110 QORTNER: A CONTRIBUTION TO TEEXII1.-A Contribution to the Study of the Oxydases.By Ross AIKEN GORTNER, Ph.D.IN 1883 Yoshida (Trans., 1883, 43, 472) discovered laccase, thefirst of the oxydases to be studied. Later Bertrand (Compt. rend.,1896, 122, 1215) found that another variety of this class waspresent in various examples of the vegetable world, for example,potatoes, bulbs of the dahlia, various mushrooms, such as Russulanigricans, etc. This oxydasc differed radically from laccase in thatit lost its vitality a t 65-70°, and was also able to oxidise aqueoussolutions of tyrosine through various colour stages (from pink torose, violet, and blue-black), ending by a deposition of a black,melanin-like substance, and leaving the supernatant liquid com-pletely decolorised.On account of this power of oxidising tyrosine,Bertrand gave the name tyrosinase t o the oxydase.Many authors* have since that time reviewed and extended theBiedermann (PJiiger’s Archic, 1898, 72, 152) ; Biedermann and Moritz (ibid.,1899, 75, 43) ; Gessard (Ann. hut. Pculezbr, 1901, 15, 593 ; Comnpt. rend., 1903,136, 631 ; 1903, 138, 774; Compt. rend. SOC. Biol., 1902, 54, 1304, 1398) ;v. Fiirth and Schneider (Beitr. C’he-ln. Physiol. Path., 1901, 1, 229) ; Durham (Proc.Roy. Soc., 1904, 74, 310) ; Wilcock (J. PhySioZ., 1906, 34, 207) ; Bouduoy ( Trav.Sci. Univ. Bennes, 1903, 2, 281 ; 1905, 4, 67) ; Gautier (ibid., 1905, 4, 287) ;Weindl (Arch. Enl’ntech., 1907, 23, 632) ; Bertrand and Roseriblntt (Compt. rend.,1908, 146, 304) ; Abd~rhalden and Guggenheim (Zczt8ch.physiol. Chem., 1908, 54,337) ; IVolff (Compt. rend., 1909, 148, 500 ; 149, 467) ; Bncli (Ber., 1909, 42,694 ; Biochem. Centr., 1909, 9, 1, 73) ; Rocques (Compt. rend., 1909, i49, 418)STUDY OF THE OXYDASES. 111original work, so much so that tyrosinases have been found to bevery widely distributed in nature, and to occur not only in manyplants, but also in numerous animal bodies.By far the greater portion of the European work has been donewith the glycerol extracts of Eussula nigricans (Bertrand, Zoc. cit .),Russula queletin (Bertrand and Rosenblatt, Zoc. cit.), Russuladelica (Wolff, Compt. rend,, 1909, 148, SOO), Russula noircissant(Bertrand, Ann. Inst. Pasteur, 1908, 22, 381), etc., but theoccurrence of tyrosinase has been demonstrated in the ink sac of thesquid (Przibram, cited by v.Furth, T’eryleichende chemischePliiysiologie der niederen Tiere, Jena, 1903, p. 372 ; Gessard, Compt.rend., 1903, 136, 631), in the hameolymph of various insects(v. Fiirth and Schneider, loc. cit.), in wheat bran (Bertrand andMutermilch, Ann. Znst. Pasteur, 1907, 21, 833), in the intestinalfluid of meal worms (Tenebro molitor) (Biedermann, Zoc. cit.),in molluscs (Biedermann and Moritz, loc. cit.), in gum arabic andmistletoe (Bonduoy, loc. cit.), and in plants which blacken duringthe process of drying (Gautier, Zoc. cit.), etc., etc.Miss Durham (loc. cit.) states that she obtained evidence of thepresence of tyrosinase in the skins of fetal and newly-born guineapigs and rabbits of black or agouti origin.Inasmuch aa her resultsdepended on the addition of a milligram of ferrous sulphate asan “activator” (no darkening occurring in a tube containing“juices ” and tyrosine but no ferrous sulphate), and, as will beshown later in this paper, a milligram of ferrous sulphate inhibi-tscoloration almost completely-also since her ‘‘ tyrosinase ” (obtainedfrom red guinea pigs) is the only known example of a tyrosineoxidising ferment which oxidises only to the orange stage (allothers progressing to black), and lastly, since the pigment-likesubstances described in her work “are readily soluble in alkalis,”unlike those produced by v. Fiirth and Schneider (Zoc. cit.), andalso by the author from the interaction of tyrosine and tyrosinase,these substances being found to be insoluble even in hot dilutesodium hydroxide or ammonia, it is apparent that her “tyrosin-ase” reaction, if not due to some section of the ferrous sulphate,is certainly due to an agent altogether distinct from that namedby Bertrand “ tyrosinase.” The author has made several att.emptsto confirm her results, but has as yet obtained no trace ofcoloration induced by an oxydase.I n all the literature cited, the tyrosinase was that obtained byextracting with either glycerol or chloroform-water. Gessard( A n n .Inst. Pasteur, 1901, 15, 601) states that the extract maybe made with either chloroform-water or glycerol, but that theglycerol extract keeps best, and has no effect on the results112 GORTNER: A CONTRIBUTION TO THEThe source of the tyrosinase in the experiments described inthis paper varied somewhat, but the major portion of the workis devoted to the description of a new variety of this ferment,which is distinguished by absolute insolubility in water, the activityof which is destroyed by glycerol, by alcohol and ether, or bydrying a t room temperature; further, it does not oxidise resorcinol,orcinol (Wolff, loc.cit.), pyramidone (Bonduoy, Zoc. cit.), or quinol,thus in most of these reactions differing radically from the knowntyrosinases.EXPERIMENTAL.Tyrosinase in t h e Intestinal Fluid of the Meal Worm (Tenebromolitor) .Biedermann (loc. cit.) in 1898’ made a detailed study of themeal worm (Tenebro molitor), and he states that “the middleintestines of three or four hungry worms were triturated withchloroform-water. On allowing the yellow solution to stand over-night with tyrosine, a violebblack coloration was produced, whilstin a solution to which no tyrosine had been added only a slightdarkening wi~s observed.”In a repetition of the work, his results have been confirmedby the author, but it has also been found that the more perfectlythe body solids were removed from the outer surface of theintestine the less rapidly did the coloration with tyrosine proceed,this being true for either hungry or well-fed larva.The body ofthe larva is filled with a white semi-solid folded in many con-volutions. If this solid is exposed to the air, it rapidly changesthrough slate to a dense grey-black, and, as will be shown later,it contains some soluble tyrosinase and a large amount of a new‘‘ insoluble tyrosinase.”The intestinal juice, obtained by removing the intestine andcleansing it as completely as possible from the body-filling, thengrinding with fine quartz in an agate mortar, triturating withchloroform-water, and filtering, does not colour appreciably intwenty-four hours, but later changes through violet to a denseblack solution. The action of the fresh extract on tyrosine isslow, but shows the presence of some small amounts of theoxydase; that the oxidising power is due t o incomplete removalof the body-filling and not to intestinal juices is the present beliefof the author.Soluble Tyrosimase in the Body-Filling of the Meal Worm.Twenty-seven grams of the larva were ground in a mortar withchloroform-water, and the milky liquid was strained througSTUDY OF THE OXYDASEB.113cheese-cloth. The grinding of the residue was repeated until thestrainings were no longer milky, and only the hulls of the larvaremained in the cloth.The milky extract, if kept a short time in the air, rapidlydarkens a t the surface, but remains white where not in contactwith oxygen.The extract was poured into a thin filter paper, and kept,covered with a witch glass, until most of the liquid had filteredthrough, dropping on solid ammonium sulphate in excess ofwhat was required t o produce a saturated solution, this processrequiring some hours.I n this manner the soluble tyrosinaseand the colloidal insoluble tyrosinase, which passed throughthe first filter, were precipitated together as a light grey,voluminous mass.* This was collected, washed with saturatedammonium sulphate solution, dissolved in distilled water andfiltered, reprecipitated with ammonium sulphate, washed with asaturated ammonium sulphate solution, and dissolved in 40 C.C.of 0.05 per cent. sodium carbonate solution, and filtered. Thesolution so obtained was light brownish-grey, and contained thesoluble tyrosinase originally present in the larva. 0.5 C.C. por-tions of this solution were added to zolutions of various reagents,with the results shown in table I. When the tyrosinase solutionhad been previously heated to 90°, no coloration appeared in anytube, excepting in that containing quinol, showing that perhapstwo oxydases were present, tyrosinase being destroyed before 90°,and laccase perhaps surviving the short heating, tyrosinase beingalmost always accompanied by a laccaselike ferment (Bourquelot,Compt. rend., 1896, 123, 315, 423).TABLE I.Action of SoZdEe Tyrosinase from Larva of Tenebro molitor.Total volume, Time,Tube.Reagent. in C.C. in hours. Results.1.2.3.4.6.6.7.8.9.10.-Tyrosine ...............Tyrosine + 0'001 gramQuinol ..................Phenol ..................p-Aminophenol.. .......Guaiacol ...............Phloroglucinol .........Resorcinol ...............Pyramidone ............FeSO,.3333333333i 2247224482424727272Unchanged.Violet-black and prc-cipitate.Unchanged.Deep red.Pink.Brownish-black.Pink,Unchanged.> )9 )* If the filtered liquid is not precipitated, i t very rapidly darkens and soonThe black pigment may be salted out with ammoninm sulphate, becomgs j e t black.and appears as a lustrous, black, amorphous mass.It will be investigated later.VOL. XCVII. 114 GORTNER: A CONTRIBUTION TO THEWhen the precipitation of the filtrate is carried out by theaddition of three volumes of alcohol instead of saturating withammonium sulphate, a grey precipitate is obtained very similar inappearance t o that produced by the ammonium sulphate, butwhen this precipitate is dissolved it shows no tyrosinase properties,and only the laccase-like ferment can be found in the alcoholicmother liquor, showing that apparently alcohol is fatal to thisvariety of tyrosinase.Insoluble Tyrosinase.The residue left on the filter from the filtration of the extract ofcrushed larva (see above) was washed on the filter with chloroform-water during several days.The washing was considered completewhen 10 C.C. of the liquid, which had been in contact with thesolid (total volume=60 c.c.) for sixteen hours, after being filteredthrough double ‘‘ barium ” filters, gave no coloration with tyrosineduring twenty-four hours.The solid so obtJained is a grey, flocculent mass, which, whendried a t 6 5 O , forms 4 to 5 per cent. of the original weight of thelive larva.It contains from 1-0 to 1.5 per cent. of ash, consistingchiefly of iron oxide, and containing no manganese which couldbe detected by the usual tests. The drying process, however,destroys all oxidising activity.The entire insoluble mass, naturally, cannot be called tyrosinase,as a large percentage of it must be other insoluble body products.The insoluble tyrosinase in this preparation is, however, very active.I f the product is washed as above, it may be kept withoutdiminution of activity for months in a tube containing enoughchloroform and water (1CHCI3: 4H@) to cover it completely.When a few drops of this suspension are added to an aqueoussolution of tyrosine, the mixture undergoes a series of colourchanges, ranging through pink, rose, violet, and blue-black to adeposition of a black, pigment-like substance, and leaving thesupernatant liquid completely decolorised.The coloration usuallybegins in from two to four minutes after the addition of tyrosine,and the series of colour changes is complete in a few hours. Ifthe colourless, supernatant liquid is then removed and moretyrosine solution added, the series of cdour changes is repeated.This continual removal and addition of tyrosine solution has beencarried out with one specimen of tyrosinase weighing approximately0.01 gram* for four days, during which the series of colourchanges was repeated seven times.* Where weights of “insoluble tyrosinase used” are given, it means that analiquot portion of the preparation was dried on a water-bath and weighed.Theweights are therefore only an approximationSTlJDY OF THE OXPDASES. 115That the entire series of colour changes is produced from contactwith the insoluble portion and not by a zymogen acting in thepresence of tyrosine to set free soluble tyrosinase, was proved bythe following experiment.A portion of insoluble tyrosinase was added to a saturatedaqueous solution of tyrosine. In a few minutes the solution hadbecome pink, changing shortly to rose. One half of this solutionwas now removed and filtered twice through “barium” filters,the other half remaining in contact with the insoluble tyrosinase.The tubes were then set aside in the dark. In a few hours thecontents of the tube containing the insoluble tyrosinase and tyrosinesolution became changed, first violet, and finally colourless, withthe deposition of a black, pigment-like substance.The filteredportion, on the other hand, remained an unchanged rose colourfor eighteen days, and was then discarded.That the tyrosinase was not present in still unruptured cellswas proved by grinding the insoluble preparation in an agatemortar with fine quartz until no grit was precipitable. This wasthen triturated with water, and filtered. The filtered portion gaveno coloration with tyrosine in twenty-four hours, whilst theinsoluble residue was as active as it was before grinding.Not only does the insoluble preparation oxidise tyrosine easily,but other phenolic compounds are also acted on to produce theseries of colour changes given in table 11:TABLE 11.[Approximately 0.01 gram of insoluble tyrosinase ( + insoluble bodyproducts) was used in each test.Volume of 5 C.C. in each.]Tube. Reagent added.- 1. .....................2. Tyrosine .....................3.4.5.6.7.a.9.10.11.12.13.14.15.Pyrogallol ..................Phloroglucinol ............Resorcinol ..................Quinol ........................Pyramidone ...............Orcinol .....................p-Aminobenzaldehyde ...p-Nitrosobenzaldehyde . , .Ethyl p-aminobenzoate ...p - Aminophenol ............Guaiacol .....................Gum gnaiacum ............2 : 4-IXaminophenol * ...Colour series.Colourless after 72 hours.Pink -+ orange-rose + rose + light red+- violet +- blue-black + insolubleblack precipitate.Colourless after 72 hours.J 9 2 , 9 s? 7 9 7 7 9* 2 9 2 2 27 ) > 7 Y99 7 > ? 9 ,9 2 $ 2 2 ,Y f $ 9 Y 22 1 Y 2 , Y Brown + reddish-brown precipitate.Orange-pink + red +- brownish-red.Rapidly blues.Pink + orange-pink + orange-brown +orange-red + deep red.* An aquecjus solution of 2 : 4-diaminophenol changes colour when exposed to theair, but not nearly so rapidly as when insoluble tyrosinase has been added.1 116 GORTNER: A CONTRIBUTION TO THEI n order to test the effect of salts on the system tyrosine-tyrosinase, 0.001 gram of various salts was added to tubes con-taining approximately 0.01 gram of insoluble tyrosinase and 5 C.C.of a saturated aqueous solution of tyrosine, and the tubes werekept for some time.I n those tubes to which had been added potassium cyanide,mercuric chloride, copper sulphate, uranyl chloride, and ferroussulphate, no coloration was observed during sixteen hours.Theaddition of manganous sulphate, potassium nit7rit>e, barium chloride,potassium oxalate, strychnine, or atropine had no effect on theprogress of the coloration, whilst sodium arsenate, starch, andstarch and pot.assium iodide had a marked effect in that the rosecoloration appeared more rapidly and remained much longer anddeeper in colour than in the case of the untreated solution. Theportion treated with starch and potassium iodide became intensered,* whilst the untreated solution was only light pink.Warmingthe solution to 7 5 O for a short time prevents all coloration when itis subsequently treated with tyrosine, starch, and potassium iodide.Action of Glycerol o n Insoluble Tyrosinase.Various attempts were made to preserve the ferment in glycerolrather than in chloroform-water since Gessard (loc. cit.) recom-mends this method, but in every instance the preparation wasrendered inactive. I n order further to test the effect of glycerol,the author proceeded as follows. One gram of live larva wasground with chloroform-water and filtered through cheese-cloth.To the filtrate (3 c.c.) was added two volumes of glycerol andsolid tyrosine. No coloration appeared in twelve hours. Thesolution was then diluted with water to 36 c.c., and kept for afurther period of twenty-four hours without a trace of colorationappearing; a further dilution to 65 C.C.caused no change inseventy-two hours more. Without the addition of glycerol thecoloration proceeds very rapidly even in the absence of addedtyrosine. Addition of glycerol to the washed insoluble tyrosinaseand keeping the mixture for a few hours caused a total loss ofthe activity of the preparation even when subsequently washedfree from glycerol.Occurrence of a Laccase-like Ferment in the Larva ofTenebro molitor.It was early noticed in the progress of this work that quinol wasrapidly oxidised by the unwashed body-filling of the larva and* No iodine was liberated in 48 hoursSTUDY O F THE OXYDASES, 117not by the washed insoluble tyrosinase.If the washings are heatedrapidly to boiling, the resulting, precipitate collected, and thefiltrate then evaporated a t 30-40°, an oily solid is depositedwhich is very active in oxidising quinol, but does not affect tyrosine.This oxydase is much more resistant to heat than tyrosinase, andmay be heated a t looo for some minutes without losing much ofit% activity. Prolonged heating, however, gradually causes it tolose its oxidising power.The Chromogen occumviryg in t h e Larva of Tenebro molitor.As has been previously stated, the body fluid of the meal worm,when exposed to the action of the air, rapidly darkens under theinfluence of the tyrosinase contained in it and oxygen, to form adense, black solution. The formation of this coloration showsthat a chromogen must be present in the body-filling of the larva,and attempts were made to isolate it.Five grams of the larva were ground with chloroform-water andfiltered through cheese-cloth. The filtrate (150 c.c.) was warmedon a water-bath to 8 5 O to destroy all tyrosinase and to coagulatethe insoluble products, albumen, etc.After a few minutes' heating,the mixture was filtered, and the filtrate precipitated with basiclead acetate, again filtered, and lead removed from the filtrate byhydrogen sulphide. The clear filtrate was evaporated to drynesson a water-bath. The product, so obtained, is a light yellow resin,completely soluble in 0.05 per cent. sodium carbonate (3 c.c.),giving a yellow solution. When three drops of this solution areadded to water, containing insoluble tyrosinase (total vol.2 c.c.),a mixture is obtained giving identical colour changes to thoseobtained from a tyrosine solution, namely, pink, through rose,violet, and blue-black, a black precipitate being finally formed.The amount o'f the chromogen so obtained was very small, butan attempt will be made to prepare larger quantities in the nearfuture. From the evidence a t hand, however, the chromogenappears t o be either tyrosine or a closely allied compound. Nocoloration is produced by the addition of laccase.Occuwence of Tyrosinase in Other Animal Bodies.Two examples of myriopods, Scalopocryptops sexpinosa andJulius canadensis, Newp., were examined for the presence oftyrosinase, and in both instances an abundance of the ferment wasfound.The entire body mas ground with sand in an agate mortar,extracted with chloroform-water, and filtered. This fluid wasdivided into three parts, one containing no added material, oni i a GORTKER: A CONTRIBUTION TO THEtyrosine, and one phenol. I n a few hours the portion containingtyrosine had changed through violet to blue-black, with thedeposition of a black, melanin-like substance. The portion con-taining phenol changed through orange to deep sepia, whilst theuntreated tubes remained colourless. Boiling prevents allcoloration.Tyrosinase was also found to be very abundant in the larva ofCucu jus cZavipes,* changing tyrosine solution through pink to rose,violet, blue-black to melanin, and phenol through rose to light redand crimson.Occurrence of a Quinol-oxidising Ferment in, Vertebrate AnimatTissues.It has long been observed (Baumann and Preusse, Zeitsch.physiol.Chem., 1879, 3, 156) that after the use of phenol theurine assumes a dark colour on exposure to the air (the so-called‘ I carbolic urine ”), from which quinol, quinolsulphonic acid, andquinol-decomposition products may be isolated. The samecoloration occurs after the administration of quinol.The author has observed that this coloration is not peculiar tothe urine, but that extracts of practically all the tissues rapidlyoxidise quinol to intensely coloured solutions. Among the animalsinvestigated were young rats, mice, albino rats, kittens, chickens,etc., and in each instance a rapid oxidation was produced withextracts of the heart, liver, lungs, brain, kidneys, spleen, pancreas,testes, ovary, skeletal muscles, skin, and blood serum; in everycase the coloration being most intense when blood serum wasused.Long-continued boiling causes a gradual loss in oxidisingpower, although a very short heating seems to increase the activity.Different extracts gave different colorations in similar circum-stances, and these changes are shown in table 111. The tissueswere ground with sand, triturated with chlorof orm-water, andfiltered. To the turbid filtrate, quinol was added, and the tube setaside in an oven a t 4OO.f-No coloration was observed in any case in “blank” tubes, orin tubes treated with tyrosine or guaiacol.I n all cases the finalcoloration obtained with quinol was either deep blood-red orreddish-brown verging on black.When quinol was injected subcutaneously in a kitten, and theanimal killed after three to four hours, the post-mortem examina-tion showed a red circle under the skin where the injection was* Identified through the courtesy of Dr. A. D. Hopkins, of the Bureau ofEntomology, The United States Department of Agriculture.t The coloration is decidedly more rapid at 40” than at room temperatureSTUDY OF THE OXYDASES. 119TABLE 111.Time,Tube. Organ. Origin. in hours.1.2.3.4.5.6.7.8.9.10.11.12.13.14.15.16.17.11.19.20.21.22.Skin ............ Young rat ... ........................,) ............,) ) ) ...Spleen ............ , ) ...Kidney ......... ), )) ...Pancreas .........Lungs ............Liver ............) , (boiled). ..Testes ............Muscle .........Brain ...........Bile ...............), ............... ), ......Liver ............ , , ......Brain ............ )) ......Ovary ............ )) ......Blood serum ... ......Pancreas ......... ), ......Spinal cord ...... Grey rat ......Brain ............ ), ......12242424242448484848484848162416161616161616Result.Claret-coloured solution.Deep red solution.Brown solution (had been firstheated to 100").Red solution.Rose ) ) (after 48 hours,red).Rose solution.L&ht pi:k solution.Deep red ,)Pink $ IDeep brown ),Brown Y YBlack 9 )Deep red ) )Reddish-brown solution.Re&ish-bl&k :: Brownish-black y )Bright red $ 9Dull reddish-brown solution.Y Y 1 39 ) $ 9made.The remainder of the under surface of the skin and theflesh were normal in colour, but on exposure to the air the skintissue and the muscles rapidly turned pink. The blood-serum alsosoon became deep red, although, when expressed from the clot, itwas of normal colour.Occurrence of Laccase and Tyrosinase in theGautier (Zoc. c i t . ) found that many plants which blacken duringthe process of drying contain a tyrosine-oxidising ferment. Thecommon Monotropa uniflora, of the north-eastern portion of theUnited States, presents this peculiarity, and an effort was madet o discover whether or not this plant could be used ils a source oftyrosinase.A chloroform-water extract of the crushed plants was pre-cipitated with five volumes of 95 per cent.alcohol. The violet-coloured precipitate so obtained was collected after twenty-fourhours, and redissolved in 0.05 per cent. sodium carbonate solution.The solution was found to have some oxidising power, slowlyoxidising tyrosine to a blue-black insoluble substance. The actionon quinol was very rapid, however, the solution becoming deepred. Boiling destroyed tihe power of oxidising tyrosine, but notIndian Pipe "(Monotrope uniflora)120 A CONTRIBUTION TO THE STUDY OF THE OXYDASES.the power of oxidising quinol, showing that the major portion ofthe oxydase is evidently laccase.Tincture of gum guaiacum israpidly turned blue, whilst guaiacol is turned pink by the actionof the solution,I f the fresh plants are crushed with three parts by weight ofglycerol, and the mixture kept for eighteen hours and then filteredby the aid of the pump, a clear, greenish-blue solution is obtained,which, after a long time, becomes an intense bluish-black.Two drops of this filtrate almost instantly turns tincture of gumguaiacum blue, a.nd oxidises tyrosine, which changes colour throughpink to rose, violet, and finally blue-black. Quinol exhibits, underits influence, colour changes from orange to brown, deep reddish-brown, and finally intense red. When the solution of the oxydasehas previously been warmed to 80°, it is without effect on tyrosinesolution. From these data, it is evident that both tyrosinase andlaccase are present in the plant, but the quantity of tyrosinase issmall compared with that found in certain of the Russula.Summary.1. A new variety of tyrosinase has been discovered and investi-gated.2. This variety is distinguished from the known tgrosinases byits insolubility in water, its loss of vitality in glycerol solutionsand on drying, and by its inability t o oxidise resorcinol, orcinol,etc.3. A chromogen has been found in the larva of Tenebro molitor,giving with tyrosinase colour reactions identical with those givenby tyrosine.4. Tyrosinase has been found in the myriopods Scalopocryptopssexphosa and Julius canadensis, Newp., and also in t.he larva ofCzcczcjus clavipes.5. It has been observed that extracts of almost all animal tissuespossess the power of oxidising solutions of quinol, and that thispower is considerably diminished by prolonged boiling.6. Tyrosinase has been found t o exist together with laccase inthe Monotropa z~niflora.THE CARNEGIE INSTITUTION OF WASHINGTON,COLD SPRING HARBOUI:,LONG ISLAND, NEW YORR, U.S.A