Iodinated amino acid chromatography on polystyrene resins

ARCHIVES

OF

BIOCHEMISTRY

lodinated

Amino

AND

BIOPHYSICS

Acid

103, 36-41 (1963)

Chromatography

on Polystyrene

Resins’

S. R. LERNER From

the Radioisotope

Service, Veteran,s Baylor University

Administration Hospital, and the Biochemistry College of Medicine, Houston, Texas

Received

March

Department,

25, 1963

The iodinated amino acids have been separated by elution analysis on ion exchange columns of Dowex 50 X 4 using an automated procedure which presents the radioactivity and iodide determinations on the effluent on a recorder chart. The iodinated amino acids are eluted using ammonium formate buffers containing 30% ethanol. The method is applicable to samples containing iodinated amino acids of concentrations as found in human plasma without carrier and quantitative recovery of 1131has been achieved. INTRODUCTION

Stein (8) offered a new route to the determinations of the iodinated amino acids under relatively mild conditions. Early experiments indicated that the iodinated tyrosines would present no difficulties, but the iodinated thyronines posed problems. This report concerns the development of an automated elution analysis scheme for the iodinated amino acids based on ion exchange separations on moderately cross linked Dowex 50 resin with alcoholic ammonium formate buffers, with recording of the separated amino acid 1131activities and a continuous flow analysis of the iodine-containing compounds by the ceric-arsenite catalysis reaction.

The separation of iodinated amino acids from thyroid hydrolysates and blood extracts has been greatly facilitated with the application of paper chromatographic procedures; the measurement of 1131 radioactivity in thyroxine (T4), triiodothyronine (T3), diiodotyrosine (DIT), monoiodotyrosine (MIT), etc., has become routine. However, the quantitation of the chemical (P) amounts of these amino acids has not been as successful; the amounts of hydrolysates, salts, and other material that can be applied to paper become limiting. Efforts to facilitate these analyses by column techniques have been limited in success and/or technically difficult as far as separation of T3 and T4 is concerned (2-Ti). Wynn et al. (6) have accomplished this separation on columns of Dowex 1, but the technique of displacement chromatography using formic acid at concentrations as high as 70-88% at elevated temperatures offers artefact possibilities (both exchange and deiodination) in compounds as labile as iodinated amino acids (7). The elegant elution analysis techniques for amino acids developed by Moore and

MATERIALS Resins. Dowex 50 W X 4 (3&35 p). The resin is prepared from minus 400 mesh Dowex 50 W (4yc cross linked) by the hydraulic sizing procedure of Hamilton (9). The fraction containing particles of 3&35 p diameter is set aside for this procedure. A suitable preparation is available commercially.2 Other resin particle sizes and types3 were prepared in a similar manner. Bu$ers. Ammonium formate buffers are prepared containing 0.2 N ammonium ion and 30% 2 AG50 W X 4 (30-35g), Bio-Rad Lab., 32nd & Griffin, Richmond, California. 3 We are indebted to Mr. David Schwartz of Bio-Rad Laboratories for several samples of these resins of various cross linkages.

1 A preliminary report of this work was presented at the Annual Meeting of the Federation of American Societies for Experimental Biology, Chicago, Ill., April 11, 1960 (1). 36

IODINATED TABLE BVFFER

AMINO

ACID

I

PREPARATION

Ethanol

Final volume (liters)

Formic acid cont. (ml.)

,(absolute:

82

2.70

9.0

28

1.20

4.0

29

2.70

9.0

4.5

(litWS)*

+0.1 0.2 ‘4.1

7.5

53.3

ZkO.1 0.2&

9.1

120

*o.ot D Adjust pH as necessary with formic (cont.) or 0.2 iV NH,OH (307, ethanol). b llivide by 0.95 if 95% ethanol is used.

acid

ethanol at nominal pH’s 9.1, 7.5, and 4.5 by the addition of sufficient formic acid to 0.2 N NHJOH in 307~ ethanol to give the required pH. These nominal pH’s are reproducible but are different from the pH obtained by dilution to lower alcohol concentration or by preparation of higher concentrations of aqueous buffer and subsequent dilution with alcohol to the final concentrations. Buffers prepared in 4- and B-liter quantities as indicated in Table I are stable indefinitely. The distilled water should be deionized by passage through a 50cm. column of Amberlite MB-3 mixed-bed ion exchange resin or redistilled in an all-glass apparatus, to reduce the iodine blank. Reagents. 2% ceric ammonium sulfate in 4 N H1S04. Dissolve 1000 ml. cont. sulfuric acid in 7 liters deionized, distilled water, add 180 g. Ce(NH,)4(SOa)4.2H20. When cool, dilute to 9 liters. One per cent sodium arsenite in 1 N H&04. Dissolve 250 ml. cont. sulfuric acid in 8.5 liters deionized, distilled water and cool to &5”. Add 90 g. sodium arsenite and stir until dissolved. At room temperature, dilute to 9 liters. .4mino acids. The iodinated amino acids MIT, DIT, T2, T3, and T4 were commercial products and all were homogeneous when chromatographed with n-butanol-dioxane-ammonia on paper (4.1-2N) (ascending) at the 50-100 rg. level. INSTRUMENTS

AND

METHODS

An instrument with a flow scheme similar to that developed by Spackman et al. (10) was built. The differences which are significant are detailed below. Ru$er selection. Buffer reservoirs of 4 or 9 liters capacity were connected by Tygon or polyethylene tubing to an automatic programmer, which changed the elution buffers on a preset time

37

CHROMATOGRAPHY

schedule. Basically, the programmer4 is a five port stopcock (Teflon and glass) which is motor driven through 90” intervals from one position to the next as required by a time schedule card (11). Each of the four available inlets is connected (through a common outlet), in sequence, to a constant volume pump (Milton Roy Chromatographic MiniPump CHMMl-B-29) set to deliver 25.0 ml. per hr. Flow calorimeter. The optical bench assembly from a spectrophotometer (Coleman Instruments, Inc., Model 6A) was mounted on a relay rack panel for support, and the photocell terminals were wired to one input channel of the recorder in parallel with a 1000 ohm ten-turn potentiometer for setting the 100% line. The lamp was operated at 6.3 v. supplied by a constant voltage transformer (Sola Electric Co., catalog No. 20-04-030, type CVE-3). A hole was drilled in the bottom of the cuvette holder (Coleman Model B-110), and a 6 mm. O.D. glass tube cuvette with sealed on capillary ends was inserted. The flow stream was upward. Columns. Columns are poured from an aqueous slurry of Dowex 50 X 4 (30-35 p particles) using the short (0.9 X 25 cm.) tubes described by Hpackman et al. (10). The column is poured initially to a height of 20 cm. and attached to the pump outlet thru Tygon or polyethylene tubing. Ammonium hydroxide (0.2 N, 3Oyc ethanol) is started and the resin cycled through the pH 4.57.5-9.10.2 N NH,OH-pH 4.5 sequence. The column is ready for use the next morning after removal of the excess resin down to the 15 cm. height. The pressures will be about 15-20 psig after a further hour to repack the top of the column. The column is operated at room temperature. Chromatography. The material to be applied is adjusted to about pH 3.5 and applied to the column after removal of the supernatant buffer. Air pressure of 10-15 psig is used to blow the solution into the column as well as two small washes with pH 4.5 buffer. The tube is filled with pH 4.5 buffer and the elution started at the pump setting of 25.0 ml. per hr. Elution schedule. The automatic programmer (11) is set to deliver pH 4.5 buffer for 5 hr., pH 7.5 buffer for the next 4 hr., pH 9.1 buffer for 8 hr., and 0.2 N ammonium hydroxide (3Oyc ethanol) for 2 hr., then to return to pH 4.5 buffer. Thus, the elution and wash are completed in 19 hr. and, after about 23 hr., the column is regenerated and the next sample can be applied. Ejtuent analysis--radioactivity. The effluent from the ion exchange column is lead by small bore polyethylene tubing (0.022 X 0.042 inch 4 Adelco Laboratories,

Box 592, Bellaire,

Texas.

38

LERNER

-.

‘c/g----

I

/ KCI Reservoir

N’ w-KC1 Agar

Bridge

FIG. 1. Recording flow-pH meter electrode system. The glass tubing is of 1 mm. bore, enlarged as little as practical to accommodate the bulb of the glass electrode. The electrodes used were part of a Beckman 39023 flow assembly. Becton, Dickinson No. PXO22) to a coil of larger bore polyethylene tubing (Clay-Adams No. PE190) (0.047 X 0.067 inch) wrapped around a shielded 1 inch diameter X 1 inch long scintillation crystal (NaI-thallium activated). A recording count rate meter is connected, whose output furnishes one channel of information to a 4 point recorder (Bristol Dynamaster, 10 mv. full scale, model 12P12H590-51-B14B-T48-T68, with one point input shorted). The total radioactivity of a peak is proportional to the area under the curve and may be approximated closely by use of the height-width (at half-height) product. When the level of I I31 is too low for accurate measurement by the rabemeter-recorder, individual tube collections may be counted in a well scintillation crystal. E&bent analysis-iodine. The effluent is led to a glass Y-tube of 1 mm. bore and the stream is split by means of a peristaltic pump5 which removes the effluent at a constant rate of about 5 ml. per hr. (0.056 inch tubing). This diverted stream is mixed with the ceric reagent (0.056 inch tubing) and the arsenite reagent (0.081 inch tubing) from additional tubes in the peristaltic pump, then 5 The Autoanalyxer proportioning pump, Technicon Instrument Corp., Chauncey, N. Y., modified by substituting t’he standard motor by a 1 rpm. synchronous motor (No. B8122E-MOOC, Bodine Elec. Co.).

flows to a micro mixing chamber (0.5 ml.) which is magnetically stirred by a glass-enclosed iron wire rotated by a small magnet (W inch diameter X W inch long Alnico V) cemented to the shaft of a small 240 rpm. motor (Cramer Controls Corp., type 117). From the mixing chamber, the st’ream passes to a 90 min. delay coil of polyethylene tubing (0.047 X 0.067 inch, Clay-Adams No. PE190) and through the recording calorimeter (420 mr) and on to a waste bottle. Efluent analysis-pH. The ion exchange column eMuent from the Y-tube (less the portion removed for iodine analysis) passes through the glass electrode apparatus shown in Fig. 1 and then to a fraction collector. The pH electrodes are connected to a recording pH meter (Beckman Zeromatic) and the output furnishes the third channel of information to the recorder. The fractions are collected on a time basis since the flow rate is constant and each fraction can be related to its analysis on the recorder for further analyses. RESULTS

A typical chromatographic separation of a mixture of synthetic amino acids is shown in Fig. 2. The MIT and DIT peaks are well separated from one another. T4 is eluted before T3; the pH 7.5 second buffer serves to accelerate T2 ahead of T4, otherwise it would elute with T4. When large (0.2.5 pmole or more) quantities are used and the ultraviolet absorbance determined, absorbance due to tryptophan appears between MIT and DIT. The time to the peak for each amino acid is reproducible to f0.2 hr. (5 ml.). Accordingly, the height of the peak optical density change of the ceric-arsenit#e reaction stream is proportional to the amount of each amino acid and this proportionality holds over the range of about 0.4 optical density change. By plotting the change in optical density (0. D.) versus the amount of each amino acid on the column, an empirical curve can be drawn for each amino acid. Since tjhe A 0. D. is different for each amino acid, the proportional region holds for O-5 nmoles of T4 and DIT, O-10 nmoles MIT, and O-l.? nmoles T3 and T2. It is evident that the availability of the iodine atom for catalysis of the reduction of ceric ion by arsenite varies according to its location on the phenolic or phenyl ether ring. The effect of elution volume on the peak height-width relationship is brought out by the finding of a higher A 0. D. for MIT than for DIT for equal io-

IODINATED

L

AMINO

I

ACID

I

CHROMATOGRAPHY

39

t--i

FIG. 2. The chromatographic separation of a synthetic mixture of iodinated amino acids labeled with 113i in the 3’ or 3’, 5’ positions, showing small iodide contaminant. 2.5 nmoles of MIT, DIT, and T4; and 7.5 nmoles of T2 and T3, were applied to the column. Note that the ceric-arsenite reaction results are printed out about 1.5 hr. later than the radioactivity measurement.

dine content. This is in keeping with the earlier elution and consequent sharper peak for MIT. The influence of pH on the elution pattern is shown by the results depicted in Fig. 3. The MIT and DIT elute with smaller delays as the pH is increased, DIT accelerating faster, until at about pH 6.5, MIT and DIT come off together and then DIT precedes MIT if the initial buffer pH is raised to pH 7. T4 and T3, however, come off in that order, with T2 emerging as a shoulder on the T4 peak at lower pH’s. At a pH higher than 9.0, the T4-T2 separation is nil and T2 elutes with T4. It was found, however, that 100 ml. of pH 7.5 buffer will preferentially wash T2 ahead and then the pH 9.1 buffer

elutes T2 as a peak ahead of T4, with T3 following, well separated from T4. While the best separations were achieved using Dowex 5OWx4 (30-35~), good results were obtained with Dowex 5OWx2 with similar particle sizes. Comparable samples of Dowex 5OWx8 and of crushed IR-120 gave much less satisfactory separations. Dowex 5OWx4 of 200-400 mesh size gave virtually no separation of the thyronines. It might be assumed that a slower flow rate would enhance the separations somewhat, since penetration of the larger iodinated amino acids into the resin seems to he important to the ion exchange mechanism as shown by the higher cross linked resins.

40

LERNER ELUTION

OF IODINATED

AMINO FORMATE

ACIDS

WITH

ETHANOLIC

AMMONIUM

BUFFERS

IO

I

50

IO0

150

300

400

500

ml to peak

FIG. 3. Influence of pH on elution of iodinated pH of the first buffer is indicated on the ordinate; (except in the case of the pH 9.5 initial buffer).

TABLE HYDROLYSIS

OF RABBIT

THYROID

amino acids from Dowex 5OW-X4. The this was followed by the pH 9.1 buffer

II TISSUE

WITH

PANCREATIN

Hydrolysis time (hours) 0:oo

Iodide (l-3) Fast U (4-7) MIT (S-20) DIT (21-30) Inter (3143) Ul (44-51) U2 (52-58) TJ3 (59-64) T4 (65-81) T3 (82- -) Resin bound Total recovery

1.5 0.7 0.6 1.0 1.0 22.4 11.6 19.9 2.4 37.8 98.9

0: 15

0:30

l:oo

2:oo

6: 12

12:40

21:oo

2.4 3.8 5.9 1.7 3.7 23.6 4.9 7.5 2.3 1.7 45.6 103.1

1.9 12.1 16.1 3.7 5.2 35.2 10.0 5.6 4.4

3.2 5.0 28.0 10.2 3.8 32.4 -

7.1 101.9

-

2.5 2.5 31.8 15.2 2.8 25.5 4.9 7.0 5.9 1.2 0.8 100.1

2.7 1.1 33.2 23.0 3.4 19.1 3.3 4.3 10.9 2.3 0.8 104.1

3.2 1.4 32.3 24.1 6.0 15.5 3.2 4.0 15.2 1.5 4.8” 111.2

4.1 1.6 33.8 25.7 5.2 13.7 3.0 2.3 12.6 3.4 1.7 107.1

Q Possible artefact from previous sample (zero time sample run immediately before). A 2.8 kg. rabbit was injected S.C. with iodide 1’31 (carrier-free) ; at 20 hr. the thyroids were removed, homogenized (cold) with pH 9.0 phosphate buffer (0.001 M thiouracil), centrifuged, and the supernatant hydrolyzed with 100 mg. pancreatin 4 X (Viobin). One ml. aliquots were removed, acidified, and frozen at the times indicated. Thawed samples were applied to a Dowex 50 W X 4 column and analyzed for radioactivity.

IODINATED

AMINO

ACID

DISCUSSION

The methods described here provide a technique for the separation of the iodinated amino acids (and peptides) which is essentially free of limitations imposed by salts, contaminating amino acids, sample volume, etc. For example, the samples applied to the column have covered the range from 0.010 ml. of a solution of synthetic amino acids in pH 3.5 buffer to 5 ml. of a 0.1 M phosphate buffered 5-min. pancreatin hydrolysate of a thyroid tissue containing much undigested, denatured protein, both ends of the sample spectrum giving good information. In the latter case, a small amount of Celite filter aid was slurried with the sample to reduce the pressure requirement on the pumping system. Artefact production by the chromatographic process has been reduced to very low levels, as demonstrated by experiments in which a mixture of synthetic iodinated amino acids was labeled with commercial T3-P3’ (containing 8.7% of the activity as T4 and 1.2 YO as inorganic I). Any incorporation of label into MIT or DIT was below the level of detection (estimated at less than 0.5 % of the sample). Similarly, the addition of the same T3-P3r solution to a 27-hr. pancreatin hydrolysate of unlabeled pork thyroid gave identical results, i.e., the activity appeared in the same ratio (1.5 % I ; 8.7% T4; 89.7% T3) with no detectable activity (est. < 0.5%) in any other fractions, which included at least 12 peaks of chemical iodine representing peptides, MIT, DIT, etc. From the data depicted as fig. 3, it would appear that MIT and DIT are strongly influenced by ionic forces. Indeed, the separation is hardly influenced by the alcohol concentration and is virtually the same in the absence of alcohol. The thyronines, however, seem to be strongly influenced by adsorptive forces and are very sensitive to the alcohol concentration, which would influence dielectric constants and molecular charges strongly, particularly in the region of the pK’s (amino) of the thyronines.

CHROMATOGRAPHY

41

However, a sample of unsulfonated 4% cross linked styrene-divinyl benzene copolymer (generously supplied by Mr. Philip B. Dewey of Dow Chemical Co., Midland, Michigan) of the same particle size range showed no separation. This is taken to indicate that ion exchange is still the dominant force. The data in Table II are illustrative of the separations achieved on thyroid hydrolysates. The samples were chromatographed in a relatively random sequence, i.e., not in order of hydrolysis times, and stored frozen while awaiting chromatography. The large amount of radioactivity recovered in unknowns (presumably peptides) Ul, U2, and U3, even at 21 hr., is notewort,hy. An adaptation of this method has been used by Reilly et al. to separate the iodinated amino acids of human serum (12) ; Block and Mandl have briefly described an additional variation which employed gradient elution and automated the iodine determination (13). REFERENCES 1. LERNER, S. R., Federation PTOC. 19, 171 (1960). 2. BRAASCH, J. W., FLOCK, E. V., AND ALBERT, A., Endocrinology 66, 768 (1954). 3. ROCHE, J., MICHEL, R., AND NUNEZ, J., Bull. Sot. them. biol. 40, 361 (1958). 4. PITT-RIVERS, R., AND SACKS, B. I., Biochem. J. 82, 111 (1962). 5. MEYNIEL, G., BLANQUET, P., MOURIER, J., AND ESTIBOTLE, M., Bull. Sot. them. biol. 40, 269 (1958). 6. WYNN, J., FABRIKANT, IRENE, AND DEISS, W. P., Arch. Biochem. Biophys. 34, IO%? (1959). 7. TAUROG, A., AND CHAIKOFF, I. L., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. IV, pp. 866-7. Academic Press, New York, 1957. 8. MOORE, S., AND STEIN, W. H., J. Biol. Chem. 192, 663 (1951). 9. HAMILTON, P. B., Anal. Chem. 30, 914 (1958). 10. SPACKMAN, 1~. H., STEIN, W. H., AND MOORE, S., AnaZ. Chem. 30, 1190 (1958). 11. LERNER, S. R., Anal. Chem. 36, 1108 (1963). 12. REILLY, W. A., SEARLE, G. L., AND SCOTT, K. G., Metabolism 10, 869 (1961). 13. BLOCK, R. J., AND MANDL, R. H., Biochem. J. 81, 37 P (1961).