr. Hypertensin is also precipitated by ammonium sulphate. Attempts at fractionating with this salt have so far been less successful because the activity was not to be found exclusively in any definite fraction. The main part of the hypertensin was usually precipitated first at full saturation with ammonium sulphate, but a certain percentage of the activity came down at 40 % saturation. THE CATAPHORETIC MOBILITY OF HYPERTENSIN The protein structure thus being the most probable one, the isoelectric properties of hypertensin were studied in cataphoresis experiments with the Tiselius apparatus. For this purpose a sample of hypertensin was used 1 mg. of which gave the same pressor effect as 2 mg. of T.P. Four observations were made between ph 6 and 7. The hypertensin behaved like an amphoteric electrolyte, and its isoelectric point was between ph 6-3 and 6-5. The electrophoretic mobility of the hypertensin could not be determined accurately because of its high diffusibility even outside the electric field. The electrophoretic behaviour of hypertensin thus supports the opinion that hypertensin is of protein nature. These experiments, however, do not allow any definite conclusions about the true isoelectric properties of the hypertensin itself, because it is a common observation that contaminating proteins may influence the cataphoretic mobility and may give misleading results. The further purification of the hypertensin preparations was not easy, mainly because of its instability. The activity was very often lost during the manipulations. Thus, drying after previous treatment with acetone or ether resulted in considerable loss. The active substance was adsorbed on permutit only to about 50 % or less. It passed through the 'Cella feinst' filter, but the filtration under pressure proceeded very slowly. Repeated attempts were made to use electrodialysis through a parchment membrane. In single small runs 80 % of the active substance was found in the cathode fluid. In others only 45 %. Acetone, cooled with solid carbon dioxide, was passed through the cooling spiral. In most of the experiments, however, the activity was lost and the process could not be applied on a larger scale. In one experiment the pressor effect of the substance obtained on drying the

5 288 P. EDMAN AND OTHERS neutralized cathode fluid was fairly high. Per mg. organic substance it corresponded to 5 mg. tyramine phosphate. The purest preparations gave no reaction for phenol with ferric chloride and no discoloration in alkaline solution. The carbazole reaction of Dische was positive, indicating tie presence of a certain amount of carbohydrate. SUMMARY The claims of Braun-Menendez et al. that the blood-pressure effect caused by renin is due to a proteose split off from the serum globulins through the action of renin have been confirmed. The active substance, hypertensin or angiotonin, is precipitated with ammonium sulphate, destroyed by pepsin and pancreatic extracts, and is of amphoteric nature with an isoelectric field between ph 6-3 and 6-5. This indicates that it is a pioteose. Its formation through an enzymic action on the biologically inactive serum globulins may throw some light upon the formation of secretin, cholecystokinin, substance P of Euler and Gaddum, and similar active substances of protein nature. REFERENCES Brbun-Menendez, E., Fasciolo, J. C., Leloir, L. F. & Munloz,'J. M. [1939].. Rev. Soc. argent. biol. 15, 420. Braun-Menendez, E., Fasciolo, J. C., Leloir, L. F. & Munoz, J. M. [1940a]. J. Phy8iol. 98, 283. Braun-Menendez, E., Fasciolo, J. C., Leloir, L. F. & Munoz, J. M. [1940b]. Amer. J. Med. Sci. 200, 608. Kohistaedt, K. G., Helmer; 0. M. & Page, I. [1938]. Proc. Soc. Exp. Biol., N.Y., 39, 214. Page, I. H. & Helmer, 0. M. [1939]. Central Soc. Clin. Bee. 12, 17 (quoted from Page & Helmer). Page, I. H. & Helmer, O..M. [1940]. J. Exp. Med. 71, 29. Page, I. H., Helmer, 0. M. & Koh1staedt, K. G. [1940]. Amer. Heart J. 19, 92. Tigerstedt, R. & Bergman, P. G. [1898]. Skand. Arch. Phy8iol. 8, 223.