1821
Sept., 1903
SIR4PLE AND COMPOUND &IETACHROMASIA1 Ikpartinent of Mrdicinc, the Stud?/Group on A g i i i g , and the Study Group jor Rheumatic Diseases, h e w York Uniwrsity School A'ew YorL, S . Y .
OJ
Jledicine,
A new phenornenon is described characterized by a sharply marked diflerence spectrum in solutions that contain cwtain pairs of metachromatic dyes in the presence of a polyanion. Some syst.ems, with difference spectra almost identically zero a t all wave lengths, behave m mixtures of simple nietachromat.ic compounds. Other systems, with difference spectra having high positive and deep negative peaks, are examples of what it is proposed to call wrnpound metachromctsia. Five examples of compound metachromasia showed regularities in the positions of the two wavc lengths at which the deviations from additivity were greatest; observed absorbance ma8 sharply iiicreitved near the p-bttnd of the dye whose p-band wm a t the shorter wave length, and sharply decrewed near thc p-band of the other dye in the system. I n the mixture, the stability of binding of the weaker dye to the polyanion W R Y raised. Dye pairs that in dilute solution with polymion showed wnipoulid metachromasia, when in concentrtited solution without polyanion showed a wide deviation from additivity of their spectra. These dcviations expressed as difference spectra are similar t o the difference spectra the same dye pairs show in compound riic?t:Lchrorri:isia. It is suggested that just as simp!e mctachronisirt is due t,o formation of liomopolyniers, cornpound metachromctsia is due t o formation of heteropolymen, both stabilized along polyanionic chains.
RIctachrornatic compounds arc forrncd of certain catioiiic dycs arid polyanions. 'lliey exist in solution i i i equilibrium with their coniporiciit cations and polyanions. Thcy havc absorption spectra in solution in the visible rangc which differ from those of dilute solutions of the dyes alone in a markcd and characteristic way. Thc metachrornatic compouiid has ail absorption peak (p-band) a t a xave lrngth usually 50 to 100 nip less than the wavc length of the peak of the dye aloiie in dilute aqueous solution (a-band). These charactcristic shifts in ahsorption from the a-bauds of the dyes alonc to thc p-bands of the mctachromatic compounds, as ]vel1 as ot1ic.r peculiar properties of thesc cornpounds, have had appliratioli in many fields: tissuc staining, tlic quantitative assay for ccrtaiii types of natural and synthetic polyanioiiic compounds (hcparin, polyglucosc sulfates), thc qualitativc distiiictioii of some groups of naturally o c c u ~ ~ i npolyaiiioriic g substances (nuclcic acids, aiiioiiic polysaccharides, phospholipids), arid thc dctcrrniriation of tlic critical conccntration of micclle format ion of anionic dctrrgeiits. With the growing interest ill thc physicochcmical propertics of' natural and syiitlictic polyaiiioris arid of anionic detrrgciits, the proprrtiw of mctachromatic compounds could havc nidrr application if tlic conditions of thcir formatioil and dcstructioii wcrc inorc exactly understood. l ' h ~prcscnt study coiiccrns a 1 1 1 ' ~and uiicxp finding that may I)c of importanc~~ iii tlic further devclopment of the theory of mrtachrornatic compounds. The fimdamcntal observation was madc that the ahsorption spectrum of a solution containing the t n o cationic metachromatic dycs, acridine orange and mrthylcne blur, togrthcr with the polyanion A-carragecnan, is strikingly differcrit from the sum of thc metachromatic spectra of separatc solutions of acridine orange arid mrthylcnc blur, cach in tlie pi'esciicc of Acarragrcnan. This effcct is S ~ O T I I Iin Fig. 1. Several othcr pairs of mctachromatic dyes have been found which when prrscnt in the sarnc solution with polyanion produce absorption spectra widely different from the sum of the mctachroniatic spectra of each dye alone 1 1 ) TIii? a o r k was 8 u r ) i ) o i t P d h y ttir Unitcd Statcs Puhllc IIralth Srrvicr Grants 111171(('1) foi ttir ~Iultlriisr~pllnary Study of Agmg. and A28(C10) troni tlir National Institutr of Arthrrtls s n d SIetaholic Diqoascs. and by a Public IIealth Srrvlce rc.sr~rrrh cwrer proeranl award. 5-KO-A-18,431-02. (2) I'elloa of the Study Qrouu on Aping.
uith thc samc polyanion. Yet not all pairs of nictachromatic dyrs in solution with polyaiiioii show this cft'ect. Cases also uill br cited in which solutions coiitaining two nirtachrornatic dycs and a polyanion have spectra almost identical with the sum of the spectra of solutions of the srparatt: dycs cach in the presence of polyanion. Cascs in u hich such additivity of spectra occurs arc regardcd as mixtures of simple nietachromatic compounds. Cascs such as that of Fig. 1, in which deviations from additivity arc largt', will be callcd examples of compouiid mctachrornasia. Experimental and Results II13 c3ac:Ii a t 1.25 x 1 0 - 6 31; 0 , siini of tho s(!pwatr s1)ccti-n of solutions o f A 0 and ,1113 c:aeh :it 1.25 X 10-6 ,If. Upper Iialf mitli ort1in:ttc~sat t lic: right: 1,sp(:ctruni of :L solution containing bot,li A 0 :ind RIIS o:ic:li :it 5.0 X 1 0 - 3 .\.I; A, siiiri of tiit: sopar:ttr: spect,rit of solutions of A!) anti 1113 c w l i a t 5.0 x 10 - 3 -11. ~rnpounds. ,1Ictliylcne b l i i c : t i i d c violet i ~ n dacridine orange. sliow c( ctr:t (JII niisirig, i n tlie a1)senc:c :)f X-c:trr:tgccn:tii, iiot only in dilute hut : t l m in concwitr:ited soliiti:)n. .inotlier nay to study t,hc interitction oi two tlyr.; w i t h it single i)olyunion wiis esplorctl, depending on a met liod3 to iric:tsure tlic: st:tt)ilitics of rnet:iclir~,iii:~ticeorripoiinds. This method is :i ronst:iiit voliinie t>itrrit,ionwitli :ilcohol o f :in :tqueous solution oi iriet:~rJiro~ii:~ti~ conipourid, :tiid z t grapliird d(~terniinatioriof t t i e inolwity of tlit: :tlcohol :it which tlie ttt)sort):tnre :it tlie ol-\):tnd of the dye h i s risw 1i:tlf w:y. (mid-pl:ttcari v:tluc, iii.p.v.) t.o its value for the dyc :done i n iilcoliol. If o:tcli dye, i n :t niisturc were t o re:ict, independently with the poly:inion, then c:icli irict:icliroinatic cornpound would \)e expected to bre:tk down indcpendent~ly :ind give i i 1n.p.v. tlic swne :ts found For it :is it Pimple nict;tclironuitic rompourid. .*ipplicatiori of this mcthod to dyo p:iirs was neccss:irily lirnite(1 to c:ises in which tlic n-l):~ndaof the dyes are far i l p r t . A series of solutions WJ,S ni:tde up, ewli cwit,:tinirig one dye (I.2,i X 1 0 - 4 .\I) or t,wo dyes (wch :Lt 1.25 X 10 - 4 A l l ) , polyariion (hy:iluronatc or chondroitin sulfate) in :I 'LO(,:, esc(?ss (cqiiiv.,~l.)of the tot:tl dye, r~ndrilcotiol (nietliiinol o r c:th:inol) at, :t series of conrelitriitions (0to 15 -11). T h e ithsorb:tiircs of tliew solutions :it tlie a - h n d of each dye i n t,lic solution wts nie:isured ethylene blue, 4!E rnp for :tcridine orange, und 590 LI violet) . From t,tie plot of absorharice :tgainst :ilrohol concciritr;ition rri.p.v. for e:ich dye wts cstiniated. I n T:tl)le I1 are collorted POIIIC results. The first, dye pair, tirridiiif: orange :tnd nicttiylcne blue, es1iibit.s cornpound mctac.hroni:tsiit and ni.p.v. h v c Ilcen determined under :t few v:trid ronditioiip. I'rider each sct of coiiditions t,he n1.p.v. for e:wh dye was determined alone :is wcll ttr: in the mixture. Under :dl conditions the 1n.p.v. for incthylenr blne tilone w:is much lower than for acridine orange alone. Lnder :dl ronditioris when niised tlict 1n.p.v. for inethylene blue nnd for :midine oraiige w t w found to I)(: tlie P:LIIIC. Tri thc niistiirrs containing hyalrironntc: tlie c o n i ~ ~ i oi nn .p.v. w t s nearly t h t , for the niorc stdilc roniponent (acridine orange) alone, b u t in the case of churidroit in
1825 sulfate the coinmon n1.p.v. was Iiigher than that for either dye done. The second dye pair of Table 11, wridirie orange aiitl crystal violet, does riot exhibit conipound rnetacIirom:i~ia (Table I ) itrid rn.p.v. for each of these dyes when mixed do not, t)econie equal, nor is either v:tlue in the mixture miseci sigriificantly above its value for the dye alonc. TABLE: (:OMPARISON
ti0
1
11
O B TlIE STABILITIICS O F COhfBIiXATION
1 ) Y E W I T H ~ ' O I . Y A N I O N A X U TIilC $rABII,lTIICS O F E.4CIi 1)YE MITI1 I-'OI.YASION I N
01.' A
sILYGl.l*i
IO
01" CO.MHISATI0.U
&fIXTL'l?l.~a COSTAINISG
T W O 1)YES
The niid-plateau values (1c.p.v.) are the concentrations (111) of alcohol a t wliicli the a-band of each dye is half way back t o ita v:tlue in alcohol 1)ye 1 Dye 2
110
...
A0 A0 , ,
.
A0 A0
...
A0
.40
... A0 A0
.., A0
...
I'olysnion
NaCl concn., A!!
Hyaluronate 0,000 M I 3 Hyaluronate ,000 1113 Hyaluronate ,000 . . . 11yaluronate .OOX5 311% Hyaluronate ,0025 MI3 Hyaluronate .00% . . . 1iy:duronat.e ,0025 MI3 Hynluronatc .OWL5 MI3 Hyaluronate ,0025 . . . Ctiondroitin ,004 sulfate n m Cliondroitin ,004 sulfate 1113 Chondroitin ,004 sulfstct Chondroitin .(NO ... sulf1ttc Chondroit,in CY sr1lfatc ,000 Chondroitin CV .000 sulfate
.4lcohol
h1.p.v. >I.{,.\..
