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js APPLIED IN/MGE Inc

ON THE GUANYLIC ACID OF THE SPEEEN

47996

W M.ll.K J0M;S as;, r., G. KOWNIKI !,

FR'IM 1H» I.\EOI!\r"RV -1 PllV^lnioolCM ClUMlvTBV INTHF [•HIS' H..PKI\« V'

!»..■

I HI

\r.l. IV. Nc «

H'^ 'LOGIC \I. CIIKMISIRV

•

ON THE GUANYLIC ACID OF THE SPLEEN.

Bv WALTER JOXES and L. G. ROWXTKKE.

(From the Laboratory oj Phviioloi^ical Chemistry in the Johns Hopkins

V ni: :rsity.)

(Receivcil for publication, February ni, n)o>.l

Ten years ago, Ivar Ban,<j'- siu^'ccdeil in isolatinL; frnin nx pan-
creas a nuelcic acid uiiich diflcrcil in a remarkable wax frmn all
kn'iwn substances nf this idass. According to Bang the coin-
pound is characterized by the following pro])ertics.

(i) It contains a pentose group.

(2) It coniaiiis a glycerine grnuj) and yields gh'cerine phos-
phoric acid. The nucleic acid thus stamling as a connecting link
between the pentosans and the lecithins estrdjlishcs a scries of
jihysiul'igical I'heniieal rclatiuns wlii*.!) can scarceh' \)v found
elsewhere in the science.

(3) It contains an aniount of nitrogen and iihosphorus rela-
ti\-ely gri'ater ihan is the ease with other nutleic acids.

(4) It _\iclds (111 hydrolysis an excessiveaniount of guanin but
no trace of cither adenin orthyiiiin. On account of this striking
difference from other nucleic acids, the substance has rieei'.'ed the
name lif "guanylic J' id."

This woj-k which if true \\'ould constitute an unmistakable
advance in the scieiice has unfortunately been the subject of
ad\'ersc critiiisin of a kind from which one would scarcelv e.\pcct
a rci o\-(.r_\'. Thus \\ Furih,^ who has gi\'cu the subject most
careful attention, ^\as able to find among the split products
neither glycerine iior a rediuing earboliyilrate; he claims that the
nucleic acid }'iclds adenin as well as guanin ami that there is no
reason for making any dist inci ion lietween guanvlic acid and
thymonucleic acid. In consideration of a number of objections
ail leading to the same coiulusion one might reasonabh- supjiose

' liang: Zcitschr. /. physiol. Client., .\xvi, p. i^^; xx\i, p. jii.
' V, Ftirtl; and Jerusalem; Hofmeister's Heitrui^e, \, ji. 174.

2.S()

290

Guanvlic Acid of the Spleen

that V. Furth's w, .rk wt niM finally dispose of the matter of Ruany-
lic aci'l. Rut SteiKlcl' in a very recent communication, takes an
entirelv«litt'ercni view. He notes that themeihod .,f y>re]'arati"n
emplovc.1 1)V V. 1-urih (propose.l by Bang and Raaschou-J leads
to a miclcic'acid cf the ordinary type, but that by followmg the
original method n{ Bang it is ].MSsible fi <.l.'ain fnan thox pan-
creas a true guanylic aci.l, i. e., an organic ranjiound containing
phosphorus which yields guanin but not adenin. Unfortunately
Steudel agrees with v. Furth that guanylic acid contains no
glvceritic group.

' It is the purpose of this eimimunication to show that not only
is a true guanylic acid obtainable from <.x pancreas but iliat sub-
stances (.f this class are conlined ncitherto a single animal species
nor t'.> a single gland.

Tin- prkp.'lR.xtion- of the xt'ci.HOPROTHiN- OF Pifi's splei;n-.

Fourteen kilos of carefully trimmed and hnely grcnind fresh
tissue were thoroughly mixed in portions with 21 liters of cdd
distilled water, heated quickly to boiling and the solution liltereil
off. It is immaterial whether the lluid be filtered hot or after
cooling; in either case a fairly clear filtrate is obtained which by
repeated filtration through the same hlter becomes almost as
clear as water but ])ossesscs a very pale reddish tinge. If the
residue be jiresscd through linen a milky fluid results which we
have not been able to render clearer, either by repeated tiltration
or bv long continued centrifugation; it is therefore advisable to
avoid disturbing the residue in any way and to use n(i greater
amount of water for the extraction than that stated. This part
of the process was exceedingly more satisfactory than was the
case with beef pancreas (see below), where we were never able
to obtain anything better than a highly opalescent filtrate,
although the solution obtained from this gland is described by
various writers as perfectly v lear.

The neutral fluid when cool was trcateil with acetic acid up
to 5 to 10 per mille and the prcciT.itated gelatinous nucleojircleid
was allowed to subside over niglit. Tins nu(le< .profcid is so

'StciK-k-l: /ritschr. j. physi,'t. Ch, it. liii. ]'■ 53'l.

: Han^' and Raastliou: llofiiinstrr's !<< ilr.n'r. iv. y. 17;.

Walter Jones and L. G. Rowntree

291

sirikm-lv dilTcrcnt in ,)hysira! vrnpcrtics fn.m the M,rrfsi,. ^ivlm-
liravv'tliieeulcnt sul>^;anfe i>i <>x pannvas that wc can scarcely
belicvc the tv/o substaiue^ i.lenii.Ml. The ch.u.ly lluid \vrs partly
(lecantc.l ami the remainder shari.ly reiuovecl after (e:itnfut;a-
tu.n. The micle.ipn-tei.l wliich in tliis c.mi.a. i form resetiibles a
preparation of staivli paste, was freed fmrn soluble impurities by
alternate solution in a minimal amount of caustic so.la and pre-
cipitation with the requisite aiiKiUnt of acetic aci^l. Suspended
matter was removed as far as possible from each alkaline solution
by Ions continued cent rifugat ion, and in the same manner the
precipi'tate.l nucleopr<,teid v/as sharply separated fr^-m the super-
natant lluid. After this operation had been repeate<l several
thnes a pro.luct was obtained which dissolved in a trace of alkali
and on precipitati(.n from the alkaline solution by acetic acid
left a perfectly clear fluid. The exceedingly gelatinous nucleo-
protei<l thus puritic.l was d.ehydratcd with alcohol and ether, but
unless this is done with the greatest care one will tmally obtain
a bnAvn sticky mass which is unsuitable for the work that t.jUows.
It IS necessary to begin with dilute alcohol (50 per cent) and to
replace this gradually until absolute alcohol is linally readied.
Th.' latter slvuld be repeatedly used and the material allowed to
stan.l for several <lays in well cooked flasks with frequent and
violent agitation. We mention the difficulty of dehydrating
this nucleoproteid because we encountered no such ditficuliy m
dealing with the nucleoproteid of ox pancreas and we regard this
very sinking diflerence as sufficient ground for assuming that the
tw.; nucleoproteids r.re not identical. This is however entirely
aside from the question of the identity of the two nucleic acids.
From 14 kilos of moist tissue after the sacrifice of relatively
large quantities of material in the interest of a pure product,
we finally obtained 64 grams of a perfectly dry pale yellow
powder.

THE l'REPAR.\TION OF Gf-^NVLIC ACID FROM THE NUCLEO-
PROTEID Ol" PIO'S SPLEEN.

The nucleoproteid was treated in portions of 12 grams each
with 150 cc. of 2 per cent caustic potash and heated for half an
hour in a vessel submerged in boiling water. The red fluid was
neutralized with acetic acid end whale hot filtered from a small

2q:

Guanvlir Acid ot tlit' Spleen

(luantitv -f i-erkTilv lihuk inatuKil; l.ni wen after stan.liriK' 12
lin.irs iheiv uas nn ,W\n,^hinu -f •^uin\yVu- acid alth<.u-b the
snlutinii was iiiurh in., re o .nrciitralcd tlian that wliidi Han;,'
pivparc.l fnim y.imvcufi luulc .vr-uM. i Wc used nnly i5o_y-
„f J iKT .cut caustic ]H,!ash v.-i-on- Han- used 400 ce.i The
perte^lh- dear vell.Av lluid was then made faintly aeid with
aectie aeid and allowed Im stand <.ver ni-ht '."it there uas still nn
deposition of Kuanylic aeid. Several days later when we had
c.mehidedthat thisnuelenpn.teid yiel Is n-. sul.stanee eorn-pc.nd-
ini,' to paiui-eas truanyhe arid and after v. e had praetieally alian-
d/med the suhjeet, a vt^ry small deposit was noticed in ilie thud
and on tlie ad'dution of a few drops of aeetie aeul there was an
iinmed.iate and eojnous preeipitati. .n of while lloeeiilenl material
wliile i.raetieidlv all the eol,,nn- mattiM' remained in solution.
Tlie preeipilate'was tillered oil, dissohv ' in hot water and tlie
solution til'ered from a small amount of ins.'luMe -ramilar maie-

,.,;,p On lin-. the lole yellow ihiid prompt ly deposited , uuany-

li^. a,i,l l,ut ..nrei.eatm- the process the yield soon became notiec-
al.lv siiudier ;is llie acetic acid was removed with the mother
li,p'i.,i-s. Tite addition of acetic a> id to anv of these tiltrates
causes tm iiiimeditiie precipitation of -uanvlic acid. This dif-
ference m liehavior of spleen ...uaiiylic aci-l ffoiil ptmcreas siKiny-
lie acid, nii-ht he explained by d.ilierences in the solvent J.ower
of the impurities in the two cases hut si-leen -uanylic acid retains
this i.ropertv after purification lo >.ucli an extent that we are
in. line 1 to t'hc opinion that tlio 1 wo nucleic acid^ are not iden-
liiaP The ori-inal neutral solution .u'enenilly tillers Mowly hut
eontmuouslv and as a deposition of -uaiiyhc acid is not likely,
the slov.ncNS of the hltnition is with.out con<e,pience. Hut it
mav hapi^cn th.at the neutral fluid cannot he iiltere.l tit ;dl. In
the one such ease which we met the fluid was markedly aciditied
with .acetic acid, and co,,1ed in ice water when cletir and rapid
nitration could he made leavin- the -uaiiyli.- acid on the filter.
This dtirk l.rown residue was l.oiled with water and a product
obtained whi,'h wtis easilv tiltertible and which deposited K"anv-
lie acid on . oolin-. All specimens of what we considered the
best products were cllected and dissolved in hot water and the
guanvlic acid which was deposited .,n coojin- the fluid was dried
in tlie ordinarv wav, witli alcohol and eth.er. From 5J -rtiiiis of

Waller Jones and L. Ci. Rnwntree

293

mu'lc'.prnteia after -cvere lussrs m the inuthcr l:(iuors f^r the
reason state.] ue imally ..btaiiie^ i.;S 1,'ram of pure jjuanylie
aeid. The suhstan> e e<msists of a perfeetly white dry j>ow.ler
soluble in hot water f..rming a transiKirent liquid whieh has an
arid reaction to litmus. It responds neither to the buiret nfir to
Millon's reaction bu' contains phosphorus and exhibi.s .ueneial
].roperties and reactions '.vlii( h closely accord with those whim
Bang describes for the tjuanylic acid of the i)ancreas.

THE PfKIX liASKS PRODfC K I) HV HYDROLYSIS or Tlli-: GUAN'YLIC
ACIU Ol' I'IC.'s SPLKHN.

Owing to the misf.irtune of not knowin- in the earlier part of
our work that larf,'e (juantities of -uanylic acid may be recovered
from mother liquors by thcadditi^ai of acetic acid, the amount of
material at our disposal was insulficient for an exhaustive exam-
ination such as we ^vould otherwise have made and as we intend
to make in the immc^iiate future; so that we decided to devoie
all of our material to a tinal decision of the very important (|ues-
tion, whether or nc^t we are here dealing with a true guanylic
aciil.

A uram and a half ..f the material was heated for three hours
^vitlil^s cc. of 5 per ( ent sulphuri..' acid in a vessel submerged in
boiling water. (Jn stan.ling over niglit the lluid deposited in
])rofusion macroscopic needles of guanin sulphate. These were
dissolved by warming and the S(rlution was first neutralized and
then treated with such an excess of ammonia that the fluid con-
tained 2 per cent of the reagent. The jiroducl after digestion
in the warm for an hour was allowed to cool and the p..-ecipitated
guanin liltered off. After thoroughly washing in turn with i
I)er cent ammonia and water the base was dissolved in i per cent
caustic s(jda and again prcciidtated by the addition of acetic
acid. The precipitate was filtered off, washed, dried ami weighed.
For tlie separation (if guanin from small quantities of adenin
there are two properties of the liases whiJi can be used ami can
be thoroughly depended ujion. First, gu.anin is almost insoluble
in 2 per cent ammonia while adenin diss.ilves in this reagent
with comparative case. Second, both bases dissolve easily m
dilute caustic soda but while guanin is quantitatively precipitated

Guanylir Acid of the Spleen

fn,m '^uJ^ a s,.lut,nn bv ac-cii-- a.ul, adcnin remain, mvhr th.^se

f these methois were ar:«Hcd u, n.rn to tl,e case wh.ch w.. a
descnbin,. The original amnv.n.aal f.Urate from .uan.n an.
the acetic aci.l tlui-l ubtame-l in its puriiicaii..n were urn: e.l an,l
treate<l with silver nitrate and ammcmia. The small silver pie-
cipitate was th..rou,l-.lv washed, suspended m boding watc^ and
Xosed with hvdrochlonc acid. Th.e a. id lUiid was .dtere,
from silver chloride, evaporated carefully just to dryness and
the last traces ..f hydrochloric acid driven ot. by moistenmg ^Mtl
■ater and n,ain caa-fully evaporating. The insignihcant amount
of esidue w;s dissolved in water at 40° and treated withammon.a.
A v" stnall precipitate of guanin was forntcd which showed no
inclination to dissolve in ammonia even afuT the ,M...... • -

,.reat excess of the reagent. The lluid was nltered off an. l-dcd
tint pcrtectlv neutral to litmus. I. will be observe.l that any
ad nirori.inallv present must now be foutid in this .lu,d ts
volume w;; <.nlv .0 cc. yet a T>ortion failed to give a distinct pie-
ciphate wi,h sih-er nitrate and ammonia whil.. in an^ 'her portion
pi,ric acid did not even produce even an opalescence. I lu
pu,v vUc nad of the sf^lccu gives no trace of ador.n.

The main vield <,f guanin together with the small amou,
obtained from the mother liqu-.rs weighed ;,„o ,m hgrams. 1
Zl issolved in hot 5 per cent hydrochloru- acid and decolorized
with a small an.ount of animal charcoal. The solution on cool ng
deposited the characteristic centimeter-long feathery nee. e
guanin hvdrochloratc. Thesal, w,.s all-.wcd .-, drv ,n the an and
analyzed' with the f..llowing results.

, o ■ - w Rrani lost ocSo gram at , 00^ and rcuirc.l 706 cc. of stand-
ard sulphuric acid >> cc. « 0.0077 gram ol ">\^"«^";. , ^^ ^,,^

,. o.'u.M pram lost o. 0,^0.. gram at loc and rciu.red ,.H,, cc. ot
same sulphuric acid.

,■ II s ., ,. IICI. .'11.. o: I.

!, ir p; 11-- tiiT cent.

\'.. .:a.:n " • ' -' '

It mav appear that our eonclusum. viz: that we are here deal-
ing uith a guanvlic acid, is base.l ..n an experiment with rath r
a ntall amount 'of material. In atiswer to such an objection .e

Walter jcno and L. C Rcwntree 295

w,mM state that ^,c sh.,uM uso rm m,,re if wo wvr. tn n^eat the
,vnrk wuh an unUnnted stn-ply "( '"^"^"='1 ^'1 V^"" '■.'^l'"';'; , ''^
arc n,.v cn.auc.' in an inve^n.ati.n of nude.c acMs M .n-
volves a lar,e't.un.bcr of ju.t such an-Oyses as tha, ,losonbcd uvl
where the amount of material is of no consi.lcrai. on to us. Kx,;t-
ri en e in these cases has tau.ht us tha, the best quantuat.v
results can bo obtah^ed by us,n, no n.oro nuokno ao.l than w,ll
pnuluoe 300 to 400 milligrams of the base sou-ht.

ON THE msTRiiu-nox or (U-anvlic Ann in i.n: okoan.sm.
We arc now ocoupicl with the oxanunatiou of . nundK," ot
.lands for substances of this typo and hav. "";'"-;>,";;';;
Substances whose ,hys.cal propert,cs curres,K:.nd ^'l'- -;
those of KuanyUc acid. Thepig's pancreas yields ^ I-^'-"> ;^;
aqueous extract from which acetic acid prccpuates a tloccu ont
,^ ,,U„,,,.,.,cid wlhch closely resembles the nucleoprotcd o ox
ancrcls and from which a ,uanvlic a, id can be prepared .huh
!,.„„„ be distinguished by anv apparent dhference from ox
,,,„,,vas ^uanvhc a.id. Wuh ox pancreas we exponencod oi-
lid:rable dhhculty. The .lands used wore perfoctlv trosh and to
„,ethod ,nvcn bv Ban. cdosely foU-.-od bu, m spue o, ex c
eilort w;were unable to prepare any Mnn, approach.n, a d. u
aqueous extra,,. However, the cloudv lUud ,u>ves a nucleop,o-
tcid which in turn yu'lds a true Kuanvhc acid.

Our results show conclusively , ha, ,uanv.. ac,d f. ,r t ho ,>,.>-
H, ands') are considc,-ablv ,n.rc w.lolv d,s,nbu,cd , ,n a

f,.rmerlv supposed and lend m -Mva, n,oasu,-c ,0 'l:o bohot h.a
,lu.se substances are conmu.n nuclear cons,„uen,s. As a . - -
,,,,n< >,f orduKU-v nu, Ice aod ln,herto prepa,-cd have boon lound
,„ ,,,,,1 „,,n,n as .oil as ,nan,n „ seems cor,a,n ,hat ,he .lands
winch veld ,uanyhca.>d nu>s, also con,a,n e.thcr adcn,hc
arid" ,',rnaclc.oa,,ds whuh pn.iuccbo,h bases.

S,ncewri,m. the above ar„.]o our a„cnt,.m has been called
t,, ,he work of Odcn.us^ who prepared .uanyhc a. .d Iron. ,he
,ni> loopnitiid .if ,he mammary .land.

difrlnccs amen, the nucleic acids of this class, thus cstahhshu., a s.rus

of jjuanvlic acids.

'Sec Mulys Jahn-sbrrulU. sss:s. u)oo.

niEK!" or WII.I.IAMK * W1I.«IN» (•.Mr»VT
BAI.TIMORR