DEMONSTRATION OF ORNITHINE IN GELATIN

BY ION EXCHANGE CHROMATOGRAPHY

BY PAUL B. HAMILTON AND ROBERTA A. ANDERSON

(Prom the Alfred I. du Pont Institute of The Nemours Foundation,
Wilmington, Delaware)

(Received for publication, June 1, 1954)

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Mild alkaline treatment partially degrades the guanidino group of argi-
nine with the formation of ammonia and citrulline (1) and more vigorous
treatment yields ammonia, urea, and ornithine (Z-5). It could be as-
sumed, therefore, that gelatin would contain some ornithine formed from
arginine during the immersion of calf skins and demineralized bone tissue
in calcium hydroxide suspension at 18.5’ for 5 to 6 weeks in the course of
the commercial manufacture of gelatin. It is well known that ammonia
and urea are produced during this liming process. Bowes and Kenton
(6) inferred that ornithine might be present in collagen treated with alkali
at pH 13 for 14 days at 20”, for they found that these conditions were
attended by liberation of ammonia, a loss of approximately 4 per cent
of the arginine, a small increase of amino nitrogen, and the production
of a small amount of urea. These authors concluded that the ammonia
was derived mainly from amide groups and that the loss of arginine was
due to the formation of small amounts of citrulline and ornithine; the for-
mation of ornithine was considered negligible.
After examining the basic amino acid composition of some proteins by
separation on 0.9 X 15 cm. columns of Dowex 50, it was found necessary to
change the pH, concentration, and sequence of some of the eluent buffers
described by Moore and Stein (7) to give a more satisfactory resolution
of histidine from hydroxylysine and particularly of ornithine from lysine.
With this improved resolution, it became of interest to examine acid hy-
drolysates in the expectation of establishing the presence or absence of
ornithine in gelatin. It was found that, depending upon its source, gelatin
contains ornithine in variable amounts ranging from a trace to 1.8 per cent
of the total amino acid nitrogen. Expressed as the percentage ratio of
ornithine to total original arginine (i.e., ornithine formed plus arginine
remaining), from a trace to 34 per cent of the arginine was degraded. Fresh
skin or bone proteins did not contain ornithine as a normal constituent,
but this substance was readily demonstrated after the proteins had been
subjected to processing in alkali. A trace of citrulline was encountered
95
96 ORNITHINE IN GELATIN

in only one of the gelatin hydrolysates examined. These findings are

reported in the present paper.’
Preparation of Columns-Dowex 50, 8 per cent cross-linked cationic
exchange resin (The Dow Chemical Company, Midland, Michigan), was
sieved while wet to remove particles larger than 200 mesh, followed by
decantation to remove particles smaller than 400 mesh, and was then pre-
pared in the sodium form according to Moore and Stein (7). Columns
0.9 X 15 cm. were poured in a single section and jacketed for temperature
control at 25”. Prior to introduction of the amino acid solution to be
analyzed, the column was equilibrated with 0.1 M citrate buffer, pH 3.4
(7). Fractions were collected on a Technicon fraction collector provided

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with a 1 ml. siphon assembly (9). The effluent was analyzed by the photo-
metric ninhydrin method (10) .2
Preparation of Buglers-The 0.1 M citrate buffers of pH 3.4 and 5.0 were
prepared according to Moore and Stein (7). The 0.4 M citrate buffer, pH
5.0, was prepared similarly. The 0.1 M phosphate buffer, pH 7.5, was
prepared from the sodium salts. Detergent and sequestering agents were
added in the same proportion to both phosphate and citrate buffers.
Preparation of Protein Hydrolysates-4.0 gm. of either gelatin or skin,
or 15 gm. of bone chips, were hydrolyzed in 200 ml. of boiling 6 N hydro-
chloric acid for 20 hours and filtered to remove insoluble humin. The
hydrolysate was reduced by evaporation to about 50 ml., transferred to a
100 ml. flask, and made to volume with 6 N hydrochloric acid; the nitrogen
content was determined by Kjeldahl. For chromatographic analysis,
portions of hydrolysate were evaporated to remove excess acid and the
residue was dissolved in water, neutralized to pH 2.0 with sodium hydrox-
ide, and diluted so that 1 ml. contained 1.8 mg. of nitrogen equivalent to
approximately 10 mg. of protein. Calcium was removed from the bone
hydrolysates; otherwise, poor resolution of amino acids was obtained and
the peaks were wide and ill defined. Portions of bone hydrolysate were
diluted 20-fold with water and the solution was neutralized with sodium
hydroxide to pH 8.2 (glass electrode). The flocculent precipitate of cal-
cium phosphate was removed by filtration with the aid of standard Filter-
1 Presented before the American Society of Biological Chemists, Atlantic City
(8).
2 Low color development was at first encountered, which was attributable to the
high peroxide content of the methyl Cellosolve diluent for the ninhydrin solution.
This solvent had been stored in a warm room. Redistilled solvent (2 liters) was
kept peroxide-free on being stored in a brown bottle containing six strips of 2 X 15
cm, of zinc foil previously immersed for 1 minute in acidified 5 per cent copper sulfate
solution (11). Solvent stored in brown or white bottles with zinc-copper couples
remained peroxide-free for as long as tested (4 months) even at room temperature
and when exposed to sunlight.
 P. B. HAMILTON AND R. A. ANDERSON 97

eel (Johns-Manville, Philadelphia, Pennsylvania). The precipitate was

washed with water until 1 ml. of effluent gave a blank value on being tested
with ninhydrin. The combined filtrate and washings were evaporated to
dryness, 10 ml. of concentrated hydrochloric acid were added to the residue,
and the insoluble sodium chloride was removed. The filtrate was evap-
orated to dryness, and the residue was dissolved in water, neutralized to

Leucine Aymonia
: Phenylalanine ;Ethanolamine
z 1.25- ! : G,lucosamine I
I I _ ,:Tryptophan
I

I, ,I Histidine \ I
I

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E I

2 l.OO- i 0,rnithine 1
I
2 I
I

6 I
I

0 0.75 : Arginine
E
0
; 0.50
a
-g 0.25
5

0 25 50 75 100 125 150 175 200 225

wpH 5.0 -----+tpH75+tpH 5.0--+
Effluent/cc.
FIG. 1. Resolution of basic amino acids on a column of Dowex 50, 0.9 X 15 cm.,
in the sodium form with pH and sequence of eluent buffers as indicated. The column
was jacketed at 25”. The dotted peak at Fraction 40 indicates the position occupied
by histidine when phosphate at pH 7.5 is introduced at Fraction 25 for twenty frac-
tions.

pH 2.0, and made to a volume so that 1 ml. contained the amino acids
from 10 mg. of protein.
Operation of Columns-2 ml. of amino acid solution were washed into
the column with two 1 ml. portions of 0.1 M citrate, pH 5.0. Develop-
ment was continued with the same solution. At Fraction 150, the eluent
solution was changed to 0.4 M citrate, pH 5.0. To elute histidine, the 0.1
M citrate buffer was interrupted at a convenient point and phosphate buf-
fer, pH 7.5, was substituted as eluent for twenty fractions. This technique
offers some advantages over the buffer sequence of Moore and Stein (7).
At pH 5.0, hydroxylysine, ornithine, and lysine are well resolved, whereas
at pH 6.8 ornithine and lysine emerge together and histidine and hydrox-
ylysine may be resolved poorly or not at all. Ammonia and arginine
98 ORNITHINE IN GELATIN

are well resolved by 0.4 M citrate and are eluted more rapidly than by 0.2
M citrate, pH 6.8. Histidine may be eluted by phosphate at pH 7.5 at
practically any position on the chromatogram without appreciably altering
the position of the other amino acids. If recoveries approximating 100
per cent are desired, introduction of phosphate, pH 7.5, at Fraction 25 for
twenty fractions places histidine between phenylalanine and hydroxylysine
as shown by the dotted peak in Fig. 1. In the present work, histidine was

Monoamino mono- Ammonia

Carboxylic acids Arginine
- ISO-

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u)
-. r-l

.pH5.0 ~~pHl!%-pH SO----+

Effluent /cc.
FIG. 2. Chromatogram of the gelatin first extracted by hot water from the residue
of ox bone chips after they have been demineralized with 5 per cent hydrochloric
acid and digested for 6 weeks in an aqueous suspension of calcium hydroxide at 18.5”.

eluted after lysine as a matter of convenience, but the recoveries were in-
variably low. In our experience, histidine is more susceptible than other
amino acids to destruction on the column at any pH with time. A typical
chromatogram of a test amino acid mixture is shown in Fig. 1, where it
can be noted that the entire chromatogram takes very little more than
the 200 fractions of the original procedure (7).
Analysis of Protein Hydrolysates-Chromatograms of hydrolysates of
bone chips, or hydrolysates of the protein residue of bone chips after de-
mineralization with 5 per cent hydrochloric acid, showed no peak ahead of
lysine in the position occupied by ornithine. Chromatograms of the res-
idue from bone chips after demineralization with acid and digestion for 6
 P. B. HAMILTON AND R. A. ANDERSON 99

weeks in saturated calcium hydroxide solution at 18.5” exhibited a small

peak ahead of lysine. This peak was also present in the gelatin extracted
by hot water from the demineralized limed material and in the insoluble
residue which remained after removal of the gelatin.3 Identification of
this peak as ornithine was made by its position on the chromatogram and
by the calorimetric ninhydrin procedure of Chinard (12). The chromato-
gram of gelatin (Kind and Knox Gelatine Company, Camden, New Jersey)
made from bone chips is shown in Fig. 2; that of a calf skin gelatin (Eastman

Monoamino mono -
Carboxylic Acids

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c I
g 0.3 I

Ornithine
2 I
1
5-O 0.4( 1 Lys,ine

r”
8 0.31
P-
2 0.2(
_-ifi Hydroxylysine
.!

l ILL\. lkli
5 O.i(

25 50 75 100 125 150 175 200 225

f--------- pH 5.0 AtpH75++pH SO-
Effluent /cc.
FIG. 3. Chromatogram of calf skin gelatin (Eastman Kodak Company, Rochester,
New York). The large amount of ornithine is probably due to the greatly increased
digestion time (4 to 8 months) in calcium hydroxide suspension usually employed for
calf skin.

Kodak Company, Rochester, New York) is presented in Fig. 3. The

greater amount of ornithine in this latter gelatin can most probably be
accounted for by the longer time of digestion of calf skins in lime which is
usually 10 to 12 weeks and often 4 to 8 months.
Ornithine rather than citrulline appears to be the major amino acid deg-
3 The authors are greatly indebted to Dr. T. B. Downey of the Kind and Knox
Gefatine Company, Camden, New Jersey, for samples of bone chips, bone demin-
eralized by 5 per cent hydrochloric acid, demineralized limed bone, gelatin, and
“cook pan” residue, representing all stages in the manufacture of gelatin from bones.
As well, the authors are indebted to him for generous samples of pork skin and calf
skin gelatin and acknowledge his help and interest.
100 ORNITHINE IN GELATIN

radation product of arginine, for the Fearon diacetylmonoxime reaction

for citrulline, as modified by Archibald (13), was negative for all hydrol-
ysates except the calf skin gelatin (see Table I) which contained little more
than trace amounts. The sum of ornithine plus arginine, therefore, repre-
sents the total arginine originally present in the protein, and the molar

TABLE I
Analysis of Protein, Hydrolysates

Ornithine
Hydrolysate of Ornithine Arginine* )mithine+ arginine
x 100
--

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CcJf PM per cent
1. Ox bone chips (cortical)t 0 10 0
2. (1) demineralized in 50/, hydrochloric
acidt................................. 0 10 0
3. (1) demineralized and limed 5-6 wks.i. 0.19 10 2
4. Gelatin extracted from (3)t.. 0.16 10 2
5. Residue from (3) after extraction of (4)t 0.25 10 2.5
6. Gelatinf................................ 0.03 10 0.3
7. Calf skin gelatin limed 4-8 mos.$ 5.1 10 34
8. Foam-compressed gelatin, limed lo-20
wks.§................................ 2.7 10 21
9. Pork skin gelatins.. 0 10 0
10. Human cortical bone (humerus) 11. 0 10 0
11. Pork skins....................... 0 10 0
12. Beef (( T[. 0 10 0
13. “ articular cartilage7.. 0 10 0

* All values computed to the arginine content in 20 mg. of gelatin (13).

t Kind and Knox Gelatine Company, Camden, New Jersey.
,$ Nutritional Biochemicals Corporation, Cleveland, Ohio.
5 Eastman Kodak Company, Rochester, New York. Citrulline was detected in
calf skin gelatin in amounts less than 0.3 per cent of ornithine + arginine. A chro-
matogram of this gelatin is shown in Fig. 3.
11Operating room specimen.
7 Slaughter-house.

ratio of (ornithine) to (ornithine + arginine) X 100 indicates the percent-

age of arginine converted to ornithine. Chromatograms of human cortical
bone (humerus), beef articular cartilage, beef tendon, beef skin, pork skin,
and pork skin gelatin did not show the presence of ornithine. A summary
of the findings is given in Table I.
It can also be inferred that a loss of arginine would disturb its quanti-
tative relationship with other amino acids, e.g. lysine. The molar ratio of
arginine to lysine computed from the data quoted by Tristram (14) for
gelatin was 1.56. We have found that all collagenous proteins investigated
 P. B. HAMILTON AND R. A. ANDERSON 101

which contained little or no ornithine had a comparable or even higher

ratio. For the calf skin gelatin (Fig. 3), the ratio of arginine to lysine was
much lower, 1.05, but, in accordance with the assumption that ornithine
gain is equivalent to arginine loss, the ratio of ornithine + arginine to
lysine was 1.59.

DISCUSSION

These findings indicate that ornithine is not a normal constituent of the

collagenous proteins examined but was demonstrable in those proteins
that had been limed, and, the longer the time in alkali, the greater the
amount of ornithine. Since the amount of citrulline found was negligible,

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the conclusion that alkaline degradation of the guanidino group of arginine
leads mainly to ornithine seemsjustified.
Since ornithine appears to be an artifact in gelatin, there is no need to
attach significance to it with respect to protein structure. It would be
anticipated, however, that the isoelectric point of gelatin would be shifted
somewhat to the acid side because of the formation of the weaker &amino
groups of ornithine from the more strongly basic guanidino groups of argi-
nine. As well, some caution in interpreting analytical values of amino
acids in gelatin is necessary; arginine may be unexpectedly low and lysine
high unless care is exercised in separating ornithine. Also, care in select-
ing gelatin as a growth medium would be necessary when an ornithine-free
medium was desired.
Attention should be drawn to the slightly skewed hydroxylysine peak
especially that shown in Fig. 3. This finding has been partially explained
by Piez (15) as a separation of hydroxylysine from allohydroxylysine and
has been more fully investigated by work from this 1aboratoryP

SUMMARY

1. The chromatographic resolution of basic amino acids on 0.9 X 15 cm.

columns of sodium Dowex 50 as described by Moore and Stein (7) was
modified to give a more satisfactory separation of histidine from hydrox-
ylysine and particularly of ornithine from lysine.
2. With the improved procedure, ornithine was found among the amino
acid constituents of some of the gelatins investigated. It was concluded
that ornithine was formed by alkaline degradation of arginine during the
immersion in calcium hydroxide suspensionfor 5 or more weeks, part of
the manufacturing process of gelatin. Ornithine was therefore present as
an artifact. Gelatin from source material that was not soaked in lime
suspensionor other alkali, i.e. pork skin, did not contain ornithine. Larger
amounts of ornithine were associated with longer times of liming.
4 Hamilton, P. B., and Anderson, R. A., in preparation.
102 ORNITHINE IN GELATIN

3. Ornithine appears to be the major degradation product of arginine.

The other degradation product expected, namely citrulline, was found in
little more than a trace in only one gelatin hydrolysate examined.
4. Since ornithine and not citrulline is formed from arginine, the ratio
of (ornithine) to (ornithine + arginine) X 100 represents percentage deg-
radation of arginine. It ranged from less than 0.1 to 34 per cent in the
gelatins examined.

The authors wish to acknowledge with grateful thanks the continued

help and interest of Dr. S. Moore and Dr. W. H. Stein of The Rockefeller
Institute for Medical Research, New York.

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BIBLIOGRAPHY

1. Fox, S. W., J. Biol. Chem., 123, 687 (1938).

2. Van Slyke, D. D., J. BioZ. Chem., 10, 15 (1911-12).
3. Plimmer, R. H. A., Biochem. J., 10, 115 (1916).
4. Boon, W. R., and Robson, W., Biochem. J., 29, 2684 (1935).
5. Hamilton, P. B., and Anderson, R. A., Biochem. Preparations, 3, 96 (1953).
6. Bowes, J. H., and Kenton, R. H., Biochem. J., 43, 365 (1948).
7. Moore, S., and Stein, W. H., J. Biol. Chem., 192,663 (1951).
8. Hamilton, P. B., and Anderson, R. A., Federation Proc., 13,224 (1954).
9. Hamilton, P. B., And. Chem., in press.
10. Moore, S., and Stein, W. H., J. Biol. Chem., 176,367 (1948).
11. Muers, M. M., and House, M. A., Analyst, 74,85 (1949).
12. Chinard, F. P., J. Biol. Chem., 199, 91 (1952).
13. Archibald, R. M., J. Biol. Chem., 166, 121 (1944).
14. Tristram, G. R., Advances in Protein Chem., 6, 83 (1949).
15. Piez, K. A., J. Biol. Chem., 207,77 (1954).
DEMONSTRATION OF ORNITHINE IN
GELATIN BY ION EXCHANGE
CHROMATOGRAPHY
Paul B. Hamilton and Roberta A. Anderson
J. Biol. Chem. 1954, 211:95-102.

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