CHROMATOGRAPHY OF ADRENOCORTICOTROPIC PEPTIDES

BY CHOH HA0 LI, ARNE TISELIUS, KAI 0. PEDERSEN, LENNART HAGDAHL, AND HANS CARSTENSEN

(From Department of Biochemistry, University of California, Berkeley, California, and the Institutes of Biochemistry, Physical Chemistry, and Physiology,

University of Upsala, Upsala, Sweden)

(Received for publication, August 16, 1950)

The preparation and properties of adrenocorticotropically active pep- tides (ACTH peptides) obtained from the pepsin digest of the protein hormone isolated from sheep pituitary glands have recently been described (l-3). The present communication concerns the results obtained by chromatographic separations of the active peptide mixture.

Paper Partition Chromatography-The apparatus used and the technique employed were similar to those described by Consden, Gordon, and Martin (4). Acetic acid (10 per cent)-butanol (in H20) was used as the solvent and was prepared as suggested by Partridge (5). Whatman No. 4 paper was used throughout. The peptide material was dissolved in water at a concentration of 20 per cent. A volume of 0.005 cc. (1 mg.) was applied on the paper with a micro pipette. Alanine was always applied on the same paper so that the alanine spot was used as the reference for the po- sitions of other spots on the paper.

As shown in Fig. 1, the peptide mixture (Preparation L-1669-CS) gave rise to a pattern in butanol-10 per cent acetic acid (in HZO) indicating the complexity of the mixture. However, six ninhydrin-reacting spots could be identified when low concentrations were used. In order to estimate the relative distribution of material and of biological activity, 50 mg. of peptides were chromatographed (50 spots; 1 mg. per spot) and the edge strip cut for testing. The positions of spots were numbered 1, 2, 3, etc., from the original. After correlating, each spot area was cut out and elu- ted with water. Elution was carried out according to Work (6). Ni- trogen was determined by the micro-Kjeldahl method. The adrenocorti- cotropic activity was estimated by the ascorbic acid depletion method of Sayers et al. (7) in hypophysectomized male rats. A dose of 1 y of N per 100 gm. of body weight of the animal was used throughout. The ACTH potency was estimated from a standard curve which was obtained from an extensive assay of a highly purified ACTH protein preparation. It may be seen in Fig. 2 that a plot of the dose against the ascorbic acid depletion gives a straight line relationship and that 0.2 y of ACTH depletes 40 mg. of ascorbic acid per 100 gm. of adrenal tissue. Since the bioassay was

317

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

318 ADRENOCORTICOTROPIC PEPTIDES

FIG. 1. Different amounts of ACTH peptide mixtures (Preparation L-166%CS) run on one-dimensional paper (Whatman No. 4) chromatogram; solvent, butanol- 10 per cent acetic acid.

b a

g a 180

.G $

.:i: //

.p 20 ’ ’ ” I t ,II I III

f 0.2 0.3 0.5 I 235 IO 20 30 50

a

0” Dose ACTH in Micrograms per

100 gms. Body Weight

FIG. 2. Standardization of a purified preparation of ACTH protein isolated from sheep glands by the ascorbic acid depletion method.

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

LI, TISELIUS, PEDERSEN, HAGDAHL, CARSTENSEN 319

employed as a guide for the location of activity, the assay results were obtained in many cases from two or three rats. Thus one cannot expect to utilize these data for exact quantitative calculation of the recovery of activity.

The results of a typical experiment are summarized in Table I. It is evident that most of the activity is in the immobile spot, which consists of approximately 30 per cent of the total original nitrogen. The eluate of the active spot was lyophilized; about 15 mg. of white powder were ob- tained from 49 mg. of the peptides. This material is called Preparation A, and should represent some purification from the original mixture.

When Preparation A, was chromatographed in phenol (in HzO), it gave

TABLE I Nitrogen and ACTH Activity Distribution in Di$erent Spots of ACTH Peptides

Obtained in Paper Chromatogram*

spot No.

1 2 3 4 5 6

Nitrogen

ntg.

2.20 0.40 0.23 0.11 0.11 0.23

Original amount of N Ascorbic acid lowering ACTH standard per 100 gm. adrenal equivalent

ger cent Y

34.4 11&t 5 6.3 1 (4) 0 3.6 5 (3) 0.2 1.7 7 (4) 0.2 1.7 0 (3) 0 3.6 33 (1) 0.3

* Butanol-10 per cent acetic acid as solvent; 49 mg. in forty-nine spots in the original (see Fig. 1).

t The figures in parentheses represent the number of rats; 1 y of N per 100 gm. of body weight was injected intravenously for the assay.

only one spot which was in the same position as the active spot reported earlier (3). On the other hand, this material in the carrier displacement experiment (see below) showed definitely more than one component.

Other solvents are being investigated to resolve Preparation A, into dif- ferent components. However, butanol-10 per cent acetic acid was used throughout the present investigation to analyze the fractions obtained by other chromatographic means.

Displacement Development Analysis-A number of experiments were per- formed by the displacement analytical technique of Tiselius (8). The runs were made in the interferometric apparatus of Tiselius and Claes- son (9), equipped with a 80 mm. cuvette. In a typical experiment, 30 mg. of peptides (Preparation L-1669-CS) were dissolved in 10.5 cc. of 0.10 N HCI and pressed into the column. The column was made with eight sections, each 785 c.mm., and with the mixers of Hagdahl (10). The adsorbent was a mixture of 1 part of Carboraffin Supra and 9 parts of

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

320 ADRENOCORTICOTROPIC PEPTIDES

Hyflo Super-Cel. The displacement development was effected with a 0.4 per cent Zephiran chloride’ (in 0.10 N HCl) solution. It was found that about 10 per cent of the material was not adsorbed and that the adsorbed material was totally displaced by the displacer. Fig. 3 gives the optical curve indicating that 65 per cent of the peptides was strongly adsorbed and was not displaced in the first 30 cc. It is also evident that the ab- sence of sharp steps in the later portion of the curve suggests that the strongly adsorbed material contains either very similar substances or too many different peptides for clear cut separation. An experiment with large amounts of the peptide mixture was carried out in order to determine the distribution of nitrogen and ACTH activity and to secure material for other analyses as well.

Ml.

FIG. 3. Displacement development of ACTH peptides (Preparation L-1669-CS). 39 mg. of the peptides were used; displacer, 0.4 per cent Zephiran chloride; adsorb- ent, Carboraffin Supra and Hyflo Super-Cel in a ratio of 1:9; column, eight sec- tions, each 785 c.mm. plus mixer.

250 mg. of the peptides were dissolved in 20 cc. of 0.10 N HCl and pressed into the charcoal column. The sections were packed with the wet adsorbent (Carboraffin Supra charcoal and Hyflo Super-Cel in a ratio of 1:3) in the manner previously described (10). The column consisted of six sections, each 1570 c.mm. and six sections, each 785 c.mm., with the largest sections in the top of the column. A solution of 1.0 per cent Zephiran chloride in 0.10 N HCl was used as the displacer. The column was driven with a pressure of approximately 4 kilos per sq. cm. Fractions (0.5 cc.) were collected in test-tubes with an automatic collector apparatus. A total of 312 fractions was obtained in this experiment; the last fraction has no peptide material. The peptide concentration was estimated by means of the ninhydrin calorimetric method (II), Kjeldahl nitrogen, or index of refract,ion.

As shown in Fig. 4, the curve is similar to that obtained when a smaller

* Zephiran chloride is an alkyldimethylbenzylammonium chloride with the fol- lowing formula: C$.H~CHZN(CH&RC1, where R = C&HIT to Cl~H37.

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

LI, TISELIUS, PEDERSEN, HAGDAHL, CARSTENSEN 321

quantity of the peptides is used (see Fig. 3). The fractions after Tube 232 were analyzed with ninhydrin for free amino nitrogen and by means of the Kjeldahl method for total nitrogen; some of the fractions were also assayed for ACTH activity in hypophysectomized rats. The results are summarized in Table II. It, is clear that the ratio of free amino nitrogen to total nitrogen varies from fraction to fraction and that the adrenal- stimulating activity occurs from Tube 255 onward to the end of displace- ment. Since bioassays were not, carried out extensively, it is difficult to ascertain the zone of maximum activity between these fractions. Never- theless, there is little doubt that the last twenty fractions have the highest activity, indicating that the ACTH peptide or peptides must be adsorbed strongly to the charcoal.

The distribution of nitrogen and ACTH activity in various fractions

210

B :z:

3. 60 a 40

0 0 20 40 60 230 250 270 290 310

Test Tube Number

FIG. 4. Displacement development of ACTH peptides (Preparation L-1669-CS). 250 mg. of the peptides were used; displacer, 1.0 per cent Zephiran chloride; adsorb- ent, Carboraffin Supra and Hyflo Super-Cel in a ratio of 1:3; column, six sections, each 1570 c.mm. plus mixer.

may be seen in Fig. 5. It is evident that eight peaks occur from Tube 253 to Tube 309. The highest activity was found in Tube 307 and it also had the highest concentration. On a nitrogen basis, it had an activity about 10 times that of the original material.

Perhaps it should be noted that Tube 237 contained definite ACTH ac- tivity, although it was very low. If the original peptide mixture con- tained only one active component, one would expect that the activity should be concentrated in a narrow displacement zone. The fact that the biological activity spreads out in many fractions suggests that the original material may consist of more than one ACTH active peptide.

One-dimensional paper partition chromatography, with butanol-10 per cent acetic acid as the solvent in Whatman No. 4 paper, was also carried out on these fractions. Aliquots of each fraction containing approxi- mately 1 mg. of peptides were dried in a vacuum desiccator before being applied to the paper. It, was found that the unadsorbed material and fractions in Tubes 1 to 230 gave very different patterns from others,

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

TABLE II

Nitrogen, Amino N, and Activity Distribution in Different Fractions of AC2’H Peptides (250 Mg.) Obtained in Di: QdC zcemhnt Development Chromatography

Fraction* or tube No.

U (unadsorbed material) A (Tubes l-55) B( “ 56-110) C( “ 111-165) D( “ 166-230) 237 239 241 243 247 249 251 253 255 257 259 261 263 265 267 269 271 273 275 277 281 283 285 287 289 291 293 295 297 299 301 303 305 307 309

- I

I

.-

Nitrogen hino Nt

mz.

5.2 0.22 0.15 0.21 0.10 0.036 0.051 0.063 0.078 0.132 0.165 0.165 0.186 0.123 0.186 0.198 0.174 0.189 0.171 0.209 0.190 0.197 0.202 0.099 0.204 0.187 0.182 0.187 0.192 0.176 0.213 0.192 0.183 0.192 0.207 0.211 0.206 0.247 0.311 0.165

w.

0.0168 0.077 0.0092 0.061 0.0155 0.074 0.0090 0.090 0.0096 0.216 0.0105 0.206 0.0112 0.178 0.0128 0.164 0.0192 0.146 0.0208 0.126 0.0224 0.136 0.0208 0.112 0.0208 0.168 0.0224 0.120 0 0224 0.113 0.0208 0.120 0.0192 0.102 0.0208 0.122 0.0224 0.107 0.0208 0.109 0.0192 0.098 0.0192 0.095 0.0176 0.178 0.0176 0.086 0.0176 0.094 0.0160 0.088 0.0144 0.077 0.0160 0.083 0.0192 0.109 0.0176 0.083 0.0176 0.092 0.0176 0.096 0.0160 0.083 0.0160 0.077 0.0192 0.091 0.0176 0.086 0.0160 0.065 0.0176 0.059 0.0124 0.075

-

1 I

--

him N Gtrogen

scorbic acid mering per 1 gm. adrena

WT.

+2 (5): 39 (4) 23 (5)

$56 (4) 9 (4)

27 (2) 31 (3) 15 (2)

4 (3) 7 (2)

15 (2) 25 (3)

+14 (3) 97 (3) 73 (2) 49 (2) 52 (3) 91 (3) 92 (3) 74 (3) 76 (3) 77 (3) 98 (3) 61 (1)

126 (3) 71 (3) 83 (3) 44 (2)

130 (2) 157 (3)

99 (3) 131 (2) 162 (3)

69 (3) 116 (3)

73 (3) 78 (2)

102 (2) 186 (3) 125 (2)

.l 6

#

ACTH standard :quivalent

7

0 0.4 0.2 0

<0.2 0.2 0.3

<0.2 <0.2 <0.2 <0.2

0.2 0 2.5 1.1 0.5 0.5 2.0 2.1 1.0 1.2 1.3 2.6 0.7 6.5 1.0 1.5 0.4 7.4

18.2 2.6 7.6

21.5 1.0 4.7 1.1 1.3 2.9

48.0 6.3

* See Fig. 4. t Estimated from the alanine standard curve $ The figures in parentheses represent the number of rats; 1 y of N per 100 gm.

of body weight was injected intravenously for the assay.

322

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

LI, TISELIUS, PEDERSEN, HAGDAHL, CARSTENSEN 323

whereas from Tube 236 onward the patterns were very similar. However, the concentration of the immobile spot appears to increase toward the end of displacement. As has been already demonstrated, the immobile spot has most of the adrenocorticotropic activity and therefore it is expected from the paper chromatogram that the hormone activity is concentrated in the strongly adsorbed fractions.

Although the displacement development does not achieve much purifi- cation as judged from paper partition chromatography, it has demonstra- ted that the ACTH peptides can be displaced quantitatively from charcoal and that 25 per cent of the original peptide mixture which has no activity can easily be removed by this technique.

Carrier Displacement Chromatography-We next investigated the possi-

237 253 269 285 301

Test Tube Number

FIG. 5. Nitrogen and ACTH activity distribution in various fractions obtained from the displacement development experiment of ACTH peptide (see Fig. 4).

bility of separating the peptides by using the new carrier displacement chromatography (12). Some preliminary experiments with homologous alcohols showed that the activity was found in the zones corresponding to alcohols with 8 to 10 carbon atoms. Thus it was decided to adopt a car- rier system consisting of n-octyl alcohol and n-nonyl alcohol with 2 per cent, n-decyl alcohol as the displacer. The solvent used to dissolve these alcohols was 50 per cent ethyl alcohol in 0.10 N HCI. The peptides were dissolved in 5 cc. of 1 per cent n-octyl alcohol and pressed into the column. It, was followed by 7 cc. of 2 per cent n-nonyl alcohol. A volume of 62 cc. of 2 per cent n-decyl alcohol was applied for displacement. The adsorbent was a mixture of 1 part Carboraffin Supra charcoal and 3 parts Hyflo Super-Cel. The column was coupled with six sections of 6280, 3140, 1570, 1570, 1570, and 785 c.mm. The effluent was delivered by a pressure of 4 kilos per sq. cm. into 2 cc. fractions after passing through the cuvette.

The data of a typical experiment with 50 mg. of the peptides (Prepara-

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

324 ADRENOCORTICOTROPIC PEPTIDES

tion 2026-MS) may be seen in Fig. 6. The fractions were collected into five main portions as Fractions A, B, C, D, and E; Fractions A and B were due to the elution with the solvent, Fraction C to the displacement with octyl alcohol, Fraction D with nonyl alcohol, and Fraction E with decyl alcohol. It should be mentioned that the presence of peaks in Fractions

,20 -A-l-B-I-C-l-D-I-E---2(

g BO-

* 60- a

40-

Fraction’

A B c D E

01 I I I I I I 0 IO 20

iI.

40 50 60

FIG. 6. Carrier displacement chromatography of ACTH peptides (Preparation 2026-MS). 50 mg, of the peptides were used; adsorbent, Carboraffin Supra and Hyflo Super-Gel in a ratio of 1:3; column, six sections of 6280, 3140, 1570, 1570, and 785 c.mm. plus mixer. Octyl alcohol, nonyl alcohol, and decyl alcohol as carriers.

TABLE III Nitrogen and Activity Distribution in Diflerent Fractions of ACTH Peptides (60 Mg.)

Obtained in Carrier Displacement Chromatography

Nitrogen Total N

w. gsr cent 1.333 21.4 0.785 12.6 2.268 36.6 1.294 20.8 0.554 8.6

Ascorbic acid lower- ing per 100 gm. ACTH standard

adrenalt equivalent

w. Y

17 0.2 19 0.2 50 0.5

116 4.7 122 5.7

* See Fig. 7. t Assayed at 1 y of N per 100 gm. of body weight; five animals in each group.

A and C is attributed to overloading in the adsorbent and hence a station- ary state has not been attained. No such peaks appeared-in Fractions D and E, but this does not mean that the stationary state has been attained. As it may be seen later, this does not cause difficulties in the separation.

The nitrogen content and assay results are given in Table III. It may be noted that a total recovery of the peptide nitrogen was achieved and that most of the activity was found in Fractions D and E. Some activity

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

LI, TISELIUS, PEDERSEN, HAGDAHL, CARSTENSEN 325

was also found in Fractions A, B, and C. Paper partition chromatography in butanol-10 per cent acetic acid revealed that Fraction E represents the purest material. It was found that each fraction gives rise to an immo- bile spot. This indicates that the immobile spot (Preparation A, of the original peptide mixture) must contain more than one component. It

0 0 20 30 40 50 60

Ml.

FIQ. 7. Carrier displacement chromatography of ACTH peptide fraction (Prep- aration AP) obtained from the active immobile spots of a paper chromatogram in butanol-10 per cent acetic acid. 16 mg. of Preparation AP were used; conditions of carrier displacement same as for Fig. 6.

TABLE IV Nitrogen and Activity Distribution in Dilerent Fractions of Preparation A, Obtained

in Carrier Displacement Chromatography

Fraction* Nitrogen Total N

fw. per cent A 0.205 9.9 B 0.189 9.1 c 0.635 30.6 D 0.697 33.6 E 0.354 16.8

Ascorbic acid lowering per 100 gm. adrenal

T(5)+ 2 (4)

26 (3) 4 (4)

131 (5)

T

ACTH standard equivalent

Y

0 0 0 0 7.6

* See Fig. 7 (16 mg. were used in this experiment). t The figures in parentheses represent the number of rats; 1 y of N per 100 gm.

of body weight was used for the assay.

will be seen in the following that Preparation A, from the paper chroma- togram can be resolved into a number of components in the carrier dis- placement chromatography.

16 mg. of Preparation Ap were pressed into a column similar to that described above. The optical data are given in Fig. 7 and the analytical results in Table IV. It is to be noted that Fractions C and D consist of

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

326 ADRENOCORTICOTROPIC PEPTIDES

60 per cent of the total material and have no ACTH activity, while Frac- tion E contains only 18 per cent of the total nitrogen and is the only frac- tion having the adrenal-stimulating action.2 In view of the limitation of our assay data, it is difficult to compute the percentage of recovery of bio- logical potency. There is, however, little doubt that ACTH activity is concentrated only in the fraction displaced by n-decyl alcohol.

If Preparation A, is a single substance, it should be expected to be dis- placed into one definite zone. For the amino acids and peptides investi- gated by Tiselius and Hagdahl (la), this was the case. Since the butanol- 10 per cent acetic acid-active immobile spot distributed itself into all displacement zones, it must be concluded that it consists of a num- ber of components.

Approximate Molecular Weights of Various ACTH Peptide Fractions-In an earlier investigation (3) it was found that the ACTH peptides prepared by peptic hydrolysis had an average molecular weight3 of the order 1200. After the fractionation described above had been made, it was of interest to determine the size of the peptides in the different fractions. The ex- periments were carried out in the same way as before (3), but the molecu- lar weights were calculated by means of the Svedberg method (3) as well as by means of a procedure described by Archibald (14). Archibald’s method has not yet been used extensively, but the model experiments so far carried out in Upsala indicate that the method is promising for sub- stances with molecular weights down to a few hundred. Details of these experiments will be published elsewhere. The method is based on the following: According to the differential equation for the combined sedi- mentation and diffusion in a sectorial ultracentrifuge cell, the flux, 4, of

2 From limited assay data in Tables III and IV, the recovery of ACTH activity appears far from complete. The reasons for the loss of activity are not clear and being investigated. However, it is likely that one or all of the following factors may be involved: (a) the activity is partially inactivated by impurities in the char- coal; (b) a cofactor has been separated and thus a decrease of specific activity in the active fraction occurs; and (c) a highly active fraction remains in the charcoal and cannot be displaced by the decyl alcohol. Since a total recovery of the peptide ni- trogen was achieved, the last factor may be ruled out.

3 Lesh et al. (13) recently obtained a highly potent fraction from the peptic digest of the swine ACTH protein, using the counter-current distribution tech- nique. They concluded that “the active moiety of the ACTH protein is of mo- lecular size considerably greater than the ACTH peptides recently reported” and that “the activity of the mixtures of peptides previously reported was due to the presence of a few per cent of a larger molecule having a very high bio- logical potency.” From the displacement and carrier displacement experiments with sheep ACTH peptides, described in the present paper, the results are evidently not in agreement with their conclusions. It is conceivable that the ACTH active peptides are different for different species.

4 Pedersen, K. O., and Hellman, E., unpublished experiments.

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

LI, TISELIUS, PEDERSEN, HAGDAHL, CARSTENSEN 327

material in 1 second throurrh a surface at the distance J: from the axis of rotation having unit are2 ven in the equation

ac + = dxcs - D z

where s and D are the sedimentation and diffusion constants respectively, c the concentration, and w the angular velocity. The first term on the right of the equation provided for the transport of material by sedimenta- tion and the second by diffusion. At equilibrium this flux becomes zero for all values of 2. Since, however, no solute can pass through the menis- cus of the solution, x = x0, nor through the bottom of the cell, x = xn,

TABLE V

Approximate Average Molecular Weights of Various ACTH Peptide Fractions

Method of chromatography Fraction

Paper Preparation AP

Carrier displace- A ment B

C D E

* The solvent was 0.2 M NaCl.

COIlCell- tration’ so0 DPO

- per cent lOa 10'

1.35 0.75 1.1 0.73 21 1.95 0.27 38 1.5 0.21 41 1.5 0.41 24 1.6 0.66 21 0.85 0.73 23

ivedberg nethodt

2800 1400 570 520 410 410

1400 1000 2500 1500 2600 1800

i The partial specific volume, VZO, was assumed to be 0.70.

the flux is also zero at these two levels at all times, i.e. even prior to the attainment of equilibrium. Thus a plot of l/w2xc X&/13x against x should extrapolate to the same value, s/D, at x0 and xn at any time for monodis- perse substance. For a polydisperse substance the molecular weight thus obtained corresponds to the weight-average molecular weight at x0 and x~, respectively, at the time of the experiment. As the experiment pro- ceeds the value by the Archibald method at x = x0 should decrease, whereas increasing values by this method should be found for x = zn. The variation by the Archibald procedure with time thus gives a measure of the homogeneity of the preparation. In our experiments we have ex- clusively made use of the values for l/w2xc X &/8x extrapolated first to x = x0 and then to t = 0. By this procedure the weight-average molecu- lar weight is obtained for the substance investigated.

In order to get a rough idea of the distribution in size of the various fractions some preliminary experiments were made in which the peptides were dissolved in pure water. The solutions could then be used directly

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

328 ADRENOCORTICOTROPIC PEPTTDES

for further chemical studies immediately after the end of the sedimentation and diffusion experiments without any further preparation. In all the later experiments the peptides were dissolved in 0.2 M sodium chloride in order to eliminate charge effects on sedimentation and diffusion (15) which tend to give too low molecular weights in pure water. The scarcity of material did not allow us to make any determination of the partial specific volumes for the different peptides, but it seems reasonable to assume that the volume is of the order of 0.70.

Table V gives a summary of the results obtained. The values given must be taken as estimates of the approximate weight-average molecular weights in various fractions. The experiments indicated greater polydis- persity in the samples isolated by the paper chromatographic technique than in the fractions obtained by carrier displacements chromatography. It may be noted that the values computed by the Svedberg equation are higher than those by Archibald’s method. It must be emphasized that none of the active peptide fractions appeared homogeneous in the ultra- centrifuge. Work on further fractionation is continuing at Berkeley and Upsala.

SUMMARY

The ACTH peptides obtained from the pepsin digest of adrenocortico- tropic hormone isolated from sheep pituitary glands have been subjected to study by various chromatographic methods. It was found that some purification can be achieved by paper partition chromatography. From the displacement development analysis, adrenocorticotropically active pep- tides of the mixture were shown to adsorb strongly on charcoal. The new carrier displacement technique of Tiselius and Hagdahl was found most applicable and the purest ACTH peptide fraction was obtained by use of normal alcohols with 8 to 10 carbon atoms as the carriers. The average molecular weight of various active fractions was determined by the ultra- centrifugal method. It was shown that the average molecular weight of the purest fraction is of the order 2000.

We are much indebted to the Swedish Natural Science and Medical Re- search Councils and to Mr. Josef Aner, Gothenburg, for generous financial aid for the investigations carried out in the Upsala laboratories.

BIBLIOGRAPHY

1. Li, C. H., Conference on metabolic aspects of convalescence, Josiah Macy, Jr. Foundation, New York, 17th meeting, 114 (1948).

2. Li, C. H., Nord. Med., 43, 363 (1950); J. Endocrinol., 6, p. xl (1950). 3. Li, C. H., and Pedersen, K. O., Ark. Kerni, 1,333 (1950).

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

LI, TISELIUS, PEDERSEN, HAGDAHL, CARSTENSEN 329

4. Consden, R., Gordon, A. H., and Martin, A. J. P., Biochem. J., 38, 224 (1944). 5. Partridge, S. M., Biochem. J., 42, 238 (1948). 6. Work, E., Biochim. et biophys. acta, 3, 400 (1949). 7. Sayers, M. A., Sayers, G., and Woodbury, L. A., Endocrinology, 42, 379 (1948). 8. Tiselius, A., Ark. Kemi, Mineral. o. Geol., 16 A, No. 18 (1943). 9. Tiselius, A., and Claesson, S., Ark. Kemi, Mineral. o. Geol., 16 B, No. 18 (1942).

10. Hagdahl, L., Acta them. &and., 2, 574 (1948). 11. Moore, S., and Stein, W. H., J. Biol. Chem., 176, 367 (1948). 12. Tiselius, A., and Hagdahl, L., Acta them. &and., 4, 394 (1950). 13. Lesh, J. B., Fisher, J. D., Bunding, I. M., Kocsis, J. J., Wolaszek, L. J., White,

W. F., and Hayes, E. E., Xcience, 112, 43 (1950). 14. Archibald, W. J., J. Phys. and CoZZoid Chem., 61, 1204 (1947). 15. Svedberg, T., and Pedersen, K. O., The ultracentrifuge, Oxford, 23 (1940).

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Lennart Hagdahl and Hans CarstensenChoh Hao Li, Arne Tiselius, Kai O. Pedersen,ADRENOCORTICOTROPIC PEPTIDES

CHROMATOGRAPHY OF

1951, 190:317-329.J. Biol. Chem. 

  http://www.jbc.org/content/190/1/317.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  tml#ref-list-1

http://www.jbc.org/content/190/1/317.citation.full.haccessed free atThis article cites 0 references, 0 of which can be

by guest on May 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from