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Tiie Journal of biological chemistry

American Society of Biological Chemists, Rockefeller Institute for Medical Research,

^

HARVARD UNIVERSITY

MORGAN HALL

FATIGUE LABORATORY

SCHOOL OF MEOICINX AND PUBLIC LIB^AfVY

HCAilH

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THE JOURNAL

"' . ' .

BIOLOGICAL CHEMISTRY

BDITBD BT C. A. HERTER

HtwTork Aflrigtant Editor, K V . RICHARDS, Chicago

WITH THB OOLIABORATIOH OP

J. J. ABBL^ BflMmora, Md. P. A. LSVBUB, If vw York.

S. H. GHTTTBBDBR, B«fr Haywi, Coaa. JACQUES LOBB« Bcfkaley, CaL

H. D. DAKnr, H«w T€ck. A. 8. LOBVENHART, MadiMn, Wii.

OTTO POUH, BMtBD, BfMt. GRAHAM LUSK, lf«w Yoik.

WnXZAM J. GIBS, B«w Tock. A. B. MACALLUM, Toronto, CaiuuIa.

BBID HUBT, WadriivtMi. D. C J. J. R. MACLBOD, Clor^Umd, Ohio.

WALTBR JOBBS, BoMmoio. Md. A. P. MATHBWS, Chicago, m.

J. H. KASn>B» WaoiiiBCloii, D. C L. B. MBHDBL, How Hatoii, Conn.

WALDBMAR KOCH. Chiciico. DL F. G. BOVT, Ana Arbor, Mieh.

W. R. ORBDORFF, Ithaca. B. T.

THOMAS B. OSBORBB, Bow Havoa, Coaa.

FRABZ PPAFP, Bottoa, Maaa.

T. BRAILSFORD ROBBRTSOB, Borkelay, CaL

A. B. TATLOR. Borlnloy, Cal.

V. a VAUGHAB, Aaa Arbor. Mich.

ALFRED J. WACEMAB, Bow York.

HBBRY L. WHEELER, Bow Havon, Coaa.

VOLUME VII

Baltimore

1909-10

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IMf

Corrncvr, 1909-10

BT

THE JOURNAL OF BIOLOGICAL CHEBOSTRT

WATXKLT mtS BALTIMOKl, MD.

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CONTENTS OF VOLUME VH.

Henry L. Whbelbr and Lafayettb B. Mendel: The io- dine complex in sponges (3, s-diiodotyrosine) 1

Gustave M. Meyer: On the preparation and properties of

iodomucoids 11

Tadasu Saixi: Lactic acid in the autolyzed dog's liver 17

Tadasu Saiki: Liqtiid extraction with the aid of Soxh-

let's apparatus 21

Tadasu Saixi: A study of the chemistry of cancer. II.

Purin bases, creatin and creatinin 23

Stanley R. Benedict and Tadasu Saixi: A note on the

estimation of purin nitrogen in urine 27

Lawrence J. Henderson : On the neutrality equilibrium

in blood and protoplasm 29

Helbn Baldwin: Observations on the influence of lactic acid ferments upon intestinal putrefaction in a healthy individual 37

H. D. Daxin: The catalytic action of amino-acids, pep- tones and proteins in effecting certain S3mtheses ... 49

H. D. Daxin : Note on the urorosein reaction 57

C. A. Hertbr: Notes on the action of sodium benzoate on the multiplication and gas production of various bacteria 59

N. E. Goldthwaite: Effects of the presence of carbo- hydrates upon artificial digestion of casein 69

Francis H. McCrudden: The quantitative separation of calcium and magnesium in the presence of phosphates and small amounts of iron devised especially for the analysis of foods, urine and feces 83

Stanley R. Benedict: A note on the estimation of total

sulphur in urinle loi

H. D. Daxin : The fate of sodium benzoate in the human

organism 103

iii

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iv Contents

M. E. Pennington: A chemical and bacteriological

study of fresh eggs 109

R. T. WooDYATT : Phlorhizin glycocholia 133

Robert E. Swain and W. G. Bateman: The toxicity

of thallium salts 137

Lafayette B. Mendel and H. D. Dakin: The optical

inactivity of allantoin 153

H. H. Bunzbl: The mechanism of the oxidation of glu- cose by bromine 157

H. Gideon Wells : The purine metabolism of the monkey . 171

Francis H. McCrudden: The effect of castration on

the metabolism , 185

Francis H. McCrudden: Chemical analysis of bone

from a case of human adolescent osteomalacia 199

Francis H. Mc Crudden : Correction of an omission 201

C. A. Herter and A. I. Kendall: The influence of die- tary alternations on the types of intestinal flora (Plates I-III) \ 203

Alice Rohde and Walter Jones: The purin ferments

of the rat 237

Victor C. Myers: On the salts of cytosine, thymine and

uracil 249

H. Gideon Wells: The presence of iodine in the human

pituitary gland 259

Tadasu Saiki : A note on the physiological behavior of im-

inoallantoin and uroxanic acid 263

M. E. Rehfuss and P. B. Hawk: Nylander's reaction in

the presence of mercury or chloroform 267

M. E. Rehfuss and P. B. Hawk: A study of Nylander's

reaction 273

J. E. Greaves: Effects of soluble salts on insoluble

phosphates 287

Andrew Hunter: The determination of small quantities of iodine, with special reference to the iodine con- tent of the thyroid gland 321

T. Brailsford Robertson: Concerning the relative magnitude of the parts played by the proteins and by the bicarbonates in the maintenance of the neutrality of the blood 351

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Contents v

T. Brailsford Robertson: On the refractive indices of solntions of certain proteins. I. Ovomucoid and ovovitellin 359

Ross Aiken Gortner: The origin of the brown pigment in the integviments of the larva of Tenebrio moli- tor 365

E. P. Lyon and L. F. Shackell: Autolysis of fertilized and

unfertilized echinoderm eggs 371

Nellis B. Foster, M. D.: Studies on the influence of various dietary conditions on physiological resis- tance. I. The influence of different proportions of protein in the food on resistance to the toxicity of ridn and on recuperation from hemorrhage 379

Otto Folin and A. H. Went worth: A new method for

the determination of fat and fatty acids in feces 421

W. R. Bloor: Carbohydrate esters of the higher fatty

acids 427

S. K. Suzuki, E. G. Hastings and E. B. Hart: The production of volatile fatty acids and esters in Cheddar cheese and their relation to the develop- ment of flavor 431

Paul J. Hanzlik: On a method for determining sodium

iodide in animal tissues 459

A. Ravold and W. H. Warren : A case of alcaptonuria .... 465

C. K. Francis and P. F. Trowbridge: Phosphorus in

beef animals 481

Charles Wilson Greene: A new form of extraction

apparatus 503

Proceedings op the American society of biological

chemists vii

Index 509

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THE IODINE COMPLEX IN SPONGES (3,5.Dn01>TYROSINE).

By henry L. wheeler and LAFAYETTE B. MENDEL.

(From the Sheffield Chemical Laboratory and the Laboratory of Physiological Chemistry, Yale University.)

(Received for publication, November 3, 1909.)

The occurrence of iodine in living organisms has been known almost since the discovery of this element.* Little attention was devoted to its possible physiological rdle until after the discovery of iodine as a normal constituent of the thyroid gland by Baumann in 1895. This classic investigation, followed by Drechsel's iso- lation of "Jodgorgosaure" from the homy axial skeleton of a Gorgonian coral, awakened a new interest in the study of the organic iodine compounds. Henze' subsequently made a study of the substance described by Drechsel; he showed that it gives a xanthoprotein reaction and therefore is not iodaminobutyric acid as had been assumed by its discoverer.' The identification of the iodgorgoic acid was completed by Wheeler and Jamieson,* who established the fact that it is identical with 3,5-diiodtyrosine synthetically prepared by them. Aside from this no halogen- containing organic compound of known structure has yet been isolated from animal tissues.

Numerous circumstances exist to encourage the study of the organic halogen derivatives associated with animal tissues. The physiological action of thyroid tissue has been related to its iodine- containing complex, numerous investigations bearing witness to the comparative inefficacy of non-iodine-containing residues.

* Cf. V. F^irth: Vergleichende physiologische Chemie der niederen Tiere, p. 445; Aron: Oppenkeimer' s Handbuch der Biochemie, i, 67.

* Henze: Zeitschrift fur physiologische Chemie, xxxviii, 60, 1903. ' Cf. Drechsel: Zeitschrift fur Biologie, xxxiii, 90, 1896.

* Wheeler and Jamieson: American Chemical Journal, xxxiii, 365, 1905; see also Henze: Zeitschrift far physiologische Chemie, \i, 64, 1907.

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2 Iodine Complex of Sponges

One recalls the observations of Reid Hunt* showing a close paral- lelism between the amount of iodine and the degree of physio- logical activity of preparations of the thyroid glands. There is evidence, furthermore, to show that iodine may be stored in organic form in animal organisms, and especially in the thyroids. Prom the biological and pharmacological point of view it is im- portant to learn how the element administered in the form of inorganic iodides as a drug or otherwise, becomes synthesized into organic complexes.

In continuing the investigation of some of the problems here involved we have begun an examination of the organic substance of the sponges. The choice of this crude material was dictated by the experience of previous investigators who have pointed out the comparative richness of the albuminoid framework of these lower forms in halogens. As early as 1848 VogpP suggested that the iodine found in sponges examined by him was present in an organic (not inorganic) form.

The iodine content of different types of sponges has been vari- ously reported. The figures for the crude products in any case have no exact value because of the inherent impurities in the materials examined. Hundeshagen* selected for his interesting investigation a number of tropical and subtropical homy sponges, (Luff aria, Aplysina, Verongia), containing as high as 14 per cent of iodine in organic combination. He also showed the occurrence of other halogens (CI, Br) in sponges and maintained that the assimilation of iodine in these organisms is a definite function, not an accident. As in the case of the corals* the rich occurrence of iodine is apparently associated with certain varieties. The bath sponges yield i to 1.5 per cent of iodine.

Hundeshagen's careful observations on the horn-like dried

* Hunt: Journal of the American Medical Association, xlix, 1323, 1907; Hunt and Seidell; Bulletin 47, Hygienic Laboratory, Public Health and Marine Hospital Service, 1909. The older literature is here reviewed in detail.

•Vogel: Gelehrte Ameigen der k. Bayerischen Akademie der Wissen- schaften, xxvii, 223, 1848.

* Hundeshagen: Zeitschrift fur angewandte Chemie, 473, 1895.

*Cf. Mendel: American Journal of Physiology, iv, 243, 1900; Cook: ibid., xii, 95, 1904; Momer: Zeitschrift fiir physiologische Chemie, li, 32, 1907.

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Henry L. Wheeler and Lafayette B, Mendel 3

sponge skeletons investigated by him are of unusual significance. He noted that neither hot water nor organic solvents extract any notable amount of iodine. Superheated water or concentrated mineral acids liberate the iodine. Dilute mineral acids act in this way only gradually. Treatment with barium hydroxide dis- solves the organic complex without liberating the halogen. The organically combined halogen is not precipitated by silver nitrate in nitric acid solution except on heating or addition of fuming nitric acid, whereupon silver iodide separates. An organic halo- gen compound can, however, be precipitated as a salt with silver, copper, or lead, if the alkaline solution of the sponge product is neutralized and the corresponding neutral salt of the metal added. Putrefaction appears to liberate iodine.

The properties here described in some detail suggest a close resemblance to the behavior of the diiodtyrosine* described by Wheeler and Jamieson and by Henze. Hundeshagen managed to obtain decomposition fractions successively richer in iodine, without attaining anything of uniform composition. He as- sumed, with unique chemical insight, that: "es mussten jodierte Amidosauren vorliegen und zwar Jodamidofettsauren oder Jodty- rosine oder beiderlei zugleich."

Hamack' examined ordinary bath sponges, containing i.i to 1.2 per cent of iodine. He demonstrated that in these, too, the halogen must exist in organic combination. In attempting to isolate this organic complex he learned that superheated steam destroys it completely so as to liberate iodine. He succeeded in obtaining a more concentrated product, albuminoid in character, which contained over 8.5 per cent of iodine and 9.4 per cent of nitrogen, and which he named **Jodspongin. " Comparabl attempts had been made by Baumann.' Rosenfeld^ prepared ae product having properties like Hamack's Jodspongin by boiling sponges with 12 per cent hydrochloric acid. The iodine content of the insoluble organic residue was 4.8 per cent.

' Wheeler and Jamieson: loc. cit.] Henze: loc. cit.

'Hamack: Zeitschrift fur physiologische Chetnie, xxiv, 412, 1898.

' Baumann: Munchener medizinische Wochenschrift, No. 14, 309, 1896; cf. Hamack: loc cit. ^ 419.

* Rosenfeld : Archiv fur experimenteUe Pathologie und. Pharmakologie, xlv, 51, 1900.

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4 Iodine Complex of Sponges

An attempt to determine the nature of the iodine-yielding complex of the gponges has also been made by Scott.* This investigator claimed that hydrolysis with mineral acids as well as barium hydroxide liberates the iodine. By an undescribed treatment with sulphuric acid Scott transformed the spongin into a soluble product so that it could be subjected to pancreatic digestion, resulting finally in the complete disappearance of compounds yielding the biuret reaction. After removal of di- amino acids with phosphotungstic acid and a leucine fraction by crystallization, an amorphous organic substance rich in iodine was obtained. The author states that it was separated from accompanying monamino acids "durch fraktionierte Krystalli- sation der Kupfersalze" and finally concentrated into a fraction soluble in alcohol. His ** copper salt" could not have been that of iodgorgoic acid, as the latter is insoluble in water and alcohol and is non-crystalline; and since iodgorgoic acid is precipitated by phosphotungstic acid, as will be noted in the experimental part of this paper, it might be assumed that Scott was dealing with another iodine complex. It is more likely in our opinion, however, that the process of cleavage used by him did not yield amino acids and that Scott perhaps obtained some polypeptide combination, i. e., an incompletely hydrolyzed complex.

Halogen protein compounds have been prepared by several investigators.* They give the biuret reaction, but fail to respond to Millon's or the Hopkins-Cole test. It is probable therefore that the tyrosine (and perhaps tryptophane) groups are attacked by the halogen. It is significant in this connection that several of the earlier investigators of the organic basis of the sponges the so-called spongin ^failed to find tyrosine among the products of the hydrolytic cleavage of the albuminoid.* Even with the refined technique of the Fischer ester method, Abderhalden and Strauss* were unable to isolate tyrosine from spongin. This

* Scott: Biochemische Zeitschrift, i, 367, 1906.

"The literature on this topic has lately been compiled by Schryver: The General Character of the Proteins, p. 6 iff., 1909.

Cf . Stfldeler: Annalen der Chemie, cxi, 16, 1859; Zalocostas: Comptes rendus de Vacad4mie des sciences, cvii, 252, 1888, reported finding a trace of tyrosine.

* Abderhalden and Strauss: Zeitschrift ftir physiologie Chemie, xlviii, 53. 1906.

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Henry L. Wheeler and Lafayette B. Mendel 5

corresponds with the statement of other investigators thatspongin fails to give Millon's test, or at best affords a faint reaction.*

Strauss* has attempted to ascertain whether the failure to obtain Millon's reaction is due to a halogen substitution in the effective aromatic group. He subjected spongin to the action of superheated water with the object of liberating iodine. The tyrosine reaction was still negative with the products of the de- composition. Strauss therefore concluded that the halogen union was effected with other groups (phenylalanine?). The formation of iodoform during hydrolysis suggested to him the further possibility that the iodine might also be substituted in aliphatic complexes. Treatment with superheated steam would, however, be expected to decompose iodgorgoic acid with- out giving tyrosine.

Preliminary trials made by the writers with the common bath sponge demonstrated that boiling with barium hydroxide dis- solves the so-called spongin xiithout liberating any noteworthy amount of iodine from the organic complexes. We therefore adopted this method for the decomposition of the sponge albu- minoids. Solutions so prepared gave no reaction with Millon's test. When treated with a solution of silver nitrate and nitric acid the clear fluid on warming suddenly becomes turbid just as the boiling point is reached ; and the yellow solid causing the turbidity soon after separates as a heavy precipitate of silver iodide. This reaction is exceedingly delicate for diiod tyrosine; />-iodphenylalanine, on the other hand, can be boiled in the same way without separating silver iodide.* This behavor of the solutions of the sponge products, together with the typical resist- ance of the iodine-compound toward treatment with barium hydroxide pointed strongly to the existence of a complex similar to, if not identical with, the iodgorgoic acid of the corals. It was found possible to separate a silver compound rich in iodine

* Cf . Krukenberg: Zeitschrift fur Biologie, xxii, 241, i886; Hamack Zeitschrift fur physiologische Chemie, xxiv, 417, 1898, pronounced '*un- sicher*' the Millon's test with the **Jodspongin" prepared by himself.

'Strauss: Siudien uber die Albuminoide mit besonderer Berucksicht- igung des Spangins und der Keratine, Heidelberg, 1904; quoted from Maly*s Jahresbericht fur Tierchemie, xxxiv, 34, 1904.

* Wheeler and Clapp: American Chemical Jounal, xl, 459-60, 1908.

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6 Iodine Complex of Sponges

and soluble in excess of nitric acid, such as was originally ob- tained by Drechsel and later by Henze. Our earliest attempts to isolate a crystalline product from this silver salt failed. The silver-free residues assumed a varnish-like character on concen- tration and did not crystallize. They were still resistant to decomposition by barium hydroxide at this stage.* By fraction- ation of the product with phosphotungstic acid we have now suc- ceeded in obtaining 3,5-diiodtyrosine in abundance in typical crystalline form. The phosphotungstic acid precipitate con- tains other amino acids besides diiodtyrosine, which prevent the crystallization of this substance after a certain amount has separated . The thick mother liquor responds to the silver nitrate- nitric acid test and gives an abundance of silver iodide appar- ently enough to account for all of the iodine present.

The ease with which iodine enters into combination with the aromatic group suggests a number of considerations respecting the function of the latter in proteins as they exist in the diverse tissues. Thus in the th)rroid the halogen is found in the globulin alone, not in the nucleoprotein of the gland. The occasional accumulation of iodine in keratin-yielding tissues (skin, hair) which are as a rule comparatively rich in the tyrosine complex is suggestive in any event.* That it is the latter group with which the halogens ordinarily react seems most probable from a purely chemical standpoint. Tyrosine readily combines with iodine while phenylalanine does not,* under conditions which might be approximated in the animal organism. The behavior of tryptophane under similar circumstances remains to be inves- tigated. Neuberg^ obtained an amorphous iodine derivative of tryptophane by treatment of the latter with iodine in alkrf line solution.

^ One of us earlier had a similar experience in attempting to isolate iodgorgoic add from Gorgonian corals. (Cf. Mendel: American JourtiaL of Physiology, iv, 243, 1900.) This behavior has doubtless led others also to assume that the iodine is present in some form different from that origi- nally isolated by Drechsel.

'Cf. Justus: Arckiv fur pathologiscke Anatomies clxxvi, i, 1904; Ho- wald: Zeitschrift fur physiologische Chemi€t xxiii, 209 (hair), 1897.

* Oswald: Archiv far experimenteUe Pathologie und Pharmakologie, Ix, 126, 1908.

*Neuberg: Biochemische Zeitschrift, vi, 276, 1907.

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Henry L. Wheeler and Lafayette B. Mendel 7

The fact that 3, 5 -diiod tyrosine is contained in sponges requires that Hamack's formula for iodospongin, C^H^INjoSaOao, must at least be doubled. It is curious that 3,s-diiodtyrosine is pre- cipitated by phosphotungstic acid, while tyrosine is not. In this connection it may be stated that we have examined 3,S-di- bromtyrosine and find that this acid is also precipitated by phos- photungstic acid. In fact, the precipitants, as far as examined, that precipitate the iodine acid also precipitate the bromine compound. The acids show a close similarity in properties, as might be expected. When dibromtyrosine is warmed with silver nitrate and nitric acid, silver bromide separates.

EXPERIMENTAL PART.

The material was composed of specimens of ordinary bath sponges, from the Florida coast. They were soaked in water, washed and again dried. An analysis showed 0.69 per cent of iodine in this crude product. After the preliminary trials al- ready mentioned the sponges were hydrolyzed as follows: 400 grams were heated to boiling with 3 liters of barium hydrox- ide solution containing about 800 grams of the hydrate; the heat- ing was continued 8 hours, the volume being kept constant. No free iodide was present at this stage; but the fluid showed an abundance of organically combined halogen which precipitated as silver iodide when the solution was heated with nitric acid and silver nitrate (after removing the silver sulphide formed.) The heating was continued about 40 hours longer. On cooling the solution was mixed with toluene and then stood for about three months before receiving further attention. Considerable sediment separated. This consisted mostly of barium hydroxide and the barium salts of aspartic acid ; it contained some iodine and was treated separately in the same manner as the solution. The latter was precipitated with silver nitrate until an excess of silver was present and the filtrate, on boiling with nitric acid, no longer gave a turbidity. The silver salts, still moist, were treated at ordinary temperature with dilute nitric acid, at first i part of concentrated nitric acid with 4 parts of water, and finally with concentrated nitric acid diluted with an equal volume of water. The acid solution was filtered from silver sulphide and

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8 Iodine Complex of Sponges

then carefully neutralized with ammonia. The precipitate, but not the filtrate, gave a residue of silver iodide when boiled with nitric acid. The precipitate was washed thoroughly with water, then suspended in water and decomposed by hydrogen sul- phide. On evaporating the solution on the steam-bath a brown tar was obtained. This was taken up in water and allowed to evaporate spontaneously. The thick syrup deposited crystals which filtered very slowly on the pump. When dried on a porous plate the material was light gray and weighed 12.0 grams. It was boiled with water and animal charcoal and then recrystallized from water. It formed colorless spikes or hard, brittle, prismatic crystals which were free from iodine and sul- phur. A nitrogen determination agreed wth the calculated con- tent for aspartic acid:

Calculated: Found:

N 10.52 per cent. "^^SZ P^^ cent.

The mother liquor from the above was allowed to stand for

some time, but no crystals separated. It was then decided to

precipitate the solution with phosphotungstic acid. For this

purpose lo cc. of concentrated sulphuric acid were diluted with

190 cc. of water and 80 cc. of this acid were taken to dissolve the

thick syrup. Then 100 grams of phosphotungstic acid in 70

cc. of the acid were added to the solution of amino acids. This

produced a doughy precipitate which it was impossible to wash

in a satisfactory manner. The whole was allowed to stand over

night. The precipitate, still doughy, was washed superficially

w^ith 50 cc. of the above acid and then heated on the steam-bath

with a solution of barium hydroxide in excess. The operation

being repeated, the solution was precipitated with carbon dioxide

and the precipitate thoroughly washed. The filtrate on evapora-

a very soluble varnish. This proved to contain the

i of the iodine compound. It was dissolved in a little

dilute sulphuric acid was carefully added until no fur-

ation of barium sulphate took place, or at least until

ill amoimt of barium remained in the solution. The

w solution was placed in a desiccator over sulphuric

[lowed to stand over night. The next morning a sandy

mass had separated which was seen under the micro-

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Henry L. Wheeler and Lafayette B. Mendel 9

scope to consist of thin, yellowish white plates. This first crop of crystals weighed exactly one gram. When a portion was warmed with nitric acid and silver nitrate, silver iodide separated in abundance. From the mother liquor 1.2 grams more of this crystalline material were obtained ; then a gum separated enclos- ing the crystals so that they could not be filtered. The first crop of crystals was recrystallized from water and Prof. George S. Jamieson very kindly made an iodine determination using the method previously described.* 0.1260 gram of substance gave 0.1360 gram Agl.

Galeulated f<Nr diodtjrrofline, Found:

C^,0,NI^ 1 58.66 per cent. 58.33 per cent.

The material, when rapidly heated, melted at 213® with strong effer\'^escence, turning brown-black. Abderhalden and Guggen- heim* give the melting point of 3,s-diiodtyrosine at 213®. A sample of synthetic 3,5-diiodtyrosine prepared in this laboratory by Dr. S. H. Clapp melted at 213° and when a portion of our material was mixed with it the melting point was not altered. The compounds are therefore identical.

The material when warmed with Millon's reagent gave no red color. A portion was dissolved in strong hydriodic acid and evaporated to dryness on the steam-bath. After this reduction the aqueous solution gave a strong test for tyrosine with the above reagent. The appearance, properties, solubility, etc . , of the above crystals agreed in every respect with those of 3. 5 -diiod tyrosine.

We therefore conclude that the above results definitely prove that the iodine complex in sponges is 3,5-diiodtyrosine, or the so-called iodgorgoic acid.

* Wheeler and Jamieson: loc. cit.

'Abderhalden and Guggenheim: Berichte der deutschen chemischen Gesellschajt, xli, 1237, 1908.

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ON THE PREPARATION AND PROPERTIES OF lODO- MUCOIDS.'

By GUSTAVE M. MEYER.

(From the Laboratory of Biological Chemistry of Columbia University, at the College of Physicians and Surgeons^ New York.)

(Received for publication, November i, 1909.)

Introduction.

In continuation of the work done in this laboratory on the mucoids, by Dr. Gies and collaborators, I have lately prepared and analyzed products formed by the action of iodin on tendo- mucoids. These iodo-products, to which the name iodo-mucoid may be given, contain about 14 per cent of iodin. Similar deriv- atives of other proteins have been prepared and described by several investigators, the iodin contents of which varied from about 8 to 22 per cent.'

It has been assumed by both Blum' and Hofmeister* that the production of a particular iodo-protein depends upon the sub- stitution of iodin for hydroxyl radicals in one or more of the num- erous aromatic nuclei in each of the molecules of the correspond- ing protein. These observers came to this conclusion because hydriodic acid is invariably produced in the process of iodo-pro- tein preparation. More recently the studies of Henze* led to the conclusion that the iodin in such a compound is substituted for hydroxyl in the tyrosin nucleus of the molecule. This prob- ability was also suggested by Hof meister on the ground that the iodized protein no longer responded positively to the Millon test. After the introduction of iodin into tendomucoids these proteins,

* An abstract of a preliminary report appeared in Science, xxv, p. 457, 1907.

* Lepinois: Journal de Pharmacie et de Chimie (6), v, p. 561, 1897. *Blum: Journcd fur praktische Chemie (2), Ivi, p. 393, 1897.

* Hofmeister: Zeitschrift fur physiologische Chemie, xxiv, p. 167, 1896. 'Henze: Zeitschrift fUr physiologische Chemie, li, p. 66, 1907.

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12 lodo-mucoids

also, no longer respond to either the Millon or the Adamkiewicz test. It is probable that, besides being substituted for hydroxyl, iodin combines with protein products in other ways.

Preparation of Tendomucoids.

The tendomucoid products for this investigation were ex- tracted from Achilles tendons of oxen with half-saturated lime water.* The tendon segments were cut into thin cross sections, which were then well washed with running water. The lime water extracts, after filtering, were slightly acidified with 0.2 per cent hydrochloric acid and the precipitated mucoids washed several times by decantation with water and finally collected on hard- ened filter papers. A large portion of this mucoid material was used for the preparation of iodo-mucoids, as described below; the rest was reserved for comparative analysis. The latter por- tion was thoroughly washed successively with 50 per cent alcohol. 95 per cent alcohol and finally with ether, and then dried at 110° C. to constant weight. Elementary analysis gave the following percentage results, the average of two closely agreeing figures in each instance (oxygen by difference) :

C H N S O

48.02 6.63 13.50 3.18 30-67

Preparation of lodo-mucoids.

After several trials the following procedure for the preparation of iodo-mucoids was found to yield the largest amount of pre- cipitable material; 50 grams of mixed tendomucoids,' obtained by the process described above, were dissolved in sufficient 0.5 per cent sodium carbonate solution to make a complete and con- tentrated though somewhat opalescent solution. Into this solu- cion was dropped from time to time and in small amounts, as often as the previously added portion vanished, a total of 16 grams of finely pulverized iodin. Frequent stirring of the mix- ture favored the reaction. The mixture was kept at 50° C. for

* Cutter and Gies: American Journal of Physiology, vi, p. 155, 190 1; also Gies and collaborators: Biochemical Researches, i, p. 163 (Reprint No. 5). 1903.

* Calculated for dry material.

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Gustave M. Meyer 13

several days, with repeated stirrings and additions of dilute sod- ium carbonate to neutralize the acidity that resulted from the formation of hydriodic acid. The clear liquid was then filtered and the iodo-mucoid products thrown down by the addition of dilute hydrochloric acid. The iodo-mucoids were thus precipi- tated in large, heavy, red flocks, much resembling freshly pre- cipitated ferric hydroxid in general appearance. The precipitate was allowed to settle, then washed several times with water by decantation and finally isolated by filtration. The precipitate was further thoroughly treated successively with 50 per cent alcohol, 95 per cent alcohol, and ether. The treatment with 95 per cent alcohol was continued till the washings showed no fur- ther evidence of the presence of free iodin in the precipitate. The product was then dried at 1 10® C. The yield was 21.5 grams.

Dry mixed iodo-mucoid, as thus obtained, is a light, yellowish powder. The product is odorless and tasteless. It readily dis- solves in dilute sodium carbonate or other alkaline liquids to form solutions that in general are as frothy as those of tendomucoid itself. Upon acidif)ring such an alkaline solution with dilute nitric acid, the iodo-mucoids are reprecipitated and the clear filtrate will not jdeld a precipitate wilth silver nitrate. The iodin content of this material cannot be due to any adherent iodid.

Analysis has shown that iodo-mucoids may be reprecipitated practically unaltered. Iodo-mucoids do not respond to either the Adamldewicz test or the Millon test. The results of the xanthoproteic and biuret tests are positive, as is also that of the Molisch test. After hydration of iodo-mucoids with dilute acid, the solution readily reduces Fehling solution. Iodin is easily detected after fusion of iodo-mucoids with sodium, or by the copper-oxid flame test.

Analysis of Iodo-mucoids,

For the quantitative determination of iodin in the products the following method was employed. The substance, previously dried to constant weight at no® C, was weighed into a platinum crucible and moistened with a few drops of a 10 per cent potas- sium hydroxid solution. The mass was then covered with 40 parts by weight of a mixture consisting of i part of sodium car-

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14

lodo-mucoids

bonate and two parts of potassium nitrate, and subjected to com- plete fusion and oxidation. The cooled mass was dissolved in warm, very dilute nitric acid, and then boiled for a minute to expel nitrous acid . After cooling, the iodin was precipitated with /jf silver nitrate solution and weighed as silver iodid. The amount of iodin was also approximately determined volumetrically as a check. Carbon and hydrogen were determined by combustion, sulfur by the caustic alkali fusion process. Analytic details are appended :

1 , a . . . . o . 3294 gram of substance gave o . 0857 gram of Agl 6.... 0.3015 « « « 0.0787 " Agl

2, a. . . .0. 1225 " **

6 O.I4S4 " "

6.

.0.3902 .0.4663

0.0787

u

Agl

0,077s

u

H,0; and 0.1965 gram of CO,

0.0915

tt

HjO; and 0.2327

gram of CO,

0 . 0480

u

BaSO,

00553

u

BaSO,

Nitrogen was determined by the Kjeldahl method. Samples weighing 0.1335 gram and 0.1912 gram were taken for two determinations of nitrogen content. Ox;/gw was calculated by difference. The percentage data are appended:

SAMPLE.

C

H

N

8

I

0

1

43.75 43.66

7.07 7.04

12.56 12.40

1.47 1.67

14.06 14.10

21.09

2

21.18

Average

43.71

7.05

12.48

1.57

14.08

21.14

In order to determine whether the iodo-mucoids suffered loss of iodin by reprecipitation, a few grams of the product referred to above were dissolved in 0.5 per cent sodium carbonate solution, reprecipitated with a 0.2 percent solution of hydrochloric acid and further purified by the process already described. Anal3rsis disclosed the following iodin content :

Substance taken. . . .0. 1200 gram -> 0.0315 Agl « 14. 18 percent. I. Substance taken. .. .0. 1268 gram « 0.0334 Agl -> 14.24 " !•

The change in the iodin figures is too slight to warrant the conclusion that much of an alteration occurred.

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Gustave M. Meyer 15

lodizatian of the Mucoids in Successive Extracts of Tendon.

In the several preparatigns of mixed iodo-mucoids, which were made by the action of iodin upon alkaline solutions of mixed tendomucoids, it was noticed that the total yield of iodo-mucoids in each case was not as great as might have been expected. It was also observed on evaporating to dryness the neutralized liquid from which the iodo-mucoid had been precipitated with acid, that a residue of an organic nature was obtained which was quite soluble in water and precipitable by alcohol. The nature of these residues has not been determined. Similar behavior on the part of tendomucoids themselves had already been noticed.*

It was thought that mucoids obtained from tendons by frcu:- tianal extraction might give rise to products with different con- tents of iodin. Accordingly, further inquiry along these lines was made as follows: A lot of Achilles tendons from oxen was prepared for extraction with lime water by the process already described. Pour successive extracts were made with half- saturated lime water, each extraction lasting 24 hours.' In this way four samples of tendomucoid material were obtained, which were separately redissolved and reprecipitated once prior to further treatment with iodin . Each purified tendomucoid product was promptly dissolved in 0.5 per cent sodium carbonate solution, treated with an excess of finely powdered iodin and kept at37®C. for 24 hours. The clear solutions were then filtered and the iodo- mucoids precipitated from the filtrates with dilute (2 per cent) acetic acid. The acid filtrates were saved for the further treat- ment to be described. The iodo-mucoid precipitates from each tendomucoid product were given identical treatment with alcohol and ether, and finally were dried at no® C. for analysis. The following percentage analytic data were obtained :

Iodised Iodin Nitrogen

Frsction. per cent. per cent.

I 14.20 14.09

a 14.26 14.03

3 14.18 13.27

4 14.00 13.46

* Gies: Science, xxv, p. 463, 1907.

* Cutter and Gies: loc. cit.

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1 6 lodo-mucoids

These variations are hardly decided enough to warrant the positive conclusion that more than one mixture of iodo-mucoids was under examination, yet they do point to the probability, in line with the deduction by Cutter and Gies, that tendon con- tains more than one mucoid.

The acid filtrates from the four iodo-mucoid preparations that are referred to above were neutralized with sodium carbonate, evaporated on a water bath to a small volume and poured into an excess of alcohol. The precipitates were washed with 95 per cent alcohol and ether to free them from water and iodin. The products closely resembled the previously prepared and cor- responding iodo-mucoids. They possessed protein properties, though they did not respond positively to the Millon or Adam- kiewicz tests. They contained iodin and, after hydration with acid, reduced Fehling solution.

These particular results suggest that in the preparation of the iodo-mucoid products, fragments of the tendomucoid molecules were split off, which in many respects resembled the original mucoids, but differed from them especially in their solubility. Whether such a cleavage is brought about by the action of iodin alone, or merely as a result of acidifying the iodized product, cannot be stated at present. Of course it is quite possible that acid reprecipitation of iodo-mucoid mixtures, such as those at hand in these experiments, may result merely in mechanical separation of the products under consideration.

Further studies of iodo-mucoids in this and other relations are in progress in this laboratory under Dr. Gies' direction.

SUMMARY.

1. Iodo-mucoid products were prepared by the action of iodin on tendomucoids in dilute sodium carbonate solutions. The products were modified proteins possessing most of the proper- ties of mucoids.

2 . Iodo-mucoids in the dry state are yellow, odorless and taste- less powders containing approximately 14 per cent of iodin in organic combination. Iodo-mucoids do not appear to be decom- posed by acid reprecipitation from dilute alkaline solutions, although this matter has not been settled.

In conclusion I wish to express my appreciation of the sug- gestions and encouragement I have received from Dr. William J. Gies.

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LACTIC ACID IN THE AUTOLYZED DOG'S LIVER.

By TADASU SAIKI. Instructor, Albany Medical College.

(From the Betider Laboratory, Albany, N, Y.)

(Received for publication, November z, 1909.)

In a previous communication* the discovery of a peculiar ana^obic bacterium in twenty-two out of twenty-four healthy dogs' livers was reported. This organism, which does not grow on usual culture media, was fotmd to exercise both a putrefac- tive and fermentative action on the liver tissue as well as liver extract, resulting in the production of a large amount of carbon dioxide and a strong acid reaction. At that time the writer raised a question* as to the primary origin of lactic aci