Biochemical and physicochemical studies on renin

Biochemical and Physicochemical Studies on Renin Erwin

Haas,Hildegard Lamfrom and Harry Goldblatt

From the Institute

for Medical Research, Cedars oj Lehrrn,on Hospital, Los Angeles, Calijornia Ilcccived

November

18, 1052

A method for the isolation of highly purified renin has been described (1). The present paper deals with some of the biochemical and physicochemical properties of renin. These were established by electrophoretic analysis and solubility studies as well as by a determination of the effects on renin of organic solvents, metals, acids and bases,dialysis, lyophilizat,ion, and storage. A knowledge of some of these properties was required in establishing maximum conditions in each of the ten steps of the procedure for the isolation of highly purified renin (1). Other properties may be of interest in future attempts to elucidate the physiological and enzymatic action of renin. A somewhat more detailed description of experimental results seems indicated because the problems encountered here differ in some respects from usual enzymatic studies. Renin is present in low concentration in the kidneys of slaughterhouse animals (less than 3 mg./kg. of tissue), and to obtain sufficient amounts of purified renin a procedure suitable for large-scale preparation had to be designed. Renin is extracted from kidneys in a state of low purity, and therefore more than 56,000-fold purification was required in the course of its isolation. Problems of st,orage under various experimental conditions required consideration, since t,ime constitutes an important factor in the processing of largescale biological preparations, and becauserenin in purified form loses its enzymatic activity rapidly at room temperature, or in the icebox, unless it is stored under specified conditions. The bio-assay of renin, published previously (2), will be discussed briefly to reiterate certain features of this method which were found 63

64

H.4.46, LAMFROM Ah'D GOLDBLATT

to be of importance in the analysis of renin and in the detection of undesirable contaminants. The enzymatic action of renin in the highly purified form has been tested by the formation of hypertensin, ita vitro, with very small amounts (0.25 pg.) ‘of renin/ml. of serum. Finally, the neut.ralization of renin by antirenin has been demonstrated for renin preparations at every level of purity. I

8

I

8

I

1

60-MM Hg 504.0-

I UNITS

2 OF FENIN

3

Fro. 1. Bioaseay of renin. Blood-pressure elevation aa a function of the renin concentration. Normal, trained, unanesthetized dogs. EXPERIMENTAL

R~ULTS

B&Assay of Renin The method, published in 1943 (a), was used for the quantitative determination of renin in all fractions, from the crudest to the most purified. The solution to be tested is injected intravenously into normal, trained, unanesthetized dogs (10-20 kg.), and the elevation of the direct mean femoral blood pressure is measured. The amount of renin required

to cause an elevation of 30 mm. of mercury has been designated a~ I dog unit. At this level at least, the effect of renin is independent of the weight of the animal, within the limits stated above. The maximum blood pressure elevation, usually reached in 2 min. after the intravenous injection of renin, has been plot.ted as a func+ion of the renin concentmtiolr, itI Fig. I. I

I

I

I

I

I

I

-

2 UNITS

c--e--o

I UNIT

\ , c

0

i-----J:-. I 0

I

I

I

40

20 TIME

I

IN

I

I 60

MINUTES

The blood-pressure elevation is directly proport’ional to the renin concentrattion, up to an elevation of 30 mm. mercury. This wnstitutes the basis for a rapid, accurate and specific test for renin. Figure 2 demonstrates the time-pressure relat,ions aft,er the injections of 1 and 2 units of renin. An increase of’ the renin conrentration beyond 1 unit increases the peak pressure, but. it has a much more pronounced effect in sustaining the pressure at an elevated level. This relationship was demonstrated in Table I.

66

HAAS,

LAMFROM

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GOLDBLA’IT

The time required for a return of the blood pressure to the base line increases proportionately to the amount of renin injected, while the time-pressure integral appears to increase disproportionately to the renin concentration. TABLE

I

Blood-Pressure Elevation as Function of Renin Concentration and Time Ihits of renin

1 2 Ratio :

2

Maximum b.p. elevation mm. Rg

Timepressure integral sp. cm.

12 33

30 45

2.7

1.5

TIME

FIG. 3. Response to repeatSed injections units of renin.

Return to base line min.

30 60 2.0

IN HOURS

of renin. Arrows show injection

of 2

A colony of trained test dogs has been maintained for the past 5 years, each of which responds with a pressure increase of 30 mm. mercury to the injection of 1 unit of renin, with a variation of less than 5 mm. Each test animal, as a routine, is used only once a day. However, as shown in Fig. 3, the injection of even 2 units of renin, at hourly intervals, for a total period of 7 hr., results in a constant response without indication of tachyphylaxis.

RENIN

Detection of Anaphylactic

STUDIES

07

and Depressor Substances

The direct method of bio-assay, under physiological conditions, proved to be of special importance in this investigation. It served as a guide for the quantitative chemical separation from tissue extracts of undesirable substances which are the cause of the pyrogenic, depressor, or anaphylactic side effects encountered so frequently as the result of repeated injections of foreign proteins. The indirect assay met,hod, namely, the production of hypertensin, is unsuitable for such an investigat’ion, since the reaction between renin and hypertensinogen is permit,ted to take place in vitro (3). This method, t,herefore, would not detect the presen e of undesirable impurities in a preparation containing renin, since all the protein and most impurities are precipit,ated in the final st,eps for the production of hypertensin. Because each dose of 4000 units of renin administered t’o a hypertensive patient represents an extract of about 3000 g. of foreign kidney substance, and some of the patients received 40-60 inject,ions in the 8 months of the immunization period (4), knowledge of t)he purity of the renin is of considerable importance. Infect of Salts and Organic Solvents on the Bio-Assay

of Renin

It seemed desirable to investigate to what extent interference could be expected from various substances introduced as a result, of fractionation procedures or added for the stabilization of renin. Considering that more than 4500 bio-assays were required in the course of this investigation, it seemed essential to avoid time-consuming and, sometimes, inactivating steps, such as dialysis, prior to testing. No effert on the blood pressure was observed after the intravenous injection of 2 mI. of the foIlowing solutions: pyrophosphate buffer 0.05 M at pH 7.0 or 0.015 M at pH 5.3; 0.008 M sodium t,ungstate; and 0.2 90 acetone. Furthermore, the standard blood-pressure elevat,ion was elicited, without any side effects, when 1 unit of renin was injected together with the substances ment,ioned above or in the presence of 2 ml. of 0.001 M solutions of metal salts such as magnesium, cobalt, nickel, copper, zinc, and cadmium sulfates; manganese chloride; and mercuric, lead, and uranium nitrates. Testing for renin is possible by this method, even in the presence of small amounts of ethanol. For example, the blood pressure rises somewhat more rapidly after the injection of 1 unit of renin dissolved in 2 ml.

68

HAAS,

LAMFROM

AND

GOLDBLATT

of 5 ‘% ethanol, but, at the end of the usual test period of 2 min., a constant value of 30 mm. Hg, corresponding to 1 unit of renin, is reached. Specific Activity of Renin The term “specific activity” expresses quantitatively the purity of the renin and is a means of following the progress of purification throughout the isolation procedure. It is defined as: Specific activity

=

units of renin mg. dry substance’

The highest value for the specific activity of the final product obtained after step No. 10 of the new isolation procedure was 780 units/mg. of dry substance (1). For the estimation of dry substance gravimetric determinations were made, after exhaustive dialysis of the renin sample and drying at 100” to a constant weight. The micro-Kjeldahl analysis of a preparation of renin, after step No. 9 of the isolat,ion procedure, gave a nitrogen content, of 15.8 $&. On this basis, the activity of the most purified renin produced was about 5000 units renin/mg. nitrogen. Renin in highly purified form was found to be very susceptible to inactivation, apparently because of the removal of protective proteins present in preparations of lower specific activity. For this reason, renin of high specific activity frequently cannot be subjected to the same treatment which may be well tolerated by less pure preparations. With this fact in mind and the necessity of manufacturing renin in large-scale batches, it seemed essential to investigate the properties of renin at various levels of purity. One of the properties of renin, however, its neutralization by antirenin, was found to be completely unaffected by variations in the purity of the renin preparation. As a result, identical antirenin titers in serum were obtained when t,ested for with renin preparations which varied in their specific activity between 3 and 470 units/mg. dry substance. Electrophoretic Analysis of Renin Preparations Two preparations of renin, with specific activities of 280 and 470 were investigated in a Perkin-Elmer Model 38 Tiselius units/mg., electrophoresis apparatus by Dr. S. J. Singer at the California Institute of Technology. The presence of two components was observed in each preparation, but the data in Table II indicate that the concentration of

the slower-moving component has been increased more than threefold with increasing purification, and that it constitutes about, 65 y0 of the total protein present in the renin preparation with a specific activity of 4i0 lmits/mg. This fact, indicat,es that the slower of the two components, characterized in Table111 by an electrophoretic: mobility of - 4.9 X lo--” h((. cm./sec./v., contains t,he renin or t,hat# it might be identical witk rcnill. E’or t,he eleckrophoretic rtnalysis, shown in Fig. 4, a renin preparation with a specific activity of 470 units,/mg. was investigated. This had been c*arried t,hrough all t,en st’eps of the isolation procedure (1) and had beet1 purified 34,OOOtimes. From the electrophoretic patterns in Fig. 4, the relative distribution of the t,\tio components and their electrophoretic mobility was c*alcalated, :tfrd the resu1t.s are summarized in Table III. TABLE II lhstrib~rction~ of Two C’omponents as a Flcnction Specdic activity of renin unils/mg. 380

470

BUffW

0.15 p phosphate 0.20 fi pyrophosphate

r/f Specific [‘M

6.68 6.13

Activity Distr,ibbtioo

of;;mtein

compixxnt per cod

component per c.w

20

80

65

35

Well-defined boundaries were obtained and enantiographic patterns in the ascending and descending limbs even at. a low prot,ein concentration of 3.8 mg./ml. Pyrophosphate buffer was used in this experiment, because of its stabilizing effect on renin. Under these experimental conditions, both components are negatively charged, the isoelectric point of renin therefore lies on the acid side of pH 6.13. LSolubility of Renin in Ammonium Sulfate Solution~s The solubility of renin at a constant pH is a function of the ammonium sulfate concenlration, the renin concentration and the temperature. For a quantitative evaluation of these factors, renin at three different concent,rations was dissolved at 0” in ammonium sulfate solutions of increasing strength. After equilibration at 25”, the amount of renin precipitated was measured, and these values have been plotted in Fig. 5 as a function of t.he ammonium sulfate concentration. Spectroscopic evidence indicates that renin precipitated by ammonium

70

HAAS,

LAMFROM

AND

C;OLDBLATl’

sulfate and redissolved in water differs from renin which haa been exposed to a salt-free environment and which has been precipitated by lyophilization. This will be the subject of the following paper.

ASCENDING

DESCENDING

ELECTRO-

BOUNDARIES

BOUNDARIES

PHORESIS ( MINUTES )

h

72

FIG. 4. Electrophoretic pattern of renin. Conditions: 0.2 P pyrophosphate buffer, pH 6.13; potential gradient, 6.76 V./cm.; current, 6.5 ma.; protein concentration, 3.8 mg./ml.; renin concentration, 1806units/ml.; specific activity of renin, 470 units/mg.

TABLE III Electrophoretic Analysis of Benin

Specific activity: 470 unitsjmg.; 0.2 B pyrophosphate buffer, pH 6.13 Component Relative concentration Mobility per cent sq.cm./s.%./v. Slow

65 35

Fast Solubility

-4.9 -7.2

x LO-6 x 10-S

of Benin in Salt-Free Solutions

Renin can be dissolved readily in a neutral, salt-free solution but, purified beyond step No. 6 of the isolation procedure, it is insoluble, in

REKIPi

STUDIES

‘72

the dialyzed form, at a reaction more acid than plI 4.8. Renin will precipitate out and it can thereby be separated from accompanying renal proteins which remain in solution under t,heseconditions. The dialysis precipitation of renin from solutions of different degrees of purity forms the basis of step No. 6 and, in a somewhat modified form of step Ko. 10 of the new procedure for t.he isolation of renin (1). It has, 7 100 “/o

77

IO

25

UNITS --&-

2 80 I-i-Q - 60 i! cL 40 z z 2 20

I

1.4

1.0 +

1.8

2.2

M

NH4 z SO4 c I

FIG. 5. So1ubilit.y of renin as a functiou of ammonium sulfatt xnd renin concentrations. Temp. = 25”; pH = 3.70.

furthermore, been of practical importance since it permits the preparation of highly concentrated solutions of renin suitable for intramuscular injection into patients. Solutions containing more than 2000 units renin/ml. have been prepared by this method which obviates precipitation by saIts or organic solvents. Finally, the fact that highly purified renin, under certain conditions, can be dialyzed for 2 days, without any decrease in activity, indicates that renin does not require an easily dissociabie prosthetic group or metal ion for its enzymatic activity. Renin, even at a relatively low level of purity, e.g., with a specific

72

EL4.46,

LAMFROM

.4ND

GOLDBLATT

act.ivity of about 22 units/mg., loses about 30% of its activity during dialysis against water for 23 hr. at a temperature of 2”. After the addition of acetate or even better of pyrophosphate buffer t$osuch a renin preparation, dialysis against water becomes possible without, loss of activity, as shown in Table IV. Renin of relatively low purity can be dialyzed without’ loss under the conditions specified in Table IV. In contrast, highly purified renin was found to be much more labile, and almost complete inactivation took TABLE Dialysis

of Renin

(Specific activity:

IV Against

Water

22 units/mg.)

Before dialysis PH

TABLE

15 0 0

V

of Renin Against Salt and Bugkr Solutions (Specific activity:

Buffer

of renin

gw Gent 30

6.0 4.5 3.8 5.3

Water 0.1 M acetate 0.1 M acetate 0.03 hl pyrophosphate

Dialysis

Inactivation

780 units/mg.)

During dialysis PH

Inactivation

of renin

pe7 cent

Water 0.10 M acetate 1.6 M (NH&SO, 1.7 M (NH&SO, 0.04 M pyrophosphate 0 .O4 M pyrophosphate 0 .OP M pyrophosphate

4.5 3.5 2.5 6.0 7.0 6.2 5.2

85 44

47 37 27 17 0

place when reniu with a specific activity of 780 units/mg. was dialyzed at 2’ for 16 hr. against distilled water. This inactivation, as shown in Table V, appears to be most pronounced in salt-free solution, but it takes place also to a considerable degree in diluted acetate or in concentrated ammonium sulfate solutions. Dialysis of the purest renin, without loss of activity, is possible against pyrophosphate buffer, especially at a slightly acid reaction. A protective action is also exhibited by dialysis against 0.1% glycine, at pH 4.3, although to a lesser degree than that afforded by pyrophosphate.

RF;NIN

STUDIES

7:~

HighIy purified renin, furthermore, is completely protected during dialysis against cold distilled water while it is suspended in a renin preparation of low specific activity. Prolonged dialysis against water results in a transformation of renin which (‘an be demonstrated by a pronounced change of its absorptioil speckurn. This transformation is reversible, as indicated by spectroscopic evideme, but no way has been found so far to reverse the loss of biological activity which is encountered during the dialysis of highly purified renin :@nst water. This will be the subject of a separate communication. TABLE Innciioation

VI

yf Renin by Metal

Strlts

Incuktation : 30 min. at 2”; pH 6.i; concentration of metal : 0.001 JI; roncentration of renin: 1 unit/ml. Spdic activity of renin unilslm~.

780 175 8 175 ri 8 175 780 17.5

Metal salt

Ferric sulfate Ferric sulfate Ferric sulfate Auric chloride Auric chloride Platinic chloride Silver nitrat,e Cupric sulfate Cupric sulfate

inactivation per G.%l

100 100 16 100 100 85 58 36 0

Infects of Metals on Renin A few preliminary observations seemed to suggest that metal traces might be the cause of the inactivation of renin, which was encountered during the isolation, storage and dialysis of renin especially in its highly purified form. The experiments summarized in Table VI indicate that, renin can be inactivated by some of the metal ions, but. thatappreciable concentrations, 10e3 M are required for complete inactivation. Renin appears to be much less sensitive in this respect than other enzymes, for example crystallized urease (6). Kenin of lower specific activity, as shown in Table VI, is less susceptible to inactivation by iron or copper, than highly purified renin; it is protected apparently by the inert, proteins present in such a renin solution. Gold was found to be more efficient in its reaction with renin, as indicated by itti ability t.o inactivate renin of both high and low specific, activit’y. Protected by increasing amounts of protein, however, for

74

HAAS,

LAMFROM

AND

GOLDBL.4TT

example, in the presence of serum, gold likewise fails to inactivate renin, which precludes the application of gold as an anti-hypertensive agent. The reaction with gold is a function of the concentration of renin and it requires incubation for a few minutes. During that time a profound inactivation of renin takes place, and the customary blood pressure responsein the dog and the diuresis, proteinuria (6), and antirenin formation in the rat are all abolished by this treatment. Renin is not inactivated under identical conditions by other metals, such as the sulfates or chlorides of magnesium, manganese, cobalt, nickel, zinc, cadmium, mercury, lead, and uranium. TABLE Inactivation

(Variation

-__ - -__ -___ specific activity _- --__ unitsfntg. 3 117 60 780 2 300 180 3 56 470

of pH, salt concentration, Solution

Water Water 1.6 M 1.6 M

(NH&SOa (NH&SO,

Water Water Water Saline Saline 0.03 M (NH&SO,

VII

of Renin temperature,

-

and specific activity)

=

1.54 2.3 2.5 2.5 11.4 11.4 9.0 7.3 6.9 3.8

-

The

PH

-

“C.

min.

0 0 0 0

12 2 90 90 10 10 60 l5 20 23

25 25 0 60 23 26

Inactivation _-__~ $50ccnf 0 50 0 73 100 100 0 100 100 1 45

It is furthermore not inactivated by iron that is bound in a protein complex such as ferritin or hemoglobin. E$ect of pH, Temperature and Organic Solvents on Renin

The effect of acid and alkaline reaction at various temperatures and on renin preparations of various specific activity is shown in Table VII. In a state of low purity, renin was found to be unusually stable toward acid reaction, and this property has been utilized in a second, simplified method to be described later for the isolation of semipurified renin. At a more advanced level of purity, rapid inactivation of renin takes place, but it can be seen that the addition of ammonium sulfate has a protective effect, permitting again acidification without loss. This provides

RENN

75

STUDIES

the basis of step No. 9 of the procedure worked out for the isolation of renin. At the highest level of purity, renin cannot be acidified to such an extent without undergoing inactivation, and it cannot be protected by salt. In alkaline solution, renin is relatively stable at pH 9, provided the temperature is low. Complete inactivation takes place in a short time at room temperature and pH 11.4. At elevated temperatures, e.g., at 60” or higher, a rapid destruction of renin was observed, even at a low state of purity. Renin, in acid solution, and at low temperature, appears to be relatively stable toward organic solvents such as ethanol and acetone, even at a fairly high level of purity. A few representative examples, shown in Table VIII, demonstrate suitable experimental conditions. Because of TABLE Effect of Ethanol

Speciik I

activity

zmits/mg. 0.9 04

117

Solvent

l_--.-..--m-m

/

10% ethanol 900%ethanol 60% acetone

1

---, I j 1 j

pH

-

VIII

and Acetone on Renin 1 Temperature

-~

2.15

3.3 3.5

-i- ~~ ~ “C. 2 ~

;

-24 0

Time

~ 28 days 12 hr. 2 hr.

/ In:::-

,~----percm1 ( 0 12

6

this stability, fractionation with ethanol and acetone has been applied in several steps of the purification procedure. Storage of Renin

The storage of renin offered considerable difEculty because of its instability. For example, highly purified renin of specific activity equal to 470 units/mg. lost 45 % of its activity during storage for 1 day, at room temperature, as shown in Table VII. A considerable loss will occur even at icebox temperature, and with renin present in a relatively crude form, unless favorable conditions have been established. These include slightly acid reaction, addition of salts, or storage in the frozen state, as demonstrated in Table IX. Storage of Renin in the Frozen State

A readjustment of the storage conditions was found to be necessary with progressive purification of renin. While renin of lower specific

76

HAAS,

L.4MFROM

.4ND

GOLDBLlTT

activity can be stored at -24”, without loss of activity, in a neutral salt-free solution, stabilization by a high salt concentration or by a slightly acid reaction was required for renin preparations of intermediate activity (see Table X). But even these conditions failed to protect renin in highly purified form and inactivation took place, not so much during the time the solution was stored in the frozen st.ate but, rather during the period of freezing and thawing. Pyrophosphate buffer of pH TABLE Storage

(Specific activity PR

Temperature “C.

6.9 6.9 6.9 6.9 4.6

2 -24 2 2 2

IX

of Renin = 3.6 units/mg.)

0.1 M (NH&SO, 0.9% N&l

TABLE Storage

(Variation

salt

X

of Renin al -24”

of pH, salt, buffer and specific activity)

SpcCiliC

activity WlilS/~~. 8

40 40 40

780 780 780

Solution

Salt-free Saline Saline 1.6 M NaC1 Saline 0.05 M acetate 0.013 M pyrophoaphate

PII

storage daygr

7.3 7.2 4.7 7.2 6.7 3.8 5.2

380 132 52 9 2x3 28 70

Inactivation pn cm1

0 33

0 0” 44 28 0”

QFrozen and thawed five times.

5.5 was finally found to permit storage of renin of the highest specific activity without any loss of activity. Lyophilization

of Renin and Storage in Dry Form

The stabilizing effect,of pyrophosphate, encountered previously during dialysis and storage of renin, was found to be applicable also, with good results, to the process of drying of renin in high vacuum and from the frozen state. This is shown in Table XT.

77

RENIN STUDIES

The vials containing the renin-pyrophosphate solution were shelffrozen and cooled continuously in a propanol-Dry Ice mixture, until the drying processwas well-established, and then held at prevailing room temperature during the remainder of the drying cycle. After 6 hr. in high vacuum, they were sealed off in dry air. The cooperation of the Research Department of the Hyland Laboratories and it,s Medical Director, Dr. M. V. Veldee, in this part of the investigation is gratefully acknowledged. Highly purified renin, lyophilized in the presence of pyrophosphate, can be stored, and it can be redissolved easily by the addition of water. 90 loss of activity was observed after storage for a period of 80 days at room temperature, or after 120 days at 2”. TABLE Lyophilization Specific activity u?ds/mg.

8 60 470

XI of Renin

Solution

PH

Saline Salt-free 0.015 M pyrophosphate

Hypertensin

Neutral Neutral 5.3

Inactivation per cd

38 60 0

Formation in Vitro

The addition of small amounts of highly purified renin, e.g., 0.2 unit, corresponding to 0.25 pg./ml. of beef serum, was found to result in the complete transformation of the hypertensinogen, present in the serum, to hypertensin, during an incubation period of 18 hr. at, 2”. Identical amounts of hypertensin were formed under these conditions at a renin concentration of 0.2 and 2.0 units/ml. serum, with renin preparations varying in their specific activity between 4 and 470 units/mg. dry substance. SUMMARY 1. Enzymatic and chemical properties of renin of various degrees of purity were investigated. 2. The stability of renin in such preparations was determined under various conditions of temperature, pH, ionic strength, and alcohol and acetone concentrations. Wit.h this information available, optimum conditions were selected for the isolation of renin in large-scale preparations, for its purification and storage, and for the separation of undesirable substances.

78

HAAS,

LAMFROM

AND

GOLDBLA’IT

3. Various aspects of the bio-assay for renin have been investigated, such as the detection of shock-producing substances and the response to increasing amounts of renin or to repeated injections. A survey has also been made to ascertain possible interference by chemicals used routinely for the fractionation of renin or for its stabilization. 4. Some of the biochemical properties of renin such as the % vitro” formation of hypertensin and the neutralization by antirenin have been considered briefly. 5. Electrophoretic analysis revealed the presence of two components in a renin preparation obtained after 34,000-fold purification, indicating a purity of about 65 $&with respect to renin, in a renin preparation with a specific activity of 470 units/mg. 6. Highly purified renin was found to be very susceptible to inactivation, but stabilization could be accomplished by pyrophosphate, by glycine, or merely by the presence of inert proteins. On this basis, incidental traces of metal were suspected as a possible cause for the inactivation, but this could not be confirmed by the direct addition of metal salts in low concentrations. 7. Metal salts, e.g., 0.001 M gold chloride, were found to abolish all of the biochemical properties of renin, the ability to elevate blood pressure, to induce formation of antirenin in animals, and to causeproteinuria and diuresis in the rat. 8. The solubility of renin as a function of ammonium sulfate and renin concentration has been investigated. REFERENCES 1. HAAS, E., LAMFROM, H., AND GOLDBLATT,

H., Arch. Biochem.

and Biophys.

42, 368 (1953). 2. GOLDBLATT, H., KATZ, Y. J., LEWIS, H. A., AND RICHARDSON, E., J. Ezptl. Med. 77, 309 (1943).

3. LELOIR, L. F., MUNOZ, J. M., BRAUN-MEN~NDEZ, E., AND FASCIOLO, J. C., Rev. sot. m gentina biol. 16, 635 (1940). 4. GOLDBLATT, H., HAAS, E., AND LAMFROM, H., Trans. Assoc. Am. Physicians 64, 122 (1951). 5. SUMNER, J. B., AND MYEBHCK, K., 2. physiol. Chem. 169,218 (1930). 6. ADDIS, T., BARRETT, E., BOYD, R. I., AND UREEN, H. J., J. Ezptl. Med. 89, 131 (1949).