A General Method of Tritium Labeling Utilizing Microwave Discharge … · · 2003-01-21A General Method of Tritium Labeling Utilizing Microwave Discharge Activation of Tritium Gas

TIIE JOURI\‘AL OF RIOLOCIC.~L CIIEMISTRY Vol. 248, No. 15, Issue of August 10, pp. 5532-5540, 1073

Printed in C.S.A.

A General Method of Tritium Labeling Utilizing Microwave

Discharge Activation of Tritium Gas

METHODOLOGY AKD APPLICATION TO BIOLOGICAL COMPOUn’DS*

(Received for publication, March 12, 1973)

WYLIE C. HEMBREE, RICHARD E. EHRENKAUFER, SEYMOUR LIEBERNAN, AND ALFRED P. WOLF

Frown the Department of Meclicine, Obstetrics and Gynecology, Biochemistry, and the International Institute for the Stucly of Human Reproduction, College of Physicians and Surgeons, Columbia University, New York, New York 10032, and Department of Chemistry, Brookhaven National Laboratory, Upton, New York 119731

SUMMARY

A method of general tritium labeling is described which is applicable to a broad range of organic compounds of biological interest, including amino acids, peptides, and proteins. The method is rapid, inexpensive, and nondestructive, thereby making the purification of labeled material relatively uncom- plicated. The reaction apparatus consists of a closed system in which tritium gas at a pressure of 4 mm Hg or less can be cycled by an internal pump. The labeling reaction(s) is initiated by microwave discharge activation of the tritium gas and the sample, cooled by liquid nitrogen, remains outside the region of the high ultraviolet and electron flux from the tritium plasma. The evidence suggests that tritium-hydrogen exchange occurs as a result of bombardment of the labeled substrate with tritium atoms. Studies of the chemistry of the labeling reaction with aromatic hydrocarbons, of the analytical requirements for radiochemical purity, of the characteristic specific activities noted for several classes of compounds, and of the application of the method to the preparation of tritium-labeled peptides are presented. Tritium- labeled adrenocorticotropin (ACTH) with a specific activity of 15 Ci per mmole and full biological activity has been prepared by this method. This represents a lOOO-fold increase in the specific activity previously obtained by Wilzbach labeling of ACTH.

The availability of high specific activity tritium-labeled compounds of biological interest has made it, possible to characterize many previously inaccessible biochemical and physiological processes. In addition, studies of tritium incorporation into organic compounds, whether by synthetic, recoil, or exchange methods, have provided new insights into both chemical struc-

* Work at Brookhaven National Laboratory was supported by the United States Atomic Enerzv Commission. and at Columbia by the Atomic Energy Comm&on Grant ATill-I)-3105.

$ Address reprint requests to Brookhaven National Labora- tory.

ture and reaction mechanisms. In general, only synthetic methods have been successful in producing tritium-labeled compounds with specific activities high enough to be suitable as biological tracers. However, these synthetic methods are not generally applicable to tritium labeling of complex biologically active molecules such as polypeptides and proteins. nlumerous attempts to modify gas exposure (Wilzbach) techniques met with only limited success and the approach was largely empirical. Therefore, the desirability of having available high specific activity tritium-labeled polypeptides and proteins led us to investigate the applicability of new nonsynthctic labeling techniques.

Tritium-hydrogen exchange induced by species derived by microwave “activation” of tritium gas was first reported by Ghanem and Westermark (1) and later modified by Gosztonyi and Walde (2). 9 limited number of compounds were studied and few, if any, of the specific activities reported were demonstrated to be at radiochemical purity. However, this approach had the advantage of being poteutially nondestructive, rapid, and inexpensive. Therefore, we sought to design a new system by which this approach could be systematically evaluated. The details of this method and the factors which determine the extent of tritium incorporation into radiochemically pure substrate are reported herein. These studies indicate that this is not an “accelerated” Wilzbach method and that tritium-hydrogen exchange is unrelated to tritium decay. Evidence for an exchange reaction initiated by tritium atoms and involving free radical intermediates is presented. Finally, biologically active [%]adrenocorticotropin has been prepared with a specific activity 1000 times greater than that previously obtained by the conven tional Wilzbach technique (3).

MATERIALS ASD METHODS

Clzemicals-Trit,ium (as 3Ha) was obt,ained from Oak Ridge National Laboratory; acridine (Eastman Kodak) was purified by filtration through silica gel in benzene followed by recrystallization from hexane; 9-methylanthracene (Aldrich) was recrystallized from ethanol; anthracene and naphthalene (East- man) ; xanthcne (Aldrich), each amino acid and androstencdione

(Schwarz-Mann) ; adrenocorticotropin (natural porcine, Up- john; synthetic, Ciba, Ltd., Basel, Switzerland) ; and peptides

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REACTION AREA

‘I i’

FIG. 1. Reaction system. A, connection to main vacuum line; II, Htatham pressure transducer, 0 to 5 p.s.i.; C, nonreturn glass valves; D, llranium tritiide storage; E, microwave cavity; F, sample area; G, copper oxide oven; H, sample dewar; I, Teflon- coat,ed magnet and external magnet.

(Schwarz-1Iann) were used without further purification. Iso- caproic acid (Mallinkrodt Chemical) was converted to its sodium salt on I)owex 1 aud benzoic acid (National Bureau of Stand- ards) was recrystallized from water. Silica gel thin layer chromatography plates were obtained from Eastman Kodak. Sepha- drs G-25 Medium, G-50 Medium and sulfoethyl-Sephadex were obtained from Pharmacia, Ltd., the latter utilized as described by Island et al. (4). Whatman carboxymethq-1 cellulose was obtained from Reeve Angel, Inc. and used as suggested by the manufacturer and by Hussa and Winnick (5). Bio-Gel P-2, Dower; 5OW-X8 and AG50W-X2 were obtained from Bio-Rad. &211 solvents were purified by standard distillation methods appropriat,e to the solvent.

Labeling Apparatus-Essential equipment for the labeling process includes the microwave cavity (Opthos Instruments, Rockville, Maryland), a Raytheon Microwave generator (PGM IO-XI, 2450 MHz), and a Bendix “MicroMatch” wattmeter, model 725.3.

A diagram of the vacuum system in which all experiments were carried out is seen in Fig. 1.l In the main vacuum mani- fold (A), an oil diffusion pump is used to avoid trace mercury contamination &hin the reaction area. All stopcocks are Teflon IYith silicon O-rings. Pressure is monitored by a pressure transducer (B), calibrated with a fused quartz pressure gauge (Texas Instrument Co.). Gases are circulated by the alternating movement of a Teflon-coated magnet within true-bore glass tubing (T). Construction is similar to a device reported by Leake (6). The cxtcrnal magnet movement is controlled by a nitrogen driven piston with the stroke length regulated by alternating microswitches and the rate is regulated by nitrogen release valves. Unidirectional flow is facilitated by nonreturn

1 Detailed drawings and a set of instructions for the labeling apparatus have been prepared and will be available upon request from the allthors.

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glass valves (C). Tritium (100 Ci) is stored (D) as uranium tritiide as described by Herber (7). The microwave generator forward power output and reflected power is constantly monitored during an experiment and the microwave cavity (E) can be placed at variable distances from the sample area (F). Excess tritium is converted to 3Hs0 by passage through a copper oxide oven (G). The temperature of the sample is controlled by addition of liquid nitrogen or Dry Ice mixtures of known temperature to the sample dewar (H). The total volume of the reaction system is approximately 50 ml and, at a pressure of 4 mm Hg, less than 1 Ci of tritium is required. “Apparent tritium atom concentration” was determined at the sample area by the platinum hot wire method of Wood and Wise (8). At hydrogen pressures of 4 mm Hg and using the geometry in Fig. 1, the response, i.e. the decrease in microwatts required to maintain constant wire resistance, was linear with respect to the square root of the power between 10 to 70 watts forward power of the microwave generator.

Procedure-Dry helium is circulated through the system before introducing the sample to reduce water accumulation on the glass walls. Samples are introduced into the reaction area (Fig. 1) on a glass reaction tray (10 X 8 X 1 mm). When possible, compounds are melt,ed on the tray to insure a uniform and reproducible distribution. Otherwise, samples are added as a dry powder or, in the case of microgram quantities of polypeptides and proteins, in an aqueous solution. Water is then removed by lyophilization within the reaction system. A sample mass of 10 mg or less can be labeled by t’his procedure.

When appropriate, liquid nitrogen is added to the sample dewar after partial evacuation to prevent sublimation. Evacua- tion is then continued for at least 1 hour prior to the introduction of tritium in order to maximize removal of any volatile material adsorbed on the sample or on the walls of the reaction system. Tritium is introduced to the desired pressure by heating the uranium reservoir (350”). The plasma discharge is initiated and maintained by a forward power of approximately 20 watts. The cavity is tuned to reduce reflected power to zero. At the end of the reaction, the t,ritium is immediately removed from the system by opening the stopcock leading to the copper oxide oven. The sample is allowed to return to room temperature and evacuation is continued for an additional hour to reduce tritium adsorption on the sample and tray. This step reduces the amount of tritium associated with the sample by as much as 90% and greatly facilitates the early purification procedures.

Purification and Demonstration of Radiochemical Purity- Purification is accomplished by several general methods: (a) removal of labile tritium, usually after addition of unlabeled carrier; (b) recrystallization or sublimination or both; (c) chromatography; and finally (d) when possible, derivatization. The chemical and physical properties of the individual compounds studied dictate t,he extent to which each of the above procedures is utilized to demonstrate radiochemical purity. Reaction parameters were studied using benzoic acid and g-methylanthracene as model compounds. Radiochemical purity was estab- lished for these two compounds by a combination of all of these procedures (see “Results”). Each of the other aromatic compounds studied was purified primarily by procedures a and b, followed by silica gel, florisil, celite or alumina chromatography as final purification steps nhen indicated. Each specific activity reported represents a minimum of eight purification steps with constancy within &5y, for the last three steps.

Androst-4-en-3,17-dione was purified by recrystallization, paper and thin layer chromat,ography. Derivatization was


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accomplished by reduction with potassium borohydride to testosterone, acetylation of the testosterone with acetic anhydride and saponification of testosterone acetate in methanolic sodium hydroxide, using a chromatographic step before and after each reaction to purify each derivative. Isocaproic acid was purified by paper and thin layer chromatography, Dowex 50 chromatography as the sodium salt, sublimation of the acid, recrystallization and formation of its anilide. After removal of labile tritium, each amino acid was chromatographed twice on AG50W-X8 in appropriate citrate buffers (9) and finally rechromatographed with radiochemically pure ‘ZC and 14C-labeled amino acid standard to demonstrate t)he radiochemical purity of the 3H-labeled amino acid. Peptides were chromatographed on Dowex 5OW-X2 in pyridine acetate buffers (10) until constant specific activity was obtained. Sephadex G-25 Medium, G-50 Medium, Bio-Gel P-2, microgranular and fibrous carboxy methylcellulose and sulfoethyl-Sephadex are used in the purification of [3H]ACTH.2 Immunoassay and bioassay methods have been previously reported (11, 12) and the details of ACTH labeling, purification, biological, and, immunological characterization are reported in another paper.3

RESULTS

Labeling and Demonstration of Radiochemical Purity

When milligram quantities of compounds were labeled, no gross destruction could be demonstrated, either by weight or by ultraviolet absorption. However, for each compound studied, the crude preparation after the removal of easily exchangeable tritium contained small quantities of high specific activity impurities which represented from 500/, to 90% of the residual tritium. Purification of 9-methylanthracene labeled under standard conditions (see below), but omitting the microwave discharge, resulted in a preparation with a final specific activity after recrystallization to constant specific activity of no more than 0.57, of that obtained with the microwave discharge. However, silica gel column chromatography showed that the sample was not radiochemically pure and further chromatographic purification was not possible due to the low counting rates. Therefore, the contribution of decay-induced labeling (“Wilzbach”) to the tritium-hydrogen exchange observed in the presence of the microwave discharge was negligible.

In most instances, at least three physical or chemical methods of purification were required to achieve radiochemical purity. Two examples are given in Table I. The three derivatives of benzoic acid were prepared separately from aliquots at the third hexane recrystallization. Fractions A and B of the methyl benzoate represent. the two halves of gas-liquid chromatography peaks and all the tritium associated with the derivative was re- covered in these two fractions. Though not demonstrated, it is likely that the final decrease in specific activity was due to aromatic tritium exchange occurring during derivative formation. Purification of 9-mcthylanthracene required recrystallization, sublimation, and silica gel column chromatography to reach a specific activity that remained constant after further chromatography on florisil and celite, using reverse phase partition chromatography with the latter support (Fig. 2). Serial tube specific activities across the peak in each case of the final chro-

2 The abbreviations used are : ACTH, adrenocorticotropin; SE-Sephadex, sulfoethyl-Sephadex; LH, luteinizing hormone; FSH, follicle-stimulating hormone.

3 W. C. Hembree, S. Lieberman, P. E. Zimmering, and A. P.

TABLE I

Radiochemical puriscation of tritium-labeled benzoic acid and Y-melhylanthracene

Benzoic acid, 4.0 mg, was labeled under standard conditions ex- cept for a 15-min reaction time. Crystallization was performed following addition of 1.0 g of benzoic acid and lyophilization. Benzoic acid derivatives were prepared by routine methods (13). Preparative gas-liquid chromatography using a column (20 feet X g inch) packed with 25% diethylene glycol succinate on hexa- methyldisilazane treated Chromosorb P (40 to 60 mesh) at 155”. Chromatography of H-methylanthracene is described in Fig. 2.

Procedure Specific activity

Benzoic acid Water recrystallization 1 Water recrystallization 3 Hexane recrystallization 1 Hexane recrystallization 3 Material for derivatization Benzamide Benzanilide Methyl ester

GLCe peak 1st half (A) GLC peak 2nd half (U)

Y-Methylanthracene Crystallization 1 Crystallization 4 Sublimation Crystallization 6 Crystallization 7 Chromatography on

Silica gel Florisil Celite partition

GLC, gas-liquid chromatography. i

20.5 10.9

8.0 7.6 6 .6 3.6 3.3

1.62 1.66

900 340

88 48 42

36 3G 36

NUMBER OF HOLD- BACK VOLUMES

FIG. 2. Specific activity calculated by reverse isotope dilution after addition of 1 g of 9.methylanthracene to the crude preparation immediately after the reaction. Silica gel and Florisil chromatography used hexane as eluting solvent. Celite partition chromatography was performed Ilsing a stationary phase of tol- uene-isooctane (1: 1) and mobile phase of methanol-Tr-propyl alcohol-water (4:1:0.5).

matographies were also constant. In the case of oleic acid and amino acids, serial specific activities were based up011 tritium- carbon-14 ratios, using commercially obtained standard car-

Wolf, manuscript in preparation. bon-14 compounds which were purified prior to use. The isotope


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TABLE II

‘I’ritium labeling of various organic substrates

Each specific activity reported represents the highest value obtained after at least three purification steps during which there was no significant change and which utilized at least two qualita- tively different analytical techniques. Oligopeptides were purified on Dowex 5OW-X2 using pyridine acetate buffers and mass was determined as described by Hirs (14). Details of ACTH purification are described in another paper.3 Luteinizing hormone (2 rg) and follicle-stimulating hormone (2 pg) were each eluted from the reaction tray with 0.1 m phosphate buffer pH 7.4,25 mg of albumin were added and samples were lyophilized twice. Specific activity was calculated from the activity in the initial peak off Sephadex G-50 fine column (40 X 1.2 cm), assuming 1007, mass recovery.

Compound PWW PCV mCi/ mole XllXlle Ci/mmole

Naphthalene Androstenedione 9-Methylanthracene.. Acridine Xanthene. Anthracene................ Oleic acid. n-Valine. Benzoic acid. Tsocaproic acid (Na+ salt). L-Leu- L-Tyr- L-Leu L-Ile- L-Ile- n-Val L-Val- L-Ala- L-Ala- L-Phe. ACTH (adrenocortico-

tropin). LH (luteinixing hormone). FSH (follicle-stimulating

hormone).

0.08 0.10 0.21 0.25 2.31 2.91 3.0 9

30 167 261 548

4,600

70-3,200 44,300

51,000

10 28 40 45

423 515 832

1.1 3.8

19.4 0.1 0.2 2.0

0.3-14.5

effects noted during Dowex 5OW-X8 chromatography with

sodium citrate buffers were identical for commercially obtained tritiated standard amino acids and experimentally labeled amino acids. Repeated chromatography was required to demonstrate radiochemical purity in each case. Constant specific activity of ACTH and other peptides was obtained after a single molecular sieve chromatographic step followed by one ion exchange step on CM-cellulose (5) or Dowex 5OW-X2 (10). Further purification of ACTH on SE-Sephadex and by polyacrylamide gel elec- trophoresis confirmed radiochemical purity.

Speci$c Activity of Compounds Studied

Table II lists some of the compounds labeled by this method. The specific activities vary widely between the groups of compounds and the values obtained did not correlate with the number of exchangeable hydrogens. With the exception of ACTH, LH, and FSH, the amounts labeled in each case ranged between 3 and 5 mg. The per cent of hydrogen exchanged varied loo-fold for the aromatic compounds and was IO-fold less than both aliphatic and aromatic amino acids and loo-fold less than isocaproic acid. Evacuation of the system for at least 1 hour prior to cooling of the sample preceded the tritiation reaction with those compounds which resulted in specific activities greater than 100 MCi per mg. In contrast, for all compounds with final specific activities less than 100 r/.Ci per mg, liquid nitrogen was added to the target dewar during evacuation at a helium pressure of approximately 100 mm Hg in order to prevent sublimation.

Most striking was the high specific activity of the peptides studied. Partial and complete hydrolysis of these compounds

5000

--E ,”

; 4000

5 k $ 3000

z a

z 2000

5 L ,: 1000

;;; -

FSH

fxi [3~1-~~~

0 UNLABELED FSH

50 too 200 300

FSH ADDED

LH ts L3~I-~H

0 UNLABELED LH

IO 50 100 200 300 ng LH ADDED

FIG. 3. Immunological activity of tritium-labeled luteinizing hormone and follicle-stimulating hormone. hFSH, 2 rg, (LER 1366) and hLH, 2 pg (LER 960) were each labeled using standard reaction conditions (see text). Labeled and unlabeled samples were treated in an identical manner prior to addition to assay tubes. These materials were also used for iodination. Incu- bation conditions for the radioimmunoassay are those utilized routinely by Dr. M. Warren of the Gonadotropin Laboratory in the Department of Obstetrics and Gynecology of Columbia Uni- versity, using a modification of the methods described by Midgely (15, 16). 1311 cpm in the double antibody precipitate for each protein are plotted on the ordinate and the mass added, assuming 100% recovery, on the abscissa. In the radioimmunoassay, the mass of immunoreactive FSH or LH is inversely related to the 1311 cpm remaining in the antigen-antibody precipitate.

followed by peptide or amino acid chromatography or both on Dowex 50 confirmed that at least SOG/, of the tritium was associated with the component amino acids.4 The extremely high specific activities reported for FSH and LH represent values obtained after only part,ial purification by lyophilization and chro-

matography on Sephadex G-50 Fine. It is possible that much of the remaining tritium is associated with the molecule as tightly bound water or other slowly exchangeable tritium. How- ever, when radioimmunoassay of these partially purified tritiated preparations was performed in parallel with unlabeled preparations, treated identically to correct for losses, it was found that the tritiation procedure was not associated with any significant loss of immunologic activity. In Fig. 3, the amount of Y-

4 W. C. Hembree and R. E. Ehrenkaufer, unpublished data.


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IO Lo

b 9

38

w k 7

2 a 6

IO 50 100 200 300

ng[3H]-LH/TUBE

FIG. 4. Antibody binding of 3H-labeled luteinizing hormone. Antigen-antibody precipitates from the radioimmunoassay of [3H]hLH were counted for tritium background 8 weeks after addi- t,ion of the /“]I]hLH tracer to ensure complete 1311 decay. Trit- ium background for control tubes with no added unlabeled hLH was 142 cpm, which ~4.~1s subtracted from the values.

gonadotropins which is antibody bound is the same in the pres-

cnce of equal amounts of either tritium-labeled or unlabeled molecules. In addition, [W]LH was shown to bind to anti-LH antibodies (Fig. 4). Antibody binding of [3H]FSH was not studied. We report these values here only to illustrate that complex proteins may be labeled in a manner similar to that of ;1CTH without structural alterations significant enough to alter the immunologic properties. Further chemical and biologic cliaracterizat~ion of these tritiated proteins is now in progress.

Parameters of Labeling Reaction

9-hlcthylanthracenc was utilized to study each of the factors which determine the optimum reaction conditions because its purification is relatively rapid and radiochemical purity can be completely characterized. Parallel studies with ACTH or benzoic acid are presented in some cases. On the basis of prelim- inary studies with bcnzoic acid, standard reaction conditions are defined as: (a) reaction time of 5 min; (b) tritium pressure of 4 mm Hg; (c) microwave discharge power of 20 watts; (d) sample cooling lvith liquid nitrogen; (e) cavity distance of approximately 10 cm from plasma center to sample; and (f) tritium gas cycling at a piston rate of approximately 120 to 150 cpm. Each parameter was varied as indicated.

Reproducihlity-Table III demonstrates the reproducibility of the labcling reaction with 9-methylanthracene under various experimental conditions. The mean specific activity obtained in four preparations labeled at approximately 6-week intervals was 56 PCi per mmole with a range from 54 to 59 PCi per mmole (Experiments 7 to 10). The difference in specific activity between paired experiments performed the same day was 8% (Experiments 1 to 7). Similar reproducibility was obtained for the labeling of anthracene (Experiments 11 and 12) and of several pcptides. Therefore, differences in specific activity between experiments of at least 207, were considered significant. This

TABLE III

Reproducibility of tritium labeling and e$ect of reaction time

Experiments 1 to 5 were conducted using the reaction system geometry as shown in Fig. 1, and Experiments 6 to 12 employed the same geometry between the microwave discharge cavity and the sample area as that shown in Fig. 6. This geometric difference resulted in a 30yC decrease in the final specific activity of preparations labeled under standard conditions (Experiment 2 versus

Experiments 7 to 10). St,andard reaction conditions applied, ex- cept as noted. Each preparation was purified by recrystallization and sublimation until the specific activity remained constant within 57, during three consecutive steps. Relative specific activities are given for those preparations (Experiments 1 to 5) in which reaction times were varied. In Experiment 3, liquid nitrogen remained in the sample dewar during the evacuation and reintroduction of tritium. Experiments 7 to 10 were performed in November, &lay, Allgust, and October, respectively, of the same year.

Paired experiments 9.Methylant,hracene

Single experiments 9.Methylanthracene

Anthracene

9 10 11 12

Conditions

1 X lmin 43, 47 1 X 5 min 90, 75 3 X 5 min 79, 84 1 X 15 min 72, 68 1 X 90 min 61, 73 2mmTz 46, 48 Standard 59, 54

Standard 54 Standard 56 Standard 57 Standard 330 Standard 320

Specific activity

Relative specific activity

1.0 18 1.8 1.6 1.5

should be vielvecl as a conservative estimate of significant differences.

Time-Maximum attainable specific activity of g-methylanthracene (melted on the sample tray) was reached in 5 min (Table III). The specific activity doubled between 1 and 5 min. No further increase was achieved by reaction times up to 90 min or by repeated 5-min reactions, between which the system is completely evacuated and fresh tritiurn introduced. In preparations labeled for longer time periods, a greater amount of tritium was associated with the crude material and more extensive purification was required to achieve constant specific activity.

Pressure-Studies of the effect of pressure upon the final specific activity, using both benzoic acid and 9-methylanthracene, demonstrate a progressive decrease when tritium pressure FLX- ceeded 4 mm Hg (Table IV). The maximum pressure at which the plasma could be maintained was 50 mm IIg. Pressures less than 2 mm Hg could not bc studied in a comparative manner because of the extensive light exposure of the sample. A tritium pressure of 2 mm Hg lvitll or without added helium did not significantly alter the final specific activity from that of the control at 4 mm Hg.

Power-Tritium incorporation into crude material after removal of easily exchangeable tritium increased as the microwave generator po\Ter was increased. A linear relation between the initial specific activity of partially purified compound and the


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TABLE IV

Tritium incorporation us a fun&on of pressure

All experiments performed as outlined in Table III.

TABLE V

SpeciJic activity of tritiated ad~encxorticotropin

Amount labeled Specific activity*

TfiiT pCi/WLg

5.0 8

5.3 57 5.1c 90 5.0 65 7.5 67 2.0 50 0.2 89~~ 0.02 308” 0.02c 550” 0.002c 1434” 4.06 68

Reaction condition9

Standard 25 min 15 min 15 min-80 watts 3 X 5 min--80 watts 15 min 15 min 15 min 15 min 15 min Standard

Compound labeled Experiment Relative specific

activity Condiths

Standard 12 mm Tz 25 mm Tz 50 mm Tz Standard 2 mm TL 2mmTz 2mmHe 29 mm Te

Benzoic acid

9-Methylanthracene

1.00 0.32 0.18 0.02 1.00 0.87

1.00 0.29

a Standard conditions used 4 mm Hg of 3Hz, liquid nitrogen, 20 watts forward power and 5 min unless otherwise noted.

b Specific activity after chromatography on Bio-Gel P-2, G-50 medium Sephadex and carboxymethyl cellulose. Rechromatog- raphy of preparations on CM-cellulose and SE-Sephadex was not associated with a change in specific activity.

c Bioassay performed in vivo by the method of Purves and Sirett

(12). d Specific activity calculated by reverse isotope dilution after

Bio-Gel P-2 and CM-cellulose chromatography.

1 I I I I II I I I I I I 25 - INITIAL SPECIFIC 24- ACTIVITY vs Pi” _

23-((l)

aa %

22-

E 21-

-2

E

20- IF/;

19-

18-

17-

4 5 6 7 8 9 IO

P”2 (WATTS)

FINAL SPECIFIC ACTIVITY vs P”2 I (b)

u

E \ y 9c

7c . 4 5c

3c

IC

TABLE VI

Tritium labeling as a function of modi$ed geometry

Effect of tritium flow and cavity distance upon final specific activity of 9-methylanthracene. Altered geometric design shown in Fig. 6. All experiments were performed within a 36.hour period. Experiment 1, a and b were the initial and final experiments, respectively, of this series. Pump rate was 135 strokes per min when cycling. Tritium flow was determined by the positions of

I I I I I Ii 5 6 7 8 9 IO

pl/2

FIG. 5. Relation of microwave power input to initial and final specific activity of 9-methylanthracene. Initial specific activity (a) was calculated by reverse isotope dilution after removal of labile tritium by ethanol exchanges. Power was read directly as forward power when the microwave cavity was tuned to 0 watts reflected power. Final specific activity (b) was calculated after 8 to 10 purification steps for each preparation.

stopcocks A, E

Experi- ment

Specific activity

C, and D (Fig. 6).

Zavity dis-

tance Stopcock position

I(

PCV mnzole

3.7 4.2 3.0 1.2 0.8 0.5 0.3

‘l?L

8.8 8.8 8.8

14.0 14.0 14.0 14.0

square root of the power (17) was noted (Fig. 5~). However, increased microwave power did not increase the specific activity

of radiochemically pure compound (Fig. 5b). The increased

proportion of high specific activity impurities associated with an

increase in power markedly increased the difficulty in demon- strating radiochemical purity. “Apparent tritium atom co11-

centration” was also linearly related to the square root of the

power. Temperature-Most experiments were performed with the

samples cooled by liquid nitrogen because of the possibility of sublimation of the compounds studied. Using ACTH, a single

study was performed during which no attempt was made to cool

the sample. There was a 5-fold increase in t,ritium incorporation into the crude preparation after removal of labile tritium. How-

ever, gel filtration demonstrated a 10ryO loss of ACTH mass and

the presence of multiple high specific activity impurities of varying molecular weight which were not. present uhen ACTH was

cooled by liquid nitrogen during the reaction. The final specific

activity after multiple chromatographies was approximately twice that obtained under control conditions. However, radio-

chemical purity could not be demonstrated by as many as 10

chromatographic steps. J4ass-When the mass of 9-methylanthracene labeled was

decreased from 4.3 mg to 0.38 mg, there n-as a B-fold increase in

Cycling Cycling No cycling Cycling No cycling No cycling No cycling

A, C, D, open; B, closed A, C, D, open; B, closed A, C, D, open; B, closed A, C, D, open; B, closed B, C, open; A, D, closed B, C, D, open; A, closed A, B, C, D, closed

la lb 2 3 4 5 6

the final specific activity. A similar effect leas noted with *%CTH

labeling (Table V).

Geometric Characteristics

Table VI lists the final specific activities of compounds under

altered geometric conditions designed to test the importance of forced tritium flow, diffusion, and cavity distance in the labeling

reaction. Fig. 6 outlines the geometry of the modified system,

designating the points at which t,he vacuum line could be closed by stopcocks. The conditions under which each experiment was

conducted and the stopcock positions are also shown in Table VI.

The basic alteration in over-all geometry resulted in a lo-fold decrease in specific activity obtained under control conditions

with a piston rate of 135 cpm (Experiment 1, a and b). Under t,hese conditions, omitting forced cycling of the gas by the piston


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FIG. t?. Effect of tritium flow and cavity distance on the labeling reaction. Modified geometric design of the reaction area. Teflon stopcocks (A, B, C, U) introduced to modify tritium gas flow (Table VI). Forced tritium flow bv internal niston numn from A to 11. Microlvave cavity distance was placid at 8:s cI$ (I) and 14.0 cm (ZZ) from the sample tray (ZZZ). Light traps were introduced between the microwave cavity and sample tray in this design.

(Esperiment 2) resulted in a 25% decrease in specific activity. The effect, of tritium diffusion upon the labeling reaction was illustrated by three experiments in which varying restrictions were placed upon gas flow. When circular flow by diffusion was eliminated (Experiment 6), the specific activity was only 7?> of the control. When circular flow was unrestricted, small increases in specific activity resulted (Experiments 4 and 5). When the microwave cavity distance from the sample was increased from 8.8 cm (I) to 14 cm (II) (Fig. 6) the specific activity was decreased by 7OyG. The hot platinum wire technique of Wood and Wise (8) demonstrated that the same increase in cavity distance was associat.ed with a 757, reduction in apparent tritium atom concentration. Using the original geometric &sign, the effects of forced cycling and cavity distance were much less marked.

Adrenocorticotropin Labeling

Table V lists the specific activities of ACTH labeled under various conditions. Bioassay of the indicated preparations showed full biological activity. A small, but significant, increase in specific activity was achieved by serial 5-min reactions. How- ever, the most important increases in specific activity could be obtained by decreasing the mass labeled. When 10 pg or less were labeled, the specific activity achieved was such that approximately 5Oc/, of the nloleculcs contained tritium, assuming not more than 1 atom per molecule. Synthetic ACTH (Ciba), when labeled and purified, completely displaced 1311-ACTH from ACTH antibodies. In vivo uptake of [3H]ACTH by rat adrenals was approximately 1 y0 of the injected dose at 15 min and its lrptake was inhibited by both csogcnous unlabeled ACTH and clldogenous ACTH. Details of the methods and results are presented in another paper.3

u1scuss10s

WC have described results obtained using microlr ave discharge activation of tritium gas to effect tritiun-hydrogen exchange in a nidc variety of organic compounds. These results establish the broad applicability of this technique as a method for obtaining high specific activity tritium labeled molecules useful as biological tracers. The essential novel features of our system

are: a closed circular flow system, recycling of tritium gas by an internal pump, a microwave discharge cavity positioned at a sufficient distance from the sample to prevent exposure to high electron and ultraviolet flux, and a removable target with which temperature, sample size, and physical state can be precisely controlled. This design has permitted us to optimize the labeling reaction by studying each variable thought to be important in obtaining high specific activity and by minimizing destruction and impurity production.

These results cannot be directly compared to those previously reported (1, 2) utilizing microwave discharge activation since, in our studies, standard reaction conditions employed substrate cooling with liquid nitrogen. In addition, the geometry of this system differed significantly from that utilized by others. How- ever, there are a number of points of general agreement with the results of Gosztonyi and Walde (2). These include the effect of mass of substrate labeled, time of exposure and tritium pressure upon the final specific activity obtained of the purified subst,rate. In contrast, we found no relationship between the microwave generator power and the final specific activity obtained, utilizing 9-methylanthracene as a model compound. However, increased power was associated with an increase in the apparent tritium atom concentration at the target area. Therefore, increasing the concentration of the tritium species presumably responsible for the exchange reaction resulted in an apparent increase in the production of high specific activity impurities, rather than in a net increase in tritium-hydrogen exchange in the substrate itself.

The specific activities of amino acids reported by Gosztonyi and Walde (2) were consistently higher than that reported here. Differences in purification may account for this and other differences. The criterion for radiochemical purity reported by these authors was the demonstration of a single tritium peak with chromatographic mobility similar to or identical to that of the pure compound. However, in our hands, most compounds at this stage of purification were not radiochemically pure. Serial specific activity determinations across the peak or simultaneous chromatography n-ith ‘Y-labeled compounds or both consistently demonstrated radiochemical heterogeneity. These impurities could be resolved by appropriate rechromatography or derivative formation with rechrornatography or both. The quantitat,ive significance of these impurities varied, accounting for from 20% to 90% of the tritiurn in the partially purified preparations.

The total amount of tritium associated with organic material never exceeded 2% of that in the system and was usually less than 0.2%. Therefore, we would not have expected and did not ob- serve any significant changes in pressure during the reaction. However, during the room temperature reaction, the pressure increased and the color of the tritium plasma was altered, suggest- ing the production of volatile impurities. The reasons for the pressure changes noted by Gosztonyi (18) and Ghanem (19) are not clear.

Characteristics of the exchange reaction(s), the proportion of high specific activity impurities produced, and the difficulty with which radiochemically pure preparations could be obtained varied considerably as the labeling conditions were varied. Require- ments for purification also varied considerably from compound to compound. Nevertheless, several general characteristics of the tritiurn-labeled preparations were evident from our studies. First, there was no detectable mass destruction of the substrate. Second, the amount of tritium incorporated into purified substrate ranged between 5 and 25cj0 of the total tritium in the crude


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preparation. This is in contrast to Wilzbach labeling in which there is both significant sample destruction and production of high specific activity impurities which usually account for more than 997;) of the total fixed tritium (20). Third, in no case was it possible to demonstrate radiochemical purity without repeated chromatography, though constant specific activity could be achieved by recrystallization prior to chromatography.

Both the structure of the substrate and the labeling conditions rnay determine whether one or more of several possible reactions predominate. An example of the importance of substrate properties is illustrated by differences between the specific activities obtained when labeling naphthalene and anthracene. Both the relative specific activities and the relative rates of methyl radical addition-elimination reactions with these compounds are the same and both are proportional to the localization energies of the compounds (21). The degree to which the system is free of oxygen and water may also determine the extent to which net tritium-hydrogen exchange occurs. For cxa,mple, peroxy radical formation could occur at sites of hydrogen abstraction instead of radiral addition. Bennett et al. (22) have reported that hydrogen abstraction by hydrogen atom bombardment of related alkenes in their rotating cryostat system could be virtually eliminated by removing all water and oxygen from the system.

Under labeling conditions which result in low specific activities, maneuvers expected to increase tritium atom concentration were associated with increases in specific activity. However, using the standard geometry and reaction conditions described, specific activities were not increased by a 16.fold increase in reaction time, by a doubling of tritium content at constant pressure, or by doubling of apparent tritium atom concentration by increases in microwave power. Therefore, for each compound, there ap- peared to be a maximum specific activity at which the exchange reaction was relatively independent of the total exposure to reactive tritium species. On the other hand, the total tritium incorporation into the unpurified sample was augmented by increased substrate exposure time. This suggests that a change in local conditions occurred during the reaction which was unfavorable for the cschange reaction and favorable for the formation of impurities. Although we have no measurement of temperature during the reaction, increased temperature could result from the poor dissipation of the energy produced by tritium recombination on the substrate surface (8) during the prolonged exposure time. A1nother factor which could limit the maximum attainable specific activity for each compound is the limited penetrat,ion of reactirr tritium species into the solid matrix of the substrate (23). This hypothesis is supported by the marked increases in the specific activity obtained by decreasing substrate ma.ss. Furt,her attempts to control the reaction temperature and the surface exposure are now under iiivestigatioii.

Thcsc studies do not establish the mechanism by which the tritium-hydrogen exchange reaction occurs. Tritium atoms were not directly measured and all reaction products were not characterized. However, several lines of indirect evidence suggest a free radical mechanism involving tritium atoms. First, with the geometry of the system, tritium atoms formed in the microwave discharge would be the major species reaching the target area (24). Second, changes in the specific activity of 9-meth?-lallthracene could be quantitatively correlated with changes in the apparent tritium atom concentration at the target area. Third, a stutly of the distribution of tritium in labeled allthracene5 revealed a greater percentage of tritium at the sites

5 11. E:. li:hrenkaufer, unpublished data.

of highest free valence values (25). Finally, hydrogen atom reactions with a variety of compounds have been shown to pro- duce ESR signals or formation of free radicals characteristic of the compound studied or both (26-29). Hydrogen atoms have been shown to be responsible for addition reactions at low tem- peratures (77°K) (30) and are also responsible for the free radical yield found in the experiments of Snipes and Schmidt (23), as well as Bennett et al. (22). The free radical yield described by Holroyd et al. (31), resulting from hydrogen atom reaction with polyamino acids and proteins at room temperature, would more than account for the observed specific activities in our system if each site were labeled.

A possible reaction mechanism, consistent with our data is the following:

R-H + 3H. 4 1~. + VI-H (1)

R. + 3H. -+ R-3H (2)

R. + 3Hz + R3H + 3H. (3)

Hydrogen atom abstraction (1) would result in the formation of free radicals at positions and with half-lives characteristic of each compound. Subsequent incorporation of tritium by recombination with %’ (2) or by abstraction from YTz (3) would be a function of the radical (R’) stability, of the tritium atom concentration and of surface availability of R’. The exchange reaction proposed by Ghanem (19) (using microwave discharge activation) involved an unstable intermediate and would not be associated with free radical formation. It cannot, therefore, be the sole mechanism responsible for tritium-hydrogen exchange. However, such a mechanism cannot be ruled out by our findings.

The relative specific activities in the series of compounds studied conform to no clearly predictable pattern and vary over a lO,OOO-fold range. In general, labeled aromatic compounds have a lower final specific activity than the aliphatic compounds. These differences might be explained if aromat,ic reduction occurred more frequently than did recombination. The absolute specific activities obtained for most compounds are similar to those reported by Wilzbach methods (20). However, lack of substrate destruction and short reaction times are important advantages.

The single most important advantage of this method is its proven applicability to the labeling of complex polypeptides of biological intcrcst and its potential use for labeling proteins at high specific activity. Radioiodinated polypcptides and proteins have served adequately as biological tracers in certain selected cases. IIowcvcr, advances in protein chemistry and biochemistry have greatly increased the need for labeled tracers with long half-life isotopes in which the chemical and biologic prop- crties of the molecule were not significantly altered. Previous attempts to obtain tritiun-labelcd pcptides and proteins have met with varying success but no approach has been generally applicable (32~38). The promising results of Ghanem and Wcstermark (1) suggested the suitability of microwave discharge activation of tritium gas as a method for labeling proteins. However, the particular technique reported by these authors was unsuitable for both polypeptides and proteins because of the inability to control either temperature of the substrate or exposure to ultraviolet light. In addition, static systems were used in which the reactive species from the discharge could reach the substrate by diffusion alone. Labeling experiments utilizing the flow technique suggested by Westermark et al. (39) were not reported, and, as proposed, would have required large amounts of tritium to sustain the reaction for only as long as a


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few minutes. Holroyd et al. (31) utilized microwave discharge of the hydrogen gas to study free radical production on proteins. Free radical sites were labeled by introducing tritiated hydrogen sulfide. The specific activities obtained were low and the chemical characterization of the intact tritium-labeled molecules was incomplete. However, the mechanism of tritium labeling may be similar to that involved in our method.

The specific activity of Wilzbach-labeled ACTH previously reported by Nishizawa et al. (3) was 1.5 PCi per mg and evidence for the biological activity of the tritiated molecules could not be obtained. Using similar quantities of material, we obtained specific activities 100 times greater than that reported by these authors. However, when microgram quantities were used, specific activities were obtained which are 1000 times greater and are such that more than 50% of the molecules, statistically, contained tritium. The specific activity of these preparations and the quantities obtained are sufficient for numerous biochemical and physiological experiments. Resolution of most impurities is comparatively easy by gel filtration and by ion exchange chromatography. At least 90% of the tritium in highly purified preparations behaves chemically like ACTH. The int,rinsic variability of any bioassay precludes its use to establish that all tritium-labeled molecules are biologically identical to unlabeled ACTH. However, chemical and biological characterization of [3H]ACTH has confirmed the near identity of the labeled molecules.3

Characterization of the chemistry of the labeling reaction with various peptides and proteins may eventually enable us to pre- dict labeling characteristics of any peptide of known amino acid composition and sequence. In addition, this met’hod may per- mit the preparation of tritium-labeled peptides and proteins of unknown sequence which will be suitable for studies that require long half-life biological tracers.

GHANEM, N. A., AND WESTERMARK, T. (1960) J. Amer. Chem. sot. 82, 4432-4433

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NISHIZA~A, E. E., BILLIAR, R. B., KARR, J., AND EIIZ-NES, K. B. (1965) Can. J. Biochem. 43, 148991498

ISLAND, D. P., SHIXIZU, N., NICHOLSON, W. E., ABE, K., OGBTA, E., .~ND LIDDLP;, G. W. (1965) J. C2i~x. Endocr. 26, 975-983

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Wylie C. Hembree, Richard E. Ehrenkaufer, Seymour Lieberman and Alfred P. WolfCOMPOUNDS

of Tritium Gas: METHODOLOGY AND APPLICATION TO BIOLOGICAL A General Method of Tritium Labeling Utilizing Microwave Discharge Activation

1973, 248:5532-5540.J. Biol. Chem.

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A General Method of Tritium Labeling Utilizing Microwave Discharge … · · 2003-01-21A General Method of Tritium Labeling Utilizing Microwave Discharge Activation of Tritium Gas