HENRYE. EVERT
478
(q10,q20)in the critical surface in Fig. 1 suggests a modification which will be found equivalent to the introduction of a transmission coefficient. Consider a dissociating molecule represented by a phase-space point crossing the boundary pl = qlo, with 4 1 positive and q 2 - 4 2 0 positive but fairly small. If the point has 4 2 negative, it is likely soon to cross the boundary q 2 = 420 downward (as shown by the broken arrow on the figure), corresponding 1o a recombination of the molecule. As much of the traffic toward dissociation will, from energy considerations, be in the neighborhood of the critical corner, this suggests that we should modify the integrals on the right of equation 1 by excluding negative values of p 2 from Jq, and of pl from J p 2 . This is equivalent to replacing l / Z u , in ( 3 ) by 1/2u,’ where (compare (16)) l/jzi,‘
=
JOm
~,P’(@I, q2)d@ldd2
(30)
P’(Q1,Q2)being the joint distribution of the two
T‘ol. 64
velocities. This could he writttii donn a- L: bivariate distribution as in the lnr solution a t two initial ionic strengths of phosphate buffer (i.e., a t 0.032 and a t 0.162) produced a change iii the ioiiir iitniosphere surrounding the central ion which resulted in an apparent “salting-in” and “salting-out,” effect, attribute(: to c h n g o s in the dipole moment of the molecule. Ultraviolet absorption spectra data indicated that thyroxine esistetl in the form of an intact molecule in the soluble phase and t h a t an increased dissociation of the phenolic hydroxyl group n-ith iricreased pH may be correlated with the increased solubility of thyroxine a t a pH near 7.4.
Introduction The extremely low solubility of thyroxine and related compounds in water has limited the st’udy of their properties by convent’ionalmethods. The early work of Kendall and Osterberg2 and the later work of Winnek and Schmidt3 demonstrated the relative insolubility of thyroxine and of thyroxine salt’sin water. It has become common laboratory practice to use the salt form or to solubilize thyroxine by the addition of alkali. The present study4 with L-thyroxine (Na)5 was prompted by the scarcity of quantitative data, particularly in the pH range 7.2 to 7.8 obtained a t defined ionic strengths and a t controlled temperature. A sensitive method was used t’o determine the soluble thyroxine which combines reduced pressure concentration with polarographic analysis. (1) C . L. Geminill, A r c h . Biochem. B i o p h y s . , 64, 3,59 (19.55). (2) E. C . Iiendall and A. E. Osterberg, J . B i d . Chem.. 40, 205 (1919). ( 3 ) P. S. Winnek and C. L. A. Schmidt, J . Gen. P h y s i o l . , 19, 773 ( 1931i). (4) h preliniinary report was presented before the Ainerican Association of Biological Cheniists a t Atlantic City, N. J., in April, 1959. See Federation Proc., 18, 224 ( 1 9 3 ) . ( 5 ) L-Thyroxine (Na) was obtained froni t h e Nutritional Biocheiiiicals Corporation, Cleveland. Ohio.
Methods Stock solutions of phosphat,e biificr, 0.01:’, :iiirl 0.03; .l/, respect’ively, were prepared a t definitc p H ~ - A I ! I O ~ to the method of Hastings and Sendroy.fi Tiit. plI ot’ tlic solutions xyas checked Tvith a Beckman Ilodcl C; pFI l I v t t , i . I n order to separate t,he thyrosine siispc~nsioninto :L so!iilil(i and an insoluble phase, 19.9 nig. of L-tli:~rosiiic~ (Sa) n,!.
ml. with water. Aliquot portions ( 4 nil. I of the, miswi suspension were placcd in it 10-m1. voiiiiiic-trir fiwk : i i i t l were analyzed pol:rrogr:tljhic,tlly. T l i o ,li1iuriiiig i ~ l i ~i ~~ . i t Iyte’ consisted of 1 mi. of Jf nmnioiiirini r-hlori~lo.! x i . ~ i ’ -V ammonium hydroxide, 0.25 nil. of 0 . 2 ‘ , w i : : t i : i , 2 . 0 ml. of rL-prop?-l slcohol dilutcd to 10 nil. ivit11 \ ~ : ~ , i i >‘1‘11~ r. re dweloprd in thr 5 p : i . I a i i p t~i i i :! I ( Y V I - :iiill trorhcniograph, t!‘pcl 1.:. Tlic, i l r r ~ l t~i n i t , ary in equiniolur 0.1 .\- : i i i i i i i o i i i i i i i i 11) 11
SOLCBILITP OF L-THYROXISE
April, 1960
479
: r i d in 0.1 .lI minionium chloride was 4.6 mass of t,hc IIg drops delivered per second The calibrstion data presented in Table I t)y dissolving 19.9 mg. of L-thyosine ( N a ) in 25 ml. of 0.013 J J phosphate buffer :tt pH 10.6. Aliquot portions (0-5 nil.) TWIYJ introduced into a 10-nil. volumetric flask :\lorig with t,Iie support,ing electrolyt,e. .L\fter dilution to 10 ml. with distillrti wtter, the polsrograms were obtained I)>. the proc.cdiir~described :tl)ovi~.
TABLE I ~ l ' I ' l ( ' . U ~ . ~ L I B R A ' I I O IIATA S USED FOR T H E POLAROGRAPHlC
R U I K A T I O N OF
L-THYROXINE (Sa) Conen. of L-thyroxine (Xa) i n ~JolaIographeds o h . (concn. X lo-. -11)
1 2 3 4
('ontrol espc~rimc~nt!:rrrealed that, the filtration aiid pre*siirc ronwntration procedures produced no :ippreri:il)le t ffwt on the c~:tlil)r:ttion. For esample, when 19.9 mg. of I.-tli:L.rosine ( S a ) was dissolved in 125 ml. iii' h i i f f ~ r:it pH 10.6 and after adjusting the pH to 7.4, folloi\-(d by filtntion iind concentration, results identical ivith the procedure described above were obt:tined xvith t,he :iiiqriot snmylt~s. Thv sum of the v-ave heights in mm. obt;;inrd in the P.V. mnge -0.9 to -2.0 bore a linear relation ti) rlic, L-th,vmxint, ( S a ) present in the solution. An in( ~ i ~ i iii ~ s pho~phute c ~ buffer strength from 0.013 to 0.066 JI tliil iiat :rItm cdihmt~iond:rta. rtdiic.cd
th(8
Results Evidence for the presence of thyroxine in the soluble phstse was oht.ained from ultraviolet absorption spectra of the concentrated filtrate (Fig. 1). The spectra were studied in the pH range 7.16 t o 13.0 at a constant ionic strength of 0.284. A characteristic maximum was observed The variation in the for thyroxine at 325 height of the absorbance curve at) 325 nip wave length with \-aried pH indicated the dissociation of the phenolic hydroxyl group to yield a phenoxide type ion. Rased 011 this evidence for dissocia,tion, the phenoxide ion is believed t'o be ma,inly responsiblc for the solubility of thyroxine in aqueous medium at a pI3 near i . 4 . Having egtablislied the presence of thyroxine in the concentrated filtrate, the results for the cpmtit:iti\ve so1ul)ility of L-thyroxine (Sa') are indicated 2 :ind ;;. In Fig. 2 the solubility of Li t (S:ii i i i moles per liter x is plotted a. a. iuiiction of the ionic strength a t pH 7.4. The initial ionic strength due to the phosphate buffer, 0.01XU was 0.032, aiid further increase in the ionic streiigth w a s ploduccd h y the addition of S n C l :it p1-I i . 4 1 a i i t l at 38' temperature. Curve 1, l i g . 2 , reprw'iits thc effect of iiicrcnsed ionic strength brought ;il)out hy increasing the phozjphatc hiiffor coni:eiit r;itioii. The maximuni soliihility cic.c.urred :i! O.Oci2 ioiiir strength. Starting with p1ii)sphiite !Juf'f('l' hai-iiig :11i ioiiic strength O.o:il! :it pI~I7.4. ;III iiirrcarc in ioiiic strmgth proclucccl tiy the, :idclitioii of S:tc'I prodnceil a 1 1 iiiciv: 9. It is 1s) -1.? ) .
11:
itit(>i*eitthat :ipproxini:ttely thc snnie
11: t e r i z i
:ii1
