THE INTERACTION OF THYROXINEWITH PLASMAPROTEINS:LOCALIZATION OF THYROXINE-BINDING PROTEIN IN

COHNFRACTIONSOF PLASMA1, 2

By NORBERTFREINKEL,3 J. THOMASDOWLING, AND SIDNEY H. INGBAR(From the Thorndike Memorial Laboratory, Second and Fourth Medical Services (Harvard),

Boston City Hospital, and the Department of Medicine, Harvard Medical School,Boston, Mass. and the Howard Hughes Medical Institute)

(Submitted for publication June 17, 1955; accepted July 28, 1955)

Recently zone electrophoretic techniques havebeen employed to characterize the site of thyrox-ine-binding in plasma (1-7). In most reports, anassociation has been described between thyroxineand a protein moiety intermediate in electropho-retic mobility at pH 8.6 between the al and the a,globulins.

A concentrated source of this thyroxine-bindingprotein (TBP) has not been available for furtherpurification or for experimental evaluation of itsphysiological role. To meet these objectives, zoneelectrophoretic techniques have been employed tolocalize TBP in subfractions of plasma preparedby Method 6 of Cohn and his co-workers (8, 9).

MATERIALS

1. Cohn Fractions: Fractions were obtained from 250milliliters of fresh plasma and from 100 liters of plasmaderived from out-dated blood-bank blood. Two sepa-rate fractionations were performed with ethanol-watermixtures of controlled pH, ionic strength, temperature,and protein concentration. Yields from the large-scalefractionation of old plasma were sufficient to permit char-acterization of the individual fractions by moving-bound-ary electrophoresis and by chemical estimation of iodine/nitrogen ratios (Table I).

2. Thyroxine: Chromatographically-pure I1"-labelledthyroxine (10), dissolved in propylene glycol was ob-tained from a commercial source.4 Gravimetrically de-

1 This investigation was supported in part by researchgrant No. A-267 from the National Institute of Arthritisand Metabolic Diseases of the National Institutes ofHealth, Public Health Service and in part by the Medi-cal Research and Development Board, Office of theSurgeon General, Department of the Army, under Con-tract No. DA-49-007-MD-412.

2 Presented in part at the 1955 meeting of the Endo-crine Society, June 2-4, 1955, Atlantic City, New Jersey.

3 Fellow of the American Cancer Society, Inc. recom-mended by the Committee on Growth of the NationalResearch Council.

4 Abbott Laboratories, Oak Ridge, Tenn.

termined amounts of unlabelled L-thyroxine sodium 5were added to vary specific activity.

METHODS

1. Free electrophoresis: 6 Cohn Fractions were dis-solved in barbital buffer, pH 8.6, ionic strength 0.1.Two gram per cent solutions were dialyzed against bar-bital buffer at 50 C. Free moving boundary electro-phoresis was performed in a Klett Electrophoresis Ap-paratus and photographs were obtained with a movingdiaphragm scanning method. Pictures were enlarged andanalyzed by dropping perpendiculars from the lowestpoints and measuring the areas under the peaks in themagnified schlieren diagrams.

2. Chemical analyses: Protein-bound iodine (PBI)was determined by a modification of the alkaline ashmethod of Barker 7 (11). Wet digestion was employed

5 This was 96.2 per cent pure on a dry basis and kindlysupplied by Dr. A. E. Heming of Smith, Kline & FrenchLaboratories, Philadelphia, Pa.

6 Analyses performed in the laboratory of Dr. John M.Newell of the Massachusetts Public Health BiologicLaboratories, Jamaica Plain, Mass.

7 Iodine analyses were performed by Bio-Science Lab-oratories, Inc., 2231 Carmelina Avenue, Los Angeles 64,Calif.

TABLE I

Moving boundary electrophoresis and iodine analysesof Cohn Fractions

Electrophoretic composition (per cent)I127

Fraction Alb. ail a2 ,6% 2 y g.lgI 10* 4* 7* 11* 51* 17* 1.3

II-III 4 - 18 15 21 42 0.9IV-1 6 - 82 8 4 3.3IV-5 8 61 22 9 - 1.3IV-6 2 18 66 14 - 3.7IV-7 5 - 23 66 6 - 1.0IV-8 79 11 3 7 - 5.4IV-9 12 - 31 57 - - 2.6V 84 14 2 - 5.8

Supernate V 47 46 7 - - - 6.1

* Averages for Fractions I prepared during the past fouryears at the Massachusetts Public Health Biologic Labora-tories. All other analyses were performed with the frac-tions derived from old plasma which were employed in thepresent studies.

1698

INTERACTION OF THYROXINE WITH COHNFRACTIONS OF PLASMA

for measurement of total iodine 7 (12). Protein concen-trations were determined by a modified biuret procedure(13); the micro-Kjeldahl technique was used for ni-trogen analyses.

3. Thyroxine-protein mtixtures: One-tenth ml. of salinecontaining the desired amounts of labelled and unlabelledthyroxine were added to 0.9 ml. of the various proteinsolutions. Mixtures of protein and thyroxine were per-mitted to stand for a minimum of thirty minutes at roomtemperature to assure binding equilibrium prior to zoneelectrophoresis. Fresh solutions of stable and radioac-tive thyroxine were prepared for each experiment. Simi-larly, freshly-drawn sera and freshly-dissolved CohnFractions were routinely employed even though thyroxine-binding was apparently not altered by storage in thedeep-freeze. In all experiments, the final absolute con-centration of organic iodine was determined by chemicalanalysis.

4. Paper electrophoresis: An apparatus designed inthis laboratory was used.8 It consisted of rectangularreservoirs of lucite which were enclosed on three sidesby raised walls of lucite. When two reservoirs wereapposed by approximating the slotted margins of theirfree side-walls and bases, a single enclosed rectangularchamber was formed which could be rendered air-tightby the addition of a peaked lucite cover. The "hangingstrip" technique of Durrum (14) could then be employedby the insertion of a lucite rod through the overlappingside-walls above the two reservoirs. On the other hand,when the reservoirs were separated and two 33 by 38 by2.5 cm. glass plates resting horizontally upon a woodensupport were placed between them, paper electrophoresisby the technique of Kunkel and Tiselius (15) could beperformed. Buffer capacity of each reservoir was twoliters and the reservoirs were subdivided into communi-cating compartments in order to separate the carbonelectrodes from the major portion of buffer.

Large sheets of Whatman No. 3 filter paper were em-ployed for zone electrophoresis. The sheets were mois-tened with buffer and blotted prior to introduction intothe electrophoresis apparatus. Two-hundredths milliliteraliquots of protein solutions were applied onto the sheetsin straight 1.6 cm. lines separated from one another bymarked intervals of 1.9 cm.

Since 33 by 50 cm. sheets were used in the Durrum sys-tem, nine specimens could be applied simultaneously.Applications were made directly upon the supporting rodor one inch beyond its anodal side. However, concur-rent electrophoresis of multiple sheets was not feasible.

When the apparatus was adapted for electrophoresisby the method of Kunkel and Tiselius, 30 by 57 cm.sheets of filter paper were employed, thus limiting thecapacity to eight specimens per sheet. Mobilities in alleight specimens were identical (Figure 1). Therefore,the "plate glass" system could be employed for simul-taneous electrophoresis of multiple sheets. This modifi-cation offered certain advantages. For example, quad-

8 Constructed by Transplastics Fabricating Co., 295Huntington Ave., Boston, Mass.

ruplicate analysis could be readily secured by successivelyapplying 0.02 ml. aliquots from eight specimens to foursheets which were directly superimposed upon one an-other. Furthermore, by interposing sheets of "Parafilm" 9between successive sheets of filter paper, four sets ofeight different solutions, or a total of thirty-two sepa-rate specimens could be electrophoresed at one time.The use of plate-glass 2.5 cm. in thickness obviated theneed for equalizing pressure upon the filter paper by theuse of clamps or other devices. Plates were coveredwith a thin coat of silicone 10 and cellophane tape wasused to seal the sides of the plates.

Test solutions were delivered onto the filter paper withcalibrated hemoglobin pipettes. Adsorption to glass wasstandardized by the use of a fresh pipette for each ap-plication. A small amount of powdered bromphenol bluewas added to the site of delivery for visualization of al-bumin during electrophoretic migration.

Barbital buffer at pH 8.6, ionic strength 0.05 or acetatebuffer at pH 4.5, ionic strength 0.03 to 0.10 was em-ployed. To minimize changes in buffer pH, no bufferwas used for more than a single experiment. In moststudies, a potential of 117 volts was placed across theelectrodes for twenty hours at room temperature. AtpH 8.6, migrations averaged 12 to 15 cm. during thisinterval. Occasionally, voltages of 100 to 400 were em-ployed for varying durations and maintained constantwith a power supply and voltage regulator. Followingelectrophoresis, sheets were suspended in a drying rackin an oven at 1300 C. for ten minutes.

5. Protein localization and radioactive assay: Stainingprior to counting resulted in sufficient smearing and elu-tion of counts to diminish the subsequent accuracy ofradioactive assay. On the other hand, satisfactory stain-ing could not be secured by reapposing the segments ofstrip which had been partitioned for counting. Therefore,in every experiment, electrophoresis was performed onduplicate sheets.

One sheet was directly radioautographed in contactwith No-Screen X-ray film and subsequently stained ac-cording to the method of Durrum (14). Where indi-cated, protein distribution was quantitated with a densi-tometer.11

The second sheet was divided into component strips.These strips were further cut into 1 cm. segments begin-ning 1.5 cm. cathodal to the point of application and ex-tending 1.5 cm. beyond the advancing albumin margin.Segments were counted in well-type scintillation counterscontaining crystal detector heads of thallium-activatedsodium iodide.12 A minimum of 3000 counts, with a

9 "Parafilm": Marathon Corporation, Menasha, Wisc.10 "Dri Film": General Electric, Silicone Products

Department, Waterford, N. Y.11 Model 501 A, Photovolt Corporation, 95 Madison

Ave., New York, 16, N. Y.12 Well-counters constructed by Macdonald Instrument

Co., Cambridge, Mass. and N. Wood Counter Laboratory,Chicago, Ill.; strip scanner by Nuclear Instrument andChemical Corp., Chicago, Ill.

1699

NORBERTFREINKEL, J. THOMASDOWLING, AND SIDNEY H. INGBAR

probable error of 2 to 3 per cent, were secured in seg-tnents which approximated background; 10,000 countsabove background were secured with segments containinghigher radioactivity. In later experiments, intact stripswere counted with an automatic strip scanner.12

Radioactivity in each segment (expressed as a per-centage of the total radioactivity present on the strip)was plotted upon arithmetic graph paper. Areas underthe radioactive peaks were delineated by techniquessimilar to those of conventional schlieren analysis. Thepercentile distribution of radioactivity was determined byweight. For final orientation, reference was made to theradioautographic and staining patterns of the specimenson the undivided duplicate sheet.

RESULTS

1. Methods

Potential errors resulting from methodologicalartifacts were evaluated.

a) Adsorption to glassware: In protein-free,saline solutions, there was considerable adsorptionof thyroxine (both labelled and unlabelled) toglassware. Adsorption was maximal with carrier-poor solutions of radiothyroxine and varied in-versely with the surface area of the beakers andpipettes which were employed during preparativedilutions. From saline solutions calculated to con-tain 5 to 200 ,ug. per cent of added thyroxine, 60 to40 per cent of the radiothyroxine was adsorbed toglassware. In the presence of proteins capable ofbinding thyroxine, adsorption was negligible.

b) Recovery of radiothyroxine: Loss of radio-thyroxine during electrophoretic migration was as-sessed by comparing the total counts on electro-phoresed strips with similar aliquots which hadbeen applied onto filter paper but not electro-phoresed. After electrophoresis of protein solu-tions for six to thirty hours at pH 8.6 and 117 to400 volts, the recovery of protein-bound radio-activity averaged 88.1 ± 13.413 per cent in 152observations with the "plate-glass system"; in 13experiments with the Durrum method, recoveryaveraged 91.6 + 10.913 per cent. In 18 studies atpH 4.5, 84.5 + 12.613 per cent of the radioactivitycould be accounted for in the area occupied by theproteins. Similar recovery experiments were per-formed with non-protein mixtures of thyroxine andsaline. At pH 8.6, in 5 studies, 67.4 ± 2.613 percent of the radioactivity remained on the filter

13 Mean ± Standard Deviation.

paper slightly anodal to the point of application;following electrophoresis of unbound thyroxine atpH 4.5, radioactivity remained principally at theorigin and recovery averaged 65.5 per cent.

c) Recovery of inorganic I131: Electrophoresis(pH 8.6, 117 volts, 18 to 20 hours) was performedfollowing the addition of NaI'31 to protein solu-tions. With serum or with saline solutions of al-bumin or Cohn Fraction IV-6, recovery of car-rier-free inorganic 1131 averaged 3.1 per cent; re-coveries averaged 5.2 per cent when the proteinsolutions were supplemented with carrier to a finalconcentration of 200 mgm. per cent of inorganiciodide. In serum, the recovered radioactivity wasprincipally confined to the albumin area. The datasuggest that the presence of inorganic iodide (asa contaminant in the radiothyroxine or as a degra-dation product arising during electrophoresis)would not materially alter the electrophoretic par-tition of radioactivity.

d) Radiothyroxine: The electrophoretic distri-bution of other possible radioactive contaminantswas not evaluated. For the following reasons, itseems unlikely that such impurities were presentto any significant extent in fresh preparations ofcommercial radiothyroxine.

First, fresh radiothyroxine satisfied chromato-graphic criteria of homogeneity. Secondly, anidentical partition of radioactivity between albuminand TBP was obtained when the specific activityof thyroxine-serum mixtures was varied five-foldand the total concentration of added thyroxine(i.e., the sum of both labelled and unlabelled ma-terial) was maintained constant at 25 ug. per cent.

However, during storage in vitro, significantdegradation of radiothyroxine occurs. To mini-mize the effects of ageing, only fresh preparationswere employed. Moreover, potential errors fromdegraded moieties were kept constant by addingequal amounts of radiothyroxine to both the con-trol and experimental specimens in all subsequentstudies.

e) Reproducibility of radioactive assay: Serumwas labelled with radiothyroxine and varyinglysupplemented with stable thyroxine. Multiple ali-quots of each specimen were electrophoresed si-multaneously and analyzed separately for the par-tition of radioactivity. At PBI concentrations of8 to 122 ug. per cent, the variability in the dis-tribution of albumin and TBP-bound radioactivity

1700

INTERACTION OF THYROXINE WITH COHNFRACTIONS OF PLASMA

in quintuplicate analyses was not greater than ± 5per cent of the respective means for each category.

2. Validation of experimental design

Attempts to localize TBP in Cohn Fractionswere based on the characteristics of the interactionbetween thyroxine and plasma proteins. There-fore, preliminary to an examination of Cohn Frac-tions, binding phenomena were studied in mixturesof serum and thyroxine.

a) Distribution of radiothyroxine: Sera fromhyper-, hypo-, and euthyroid subjects were en-riched with radiothyroxine and electrophoresed atpH 8.6. As others have shown (1-7), most of thelabelled thyroxine localized with a moiety inter-mediate in mobility between the a1 and a2 globu-lins (Figure 1). A small but definite percentageof the radioactivity migrated with albumin evenat the lowest concentrations of thyroxine. Eighty

to 95 per cent of the radioactivity could be ac-counted for under the TBP and albumin peaks.Traces of radioactivity usually migrated ahead ofthe albumin area. The remaining radioactivitywas varyingly distributed with the other anodalproteins and was concentrated principally in a nar-row band, immediately ahead of the 18 globulins.Cathodal migration with y globulin was never ob-served. In all sera, albumin-bound thyroxineprogressively increased as the concentration ofadded thyroxine was augmented.

For the present, the significance of the apparent"spill-over" of thyroxine onto anodal componentsother than TBP or albumin cannot be assessed.Both in magnitude and pattern, it varied enor-mously. It could not be consistently augmentedby increasing the concentration of thyroxine, al-though occasionally, it rose in parallel with theprogressive elevation of albumin-bound radioac-

3

S

6

FIG. 1. PROTEIN BOUNDRADIOACTIVITY AT VARIOUS CONCENTRATIONSOF THYROXINE

Serum from a myxedematous subject (PBI 1.4 ug. per cent) was labelled with radiothyroxine,and PBI concentrations of 3.2, 3.6, 6.7, 10.4, 19.3, 62, 112, and 194 yg. per cent were obtained inspecimens 1 to 8, respectively, by the addition of unlabelled L-thyroxine. Following electro-phoresis at pH 8.6, proteins were localized by staining with bromphenol blue. These are de-picted on the left. The distribution of radioactivity is presented in the radioautographs on theright. The photograph does not clearly reproduce the definite amounts of albumin-boundradioactivity which could be discerned in specimens 1, 2, and 3 in the original radioautograph.As the PBI increases, progressive displacement of radioactivity from TBP to albumin occurs.

1701

.:..X,

-- 7-7

'Y.

NORBERTFREINKEL. J. THOMASDOWLING, AND SIDNEY H. INGBAR

Pal aO.IMGM%

12.17% TBP hA3 7.3% _ _ALUI

ADDED

45.0O TBP33&2% mALBUIlN _

ADDED

FIG. 2. EXCHANGEOF PROTEIN-BOUNDTHYROXINE

The upper two illustrations depict the distribution ofradioactivity in serum to which radiothyroxine had beenadded in vitro; the serum shown in the lower illustra-tions was labelled endogenously with radiothyroxinewhich had been synthesized in vivo. In both sera, theconcentrations or protein-bound iodine were adjusted bysupplementation with stable L-thyroxine.

tivity (Figure 1). In some respects the phenome-non simulates the reported "trailing" due to ad-sorption of albumin during paper electrophore-sis (16-18), and hence, it may merely representmethodological artifact. Despite these variations,the data indicate that albumin serves as the prin-cipal secondary carrier during the transport ofthyroxine in serum.

b) Exchange of endogenous and exogenous

thyroxine: Sera wxere obtained immediately beforeand four days after the administration of therapeu-tic doses of inorganic 1131 for intractable anginapectoris. The thyroidal radiation was insufficientto liberate thyroglobulin into the circulation (19).Radiothyroxine was added to the non-radioactivesera obtained prior to therapy; the post-therapysera were adjusted with stable L-thyroxine toidentical concentrations of protein-bound iodine.Higher PBI levels were achieved by adding in-creasing amounts of stable thyroxine to aliquotsof both the exogenously- and the endogenously-labelled sera. A typical experiment can be seen inFigure 2. At a PBI concentration of 4.9 jug. per

cent, an apparent difference may be noted in thepartition of radioactivity following exogenous andendogenous labelling of sera. This difference isprobably not significant. Concentrations of ra-

dioactivity in endogenously labelled sera were

considerably less than in sera labelled exogenously.In the former group, albumin-bound radioactivityNvas not (Ictcctc(1 1)v direct cotinting. b)ut colil(1

be clearly discerned in radioautographs. Thus,the percentage of TBP-bound radioactivity in theendogenously labelled serum at a PBI 4.9 ug. percent is probably artifactually increased.

c) Reversibility of albumin-binding: Varioustechniques were employed to demonstrate that thedistribution of thyroxine between TBP and al-bumin conforms to a reversible binding equi-librium.

Serum was labelled with radiothyroxine; analiquot of this serum was supplemented with stableL-thyroxine to yield a PBI level of 201.5 ,ug. percent. Intermediate concentrations of protein-bound iodine were obtained by combining the en-riched and the unenriched specimens in variousproportions. The mixtures were electrophoresedand the distributions of radioactivity were quan-titated. The results indicated that thyroxine whichis bound to albumin can be recovere(d onto TBPby simply decreasing the PBI without changingthe absolute concentration of either TBP or al-bumin (Table II).

By modifying conventional electrophoretic tech-niques, the concentrations of TBP, albumin, andPBI within a single serum could be altered inde-pendently or in combination to assess the effectsof each variable upon binding phenomena.

Sufficient amounts of L-thyroxine were addedto serum so that radiothyroxine was equally par-titioned between albumin and TBP. Electro-phoresis was performed at pH 8.6 in the "plate-glass" system. The albumin zone was detachedfrom the less rapidly migrating components. Un-labelled serum of similar or lower PBI contentwas applied onto the albumin spot and a secondelectrophoresis was performed. In each instance,partial displacement of albumin-bound radioac-

TABLE II

The effect of PBI dilution on the distribution ofradiothyroxine in plasma *

PBI %with %witlhSpecimen (gg. %) TBP albumin

1 6.5 87.2 7.72 12.6 80.6 11.53 18.4 74.8 16.84 30.9 69.9 25.35 55.2 46.9 41.96 104.0 28.5 50.37 201.5 24.5 49.6

* Specimens 1 and 7 mixed in varying proportions toobtain specimens 2-6.

PRERx

PBI 41.9 6GM1%

76.2. %TBP

5.3 toALBUtIlN _

LABELLED TX

FOUR ftDAYS aTO%TPT

POSTIt _ _ _A

UNLABELLED Tx

LWL-

1702

INTERACrION OF THYROXINE WVlTII COHNFRACTIONS OF PLASMA

bPB ST^"S... _..

:.

*: _

..e f.

..... i2,0pga:*. '.S2ijekS:.,..... ^:..... :.^ :..... : ,,o.,.'

* ^.o 2.> |... o.. ?<;z.......

R^SA| oBWTO6R^Pt¢S:........ :.N* . . : . . :-: .. t B g* . ::.::., ... : , g ,:.: .............. ' .: ,,,,,,: . .D

_wl B l . ......S_.- ...

-a_ -8: wb j.. ra r

:..:': :.: 2. g ..

A.

:

iY..

a....... .:

' . ...

FIG. 3. Two-DIMENSIONAL ELECTROPHORESISIllustrations 1 and 3 represent the respective distributions of proteinis and radioactivity in a

labelled specimen of serum which was electrophoresed in two dimensions. Illustrations 2 alnd4 represent a similar two-dimensional electrophoresis in which additional unlabelled serum wasapplied at sites corresponding to albumin and TBP prior to electrophoresis in the second di-mension. The latter manipulation was performed by localizing albumin (A and C) and TBP(B and D) with the aid of the two narrow strips of filter paper which are depicted in thecenter.

tivity onto the TBP of unlabelled serum could bedemonstrated. No such partition occurred whenthe albumin was re-electrophoresed without theaddition of unlabelled serum.

Similar results were obtained with "two-di-mensional electrophoresis" (Figure 3). Herein,serum labelled with radiothyroxine was appliedonto two sites spaced 13 cm. apart on a 26 by 57cm. sheet of filter paper. For purposes of control,similar aliquots were applied onto two appended2.5 by 57 cm. strips of filter paper. One dimen-sional electrophoresis was performed by theKunkel-Tiselius method following which the ap-pended strips were removed. One of them wasradio-autographed. The second strip was rapidlydried and stained. With the stained strip for ref-erence, protein moieties in the two bands on thelarge sheet of filter paper could be localized. Ac-cordingly, the areas corresponding to TBP andalbumin in one of these bands were supplementedwith 0.020 ml. specimens of unlabelled serum.

Electrophoresis in the secon(l dimension was thenperformed by turning the entire sheet 90 degreesand by bridging it to the buffer reservoirs withfresh filter paper. It was found that TBP of theadded unlabelled serum could abstract most of theradioactivity which, initially, had migrated withalbumin. On the other hand, a shift in the distri-bution of radioactivity was not observed in the con-trol band which had not been supplemented withserum prior to the second electrophoresis.

d) Conclusion: In serum, endogenous and ex-ogenous thyroxine are in ready exchange; thyrox-ine is distributed among plasma proteins accord-ing to a reversible binding equilibrium betweenTBP and secondary carriers of lesser affinity; dis-placement onto secondary carriers (principally al-bumin) occurs with increasing thyroxine concen-trations; and displaced thyroxine can be recoveredonto TBP by decreasing the concentration of thy-roxine or by increasing the availability of TBP.

1703

.....-.-

.....

NORBERTFREINKEL, J. THOMASDOWLING, AND SIDNEY H. INGBAR

TABLE III

The effect of added Cohn Fractions on the distribution of radiothyroxine in serum

Fractions from fresh plasma Fractions from old plasma

Fraction %lo with TBP %with alb. %with TBP %with alb.

Serum control 42.6 i 3.0 (7)* 44.4 ± 2.1 (7)* 34.2 4- 0.9 (5)t 49.3 i 2.9 (5)t

Serum + I 40.9 44.9 32.4 54.1Serum + II-IlI 42.1 43.9 34.3 51.7Serum + IV-1 45.6 44.7 34.5 55.6Serum + IV-5 57.9 31.5 58.9 30.3Serum + IV-6 78.8 15.0 74.4 18.0Serum + IV-7 54.3 37.0 47.2 41.4Serum + IV-8 55.7 34.3 51.8 39.0Serum + IV-9 65.6 26.6 61.4 30.4Serum + V 43.8 47.2 33.5 54.6Serum + Super. V 39.8 46.7 32.9 53.6

* The concentration of PBI in the serum of an euthyroid subject (J. P.) was adjusted to 50.0,ug. per cent. Thefigures in parentheses denote the number of simultaneous electrophoreses which were performed to derive standarddeviations for the distribution of radioactivity prior to the addition of Cohn Fractions which had been obtained fromfresh plasma.

t The concentration of PBI in the serum of an euthyroid subject (N. F.) was adjusted to 46.6 ,ug. per cent. AddedCohn Fractions were prepared from outdated blood-bank blood.

3. Cohn Fractions

The foregoing observations formed the basisfor testing Cohn Fractions.

a) Mixtures of Cohn Fractions and serum:Labelled and unlabelled thyroxine were added tosera to yield concentrations of protein bound io-dine ranging from 26 to 90 ,ug. per cent. Atthese PBI levels, radiothyroxine was varyinglypartitioned between TBP and albumin. Twenty-seven mgm. amounts of lyophilized Cohn Frac-tions were dissolved in 1 ml. aliquots of the sera; 14no attempt was made to correct for insoluble ma-terials. Concurrent paper electrophoreses wereperformed with sera containing added Cohn Frac-tions (circa 2.7 Gm. per cent) and with at leastfour control specimens to which extra protein hadnot been added. It was anticipated that albumin-bound thyroxine might be recovered from albuminonto TBP by Cohn Fractions containing ratiosof TBP/albumin greater than the normal serumproportions.

The partition of radioactivity was not alteredby Cohn Fractions I, II-III, IV-I, V, and Super-nate V 15 (Table III). However, all sub-groups

14 Heparin was added to prevent clotting in all ex-periments involving Cohn Fraction I.

15 Currently, Fraction VI is concentrated with zinchydroxide (28) from the supernatant solution which re-mains after the precipitation of Fraction V according tothe low temperature-low salt-ethanol fraction method(8). Preliminary evidence would indicate that zinc re-

of Fraction IV-4 (i.e., IV-5, IV-6, IV-9; andIV-7, 1V-8) significantly diminished the radio-activity in the albumin area and enhanced the ap-parent binding of thyroxine by TBP. FractionsIV-6 and IV-9 produced the most pronouncedaugmentation of TBP activity. Age of the parentplasma did not affect results. Findings weresimilar with Cohn Fractions derived from out-dated blood bank plasma as well as from freshplasma (Table III).

b) Mixtures of Cohn Fractions and saline:Lyophilized Cohn Fractions were dissolved inisotonic saline 14 and repeatedly centrifuged toremove all insoluble denatured material. Biuretanalyses were performed to adjust the proteinconcentration in all solutions to 2.0 gram per cent.Control specimens of serum were diluted to acomparable extent. At pH 8.6, the components ofthe various Cohn Fractions retained their charac-teristic electrophoretic mobilities. Thus, for pur-poses of reference, the migration of radioactivitycould be related to simultaneously electrophoresedspecimens of serum.

duces the quantity of thyroxine bound by TBP (20).Therefore, attempts were made to obtain the proteins ofFraction VI without zinc precipitation.

Excess salts and ethanol in Supernate V were removedby dialysis against repeated changes of distilled water at40 C. The dialyzed Supernate V was lyophilized and therecovered proteins were employed in subsequent experi-ments. Similar dialysis and lyophilization did not di-minish the binding of thyroxine by Cohn Fraction IV-6.

1704

INTERACTION OF THYROXINE WNrITH COHN FRACTIONS OF PLASMA

Studies were designed to demonstrate traceamounts of thyroxine-binding proteins and tocompare their relative albundances on the basis ofsaturation characteristics. Low concentrations ofcarrier thvroxine were employed to meet the firstobjective. The PBI concentration in all solutionswas adjusted to 11.4,ug. per cent with stable andlabelled L-thyroxine. Paper electrophoresis atpH 8.6 was performed and the distribution ofradioactivity was quantitatively assessed (Figure4, Column 1). Radioactivity in saline solutionsof Cohn Fractions I and II-III extended from theorigin to a zone immediately ahead of the 8 globu-lins. ContrariwNise, some of the radioactivity inFractions IV-1, IV-7, V and Supernate V, and allof the radioactivity in Fractions IV-5, IV-6, IV-8,and IV-9 migrated in parallel with the TBP ofserum. Staining with bromphenol blue was notappreciable in any of these areas of concentratedradioactivity.

For comparison of relative binding capacities,the concentration of protein-bound iodine in allsolutions was augmiiented to 71.4 )g. per cent withunlabelled thyroxine (Figure 4, Column 2).Such loading did not alter the diffuse distributionof radioactivity in solutions containingt, either Cohn

Ao, tz 13I 6t l

SERUM

IV -1

Iv - 6

Iv. 7

IV-B

V9

ALBUMIN:PBI: (uq %)

lL

11

2 3Aa, k ,sIz14

A

O.O 1.45m %TIA4 1.4

Fractions I or II-III. On the other hand, in thecontrol sample of seruimi, most of the radioactivitywas displaced from the TBP onto albumin. Simi-larly, in all Cohn Fractions other than I and II-III, the addition of large amiiounts of thyroxinecaused partial or complete "spill-over" of radio-activ ity- onto secondary carriers. Proteins otherthan albumin served as secondary carriers in frac-tions which wNere deficient in albumin. Displace-ment was least in solutions containing IV-6 orIV-9 and most of the radioactivity remained con-fined to a discrete zone corresponding in electro-phoretic mobility at pH 8.6 to the TBP of serum.

The specificity of thyroxine-binding in IV-6and IV-9 was assessed by electrophoresis in ace-tate buffer at pH 4.5 (Figure 5). At this pH,free radiothyroxine or radiothvroxine combinedwsith Cohn Fraction II-III remuained at the origin.Labelled TBP in thyroxine-serum mixtures mi-grated towards the anode in confirmation of the re-port of Robbins, Petermann, and Rall (21). Theradioactivity in solutions containing FractionsIV-6 and IV-9 also migrated slowly towards thepositive pole despite the predominant cathodalmovement of most of the other proteins.

c) Ilixtitrcs of Cohin Fr-actions, saline and

SERUM

m

y

SUPER.y

'A

SALINETx

i'

-, L

0.011.4

2A a, azaI'"4

A

3

A a, 1i;

0.0 1.5Gm %714 11.4

FIlG. 4. THYROXINE BINDING BY COHN FRACTIONS

TFle darkened areas represent the distribution of radioactivity following paperelectrophoresis.

1705

I

NORBERTFREINKEL, J. THOMASDOWLING, AND SIDNEY H. INGBAR

BP STAIN±

RADIOAUTOGRAP14+

SERUM

-T- + SALI NE

7 mfli Si

IV-5

FIG. 5. ZONEELECTROPHORESISAT PH 4.5The similarities in the anodal migrations of serum TBP and the thyroxine-

binding moieties in Fractions IV-6 and IV-9 can be seen in the radioauto-graphs on the right.

mercaptalbumin: To simiulate the inter-relation-ships of proteins in serum and to compensate forthe variable content of albumin in Cohn Fractions,attempts were made to electrophorese Cohn Frac-tions in association with constant amounts ofadded albumin. It was hoped that such combina-tion might provide further quantitative compari-son of the thyroxine-binding moieties in CohnFractions. Albumin preparations whose homo-geneity ranged from 94 to 100 per cent by movingboundary electrophoresis were tested for the pres-ence of non-albumin contaminants capable of bind-ing thyroxine. Only mercaptalbumin (22) 16 didnot contain detectable traces of a material whichresembled serum TBP during paper electrophore-sis at pH 8.6. Therefore, saline solutions of CohnFractions (Protein: 1.85 Gm. per cent; PBI: 11.4,^g. per cent) were supplemented with weighedamounts of lyophilized mercaptalbumin (15 mgm.

16 Mercaptalbumin whiclh lhad been dialyzed againstcysteine to remove mercury was kindly provided by Dr.Margaret J. Hunter of the Department of BiophysicalChemistry of Harvard Medical School. The albuminpreparations which contained TBP-like impurities in-cluded crystalline bovine albumin and various reworkedpreparations of Fraction V.

per ml.) prior to paper electrophoresis at pH 8.6.All of the radioactivity in solutions containingFractions I, II-III, V, and Supernate V migratedwith the added albumin (Figure 4, Column 3).Lesser changes were effected in mixtures con-taining the other Cohn Fractions. The distribu-tion of radioactivity was least altered in the pres-ence of IV-6 or IV-9.

d) Conclusions: All Cohn Fractions which areprecipitated subsequent to Fraction III containdetectable amounts of material which binds thy-roxine and which simulates the electrophoreticmobility of serum TBP at pH 8.6. This materialis most abundantly present in Fractions IV-6 andIV-9. On the basis of electrophoresis at pH 4.5,it may be inferred that the thyroxine-bindingmoieties in IV-6 and IV-9 are indeed the sameas the TBP of serum.

DISCUSSION

Earlier attempts to localize TBP in Cohn Frac-tions of plasma have followed several lines. Bydialysis, it has been shown that thyroxine-bindingis minimal with gammaglobulin (23). However,

1706

I., .0

i::

I*.

INTERACTION OF THYROXINEWITH COHNFRACTIONS OF PLASMA

since thyroxine is bound, at least partly, by allproteins, the dialysis technique affords limitedspecificity. Other investigators have relied uponmeasurements of iodine/nitrogen ratios (23, 24).With this approach, Salter meticulously localizedthe thyroxine-transport protein in Fractions IVand V, and even succeeded in specifically impli-cating the subfractions of IV which were rich inalpha globulins (i.e., IV-5 and IV-6) (25).However, the differences among various fractionswere small and inconsistent, and it is likely thatsome of the variability may have resulted fromartifacts intrinsic to the fractionation technique.During fractionation by Method 6 of Cohn, dis-ruption of thyroxine-protein bonds and reagentcontamination may progressively increase the con-tent of unbound inorganic and organic iodine inthe alcohol-protein-electrolyte mixtures. Thatcoprecipitation and occlusion of these iodinemoieties does occur is suggested by the routinedemonstration of significant amounts of protein-bound iodine in fractions which do not containdetectable amounts of TBP such as Fractions I orII-III (23-25). Errors thereby introduced wouldbecome especially manifest in fractions which areprecipitated last, either in great bulk (cf. FractionV) or in small amounts from large volumes ofsuspending solution (cf. Fraction VI). It is ofinterest in this connection that high iodine/nitro-gen ratios have been reported for Fraction V (23-25) and for Fraction VI (25, 26) although theircontent of TBP would appear to be small.

Obviously, it would be most desirable to meas-ure TBP directly. As yet, no such technique hasbeen evolved. In the present experiments, at-tempts were made to minimize the difficultieswhich are inherent in indirect approaches. Frac-tions prepared by Methods 6 of Cohn were com-pared by a variety of saturation and competitive-binding techniques; zone electrophoresis was em-ployed to provide specificity.

It may be argued that saturation characteristicscannot be used for intercomparisons since thefractionation technique, per se, may varyingly de-saturate or remove endogenous thyroxine fromthe different Cohn Fractions. Although this pos-sibility cannot be excluded, it would seem unlikelythat variation in endogenous hormone (e.g., io-dine/protein ratios; Table I) could significantlyaffect the results obtained in systems wherein the

concentration of added carrier thyroxine wasgreater than 100 ,g. per cent (v.s.).

Certain properties of TBP were noted in thecourse of the present experiments. It may be in-ferred that the TBP of Fraction IV-6 is not alipoprotein; multiple freezings and thawings didnot alter its binding characteristics. TBP ishighly soluble in water, and neither solubility northyroxine-binding were diminished by dialyzingIV-6 against repeated changes of distilled waterto remove occluded inorganic moieties. The sta-bility of TBP is evidenced by the observation thatfractions derived from fresh and from outdatedblood did not appreciably differ in activity.

Zinc interferes with the interaction of thyroxineand TBP in serum and in Fractions IV-6 andIV-9. Conceivably, therefore, the techniquesdescribed above may not be applicable to proteinswhich have been prepared by zinc precipitation.Among these proteins may be included the prod-ucts of Method 10 of Cohn and his co-workers(27) and the pure a glycoproteins of Schmid (28,29).

Throughout this report, the term TBP has beenemployed to designate the thyroxine-binding pro-tein of plasma. It should be stressed that nomen-clatural selection of the singular is arbitrary andintended solely for convenience. By labelling theTBP in serum with radiothyroxine, Petermann,Robbins, and Hamilton have derived an ultra-centrifugal sedimentation constant for TBP (30),and Robbins, Petermann, and Rall (21) haveshown that the electrophoretic mobility of TBPat pH 4.5 simulates that of the M-2 glycoproteinsof Mehl, Golden, and Winzler (31). However,to date, TBP has not been isolated. Thus, de-spite all physicochemical measurements, it can-not be said whether TBP represents only one pro-tein or several proteins which, in protein mixtures,exhibit similar electrophoretic and ultracentrifu-gal characteristics. Furthermore, if TBP is asingle protein, the possibility of multiple sites forthyroxine-binding cannot be excluded. Becauseof these considerations, the present data have beensubjected only to descriptive analysis. It was feltthat available information did not justify morequantitative treatment, and that precise stoichio-metrical formulations must await the availabilityof pure TBP.

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NOtBART FRtINKEL, 3. TlOMAS DOWLING, AND SIbNEY A. I11GBAR

SUMMARY

The interactions between thyroxine and plasmaproteins have been investigated with paper elec-trophoresis. The observations furnished an ex-

perimental basis for multiple techniques designedto localize the thyroxine-binding protein (TBP)in fractions of plasma prepared by Method 6 ofCohn. It has been demonstrated that Cohn Frac-tions IV-6 and IV-9 constitute the most abundantsources of TBP.

ACKNOWLEDGMENTS

The technical assistance of Miss Barbara Fine isgratefully acknowledged. The authors are greatly in-debted to Dr. Robert B. Pennell and Dr. John L. Oncleyof the Department of Biophysical Chemistry of HarvardMedical School and to Dr. John M. Newell and Mr.Roderick C. Dwyer of the Massachusetts Public HealthBiologic Laboratories. This study could not have beenundertaken without their constant encouragement andgenerosity in making laboratory space available to us.

REFERENCES

1. Gordon, A. H., Gross, J., O'Connor, D., and Pitt-Rivers, R., Nature of the circulating thyroid hor-mone-plasma protein complex. Nature, 1952, 169,19.

2. Larson, F., Deiss, W. P., and Albright, E. C., Lo-calization of protein-bound radioactive iodine byfilter paper electrophoresis. Science, 1952, 115,626.

3. Winzler, R. J., and Notrica, S. R., Association ofthyroxine with plasma proteins. Federation Proc.,1952, 11, 312.

4. Deiss, W. P., Albright, E. C., and Larson, F. C., Astudy of the nature of the circulating thyroid hor-mone in euthyroid and hyperthyroid subjects byuse of paper electrophoresis. J. Clin. Invest.,1952, 31, 1000.

5. Robbins, J., and Rall, J. E., Zone electrophoresis infilter paper of serum I" after radioiodide adminis-tration. Proc. Soc. Exper. Biol. & Med., 1952, 81,530.

6. Horst, W., and Rosler, H., Der Transport desHormonjods im menschlichen Serum untersuchtmit Papierelektrophorese und Radiojod. Zugleichein Beitrag zur Frage der Existenz von sog.Zwischenfraktionen. Klin. Wchnschr., 1953, 31,13.

7. Larson, F. C., Deiss, W. P., and Albright, E. C.,Radiochromatographic identification of thyroxinin an alpha globulin fraction of serum separatedby starch zone electrophoresis. 3. Clin. Invest.,1954, 33, 230.

8. Cohn, E. J., Strong, L. E., Hughes, W. L., Jr., Mul-ford, D. J., Ashworth, J. N., Melin, M., and Taylor,H. L., Preparation and properties of serum andplasma proteins. IV. A system for the separationinto fractions of the protein and lipoprotein com-ponents of biological tissues and fluids. J. Am.Chem. Soc., 1946, 68, 459.

9. Surgenor, D. M., Strong, L. E., Taylor, H. L., Gor-don, R. S., Jr., and Gibson, D. M., The separationof choline esterase, mucoprotein, and metal-com-bining protein into subfractions of human plasma.J. Am. Chem. Soc., 1949, 71, 1223.

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11. Barker, S. B., Humphrey, M. J., and Soley, M. H.,The clinical determination of protein-bound iodine.J. Clin. Invest., 1951, 30, 55.

12. Barker, S. B., Determination of protein-bound io-dine. J. Biol. Chem., 1948, 173, 715.

13. Mehl, J. W., The biuret reaction of proteins in thepresence of ethylene glycol. J. Biol. Chem., 1945,157, 173.

14. Durrum, E. L., A microelectrophoretic and micro-ionophoretic technique. J. Am. Chem. Soc., 1950,72, 2943.

15. Kunkel, H. G., and Tiselius, A., Electrophoresis ofproteins on filter paper. J. Gen. Physiol., 1951, 35,89.

16. Slater, R. J., and Kunkel, H. G., Filter paper electro-phoresis with special reference to urinary proteins.J. Lab. & Clin. Med., 1953, 41, 619.

17. Freinkel, N., Schreiner, G. E., Athens, J. W., Hiatt,C. W., and Breese, S., Artifactual differences inthe distribution of T-1824 and I't-labeled albuminresulting from mixing prior to administration. J.Lab. & Clin. Med., 1954, 43, 215.

18. Gabrieli, E. R., Goulian, D., Jr., Kinersly, T., andCollet, R., Zone paper electrophoresis studies onradio-iodinated human serum albumin. 3. Clin.Invest., 1954, 33, 136.

19. Robbins, J., Rall, J. E., Becker, D. V., and Rawson,R. W., The nature of the serum iodine after largedoses of I"f. J. Clin. Endocrinol., 1952, 12, 856.

20. Freinkel, N., Dowling, J. T., and Ingbar, S. H.,Unpublished observations.

21. Robbins, J., Petermann, M. L., and Rall, J. E., Elec-trophoresis of the thyroxine-binding protein of se-rum at pH 4.5. J. Biol. Chem., 1955, 212, 403.

22. Hughes, W. L., Jr., An albumin fraction isolatedfrom human plasma as a crystalline mercuric salt.J. Am. Chem. Soc., 1947, 69, 1837.

23. Taurog, A., and Chaikoff, I. L., The nature of thecirculating thyroid hormone. J. Biol. Chem., 1948,176, 639.

24. Salter, W. T., The metabolic circuit of the thyroidhormone. Ann. New York Acad. Sc., 1949, 50,358.

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INTERACTION OF THYROXINE WITH COHNFRACTIONS OF PLASMA

25. Salter, W. T., Unpublished data included in the min-utes of the weekly luncheon meetings of the Har-vard University Laboratory of Physical Chem-istry Related to Medicine and Public Health, 1944-1950.

26. Schmid, K., Preparation and properties of serum andplasma proteins. XXIX. Separation from humanplasma of polysaccharides, peptides and proteinsof low molecular weight. Crystallization of an

acid glycoprotein. J. Am. Chem. Soc., 1953, 75, 60.27. Cohn, E. J., Gurd, F. R. N., Surgenor, D. M., Barnes,

B. A., Brown, R. K., Derouaux, G., Gillespie, J.M., Kahnt, F. W., Lever, W. F., Liu, C. H., Mit-telman, D., Mouton, R. F., Schmid, K., and Uroma,E., A system for the separation of the componentsof human blood: quantitative procedures for the

separation of the protein components of humanplasma. J. Am. Chem. Soc., 1950, 72, 465.

28. Schmid, K., Isolation of a group of a2-glycoproteinsfrom human plasma. J. Am. Chem. Soc., 1955, 77,742.

29. Schmid, K., Isolation and characterization of glyco-proteins from human plasma. J. Am. Chem. Soc.,1953, 75, 2532.

30. Petermann, M. L., Robbins, J., and Hamilton, M. G.,Sedimentation of the thyroxine-binding protein ofserum in the partition cell. J. Biol. Chem., 1954,208, 369.

31. Mehl, J. W., Golden, F., and Winzler, R. J., Muco-proteins of human plasma. IV. Electrophoreticdemonstration of mucoproteins in serum at pH 4.5.Proc. Soc. Exper. Biol. & Med., 1949, 72, 110.

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