This and other experiences with the tryptophane method of Fürth and Nobel led us to doubt seriously the reliability of quantitative data obtained by its application. When, therefore, just as we completed our work with it, Folin and Looney (6) described another and apparently better method of determination, a method based upon a different color reaction and capable moreover of convenient combination with a quantitative procedure for tyrosine, it seemed to us worth while to review the problem again. With the aid of this newer method we have now determined the tryptophane and tyrosine content of two series of globin preparations, and have, we believe, settled fairly decisively the proportion of these amino-acids yielded by the pure protein. We have also taken occasion to determine by the method of Van Slyke the general distribution of nitrogen in the globin molecule

Borsook, Henry

Hunter, Andrew

English

Caltech Authors - Main

NITROGEN DISTRIBUTION IN GLOBIN. 
BY ANDREW HUNTER AND HENRY BORSOOK. 
(From the Department of Biochemistry, University of Toronto, 
Toronto, Canada.) 
(Received for publication, July 2, 1923.) 
In endeavoring to isolate any single component from a naturally 
occurring mixture of proteins, one is almost certain to be embar- 
rassed by the lack of some safe yet convenient criterion which 
shall guarantee the completeness of the separation. The writers 
lately experienced this difficulty in attempting to obtain prepa- 
rations of globin which should be demonstrably uncdntaminated 
by the other proteins of the blood. They had barely encountered 
the problem when its solution seemed to be offered. Fiirth and 
Lieben (1) reported in 1920 the entire absence of the tryptophane 
radicle from the molecule of hemoglobin. Here appeared to be 
the desired criterion. The other blood proteins are known to be 
fairly rich in tryptophane, so that the problem apparently resolved 
itself into one of purifying hemoglobin, or its globin component, 
until it gave a negative glyoxylic reaction. 
We have already feported elsewhere the failure of the expecta- 
tions thus raised (2). It has been found that not even five recrys- 
tallizations of hemoglobin made any appreciable difference in 
the intensity of the glyoxylic reaction as given either by the pig- 
ment itself or by the globin prepared from it. Not only is this 
so, but the method of tryptophane estimation based by Fiirth and 
Nobel (3) upon the Voisenet reaction-the very method the use 
of which had led IGirth and Lieben to conclude that hemoglobin 
is free from tryptophane-indicated in our hands the presence 
in globin of 2.6 per cent of that amino-acid. This proportion was 
not in the least diminished by successive purifications. We were 
forced to the conclusion-quite in accord with earlier belief 
(4,5)-that tryptophane is an integral part of the globin molecule; 
and could only suppose that Fiirth and Lieben had been misled 
by the use of globin solutions altogether too dilute to give the 
Voisenet reaction. 
507 
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508 Nitrogen Distribution in Globin 
This and other experiences with the tryptophane method of 
Fiirth and Nobel led us to doubt seriously the reliability of quan- 
titative data obtained by its application. When, therefore, just 
as we completed our work with it, Folin and Looney (6) described 
another and apparently better method of determination, a method 
based upon a different color reaction and capable moreover of 
convenient combination with a quantitative procedure for tyrosine, 
it seemed to us worth while to review the problem again. With 
the aid of this newer method we have now determined the trypto- 
phane and tyrosine content of two series of globin preparations, 
and have, we believe, settled fairly decisively the proportion of 
these amino-acids yielded by the pure protein. We have also 
taken occasion to determine by the method of Van Slyke the 
general distribution of nitrogen in the globin molecule. 
Meanwhile, our original conclusion has been independently con- 
firmed in Fiirth’s own laboratory. Kiyotaki (7), still using the 
method of Fiirth and Nobel, but observing certain precautions 
neglected by Fiirth and Lieben, estimates the tryptophane content 
of globin at the rather high figure of 3.6 per cent. He has also 
determined the tyrosine content, which he places at 3.5 to 4 per 
cent. Since in each determination his result as well as his method 
is different from ours, the presentation of our data would still 
seem to be worth while. 
Globin Preparations. 
First Series.-A large quantity of crystalline oxyhemoglobin 
was obtained from horse blood by the method of Zinoffsky (8). 
The preparation was recrystallized four times. Of each of the 
fist four crops of crystals a portion was reserved. From each 
reserved portion, and from the whole of the fifth and final crop, 
globin was separated by a slight modification of the method of 
Robertson (9). Five successive specimens of globin, of presumably 
increasing purity, were thus obtained. The first and fourth were 
somewhat contaminated with hematin; but the second, third, and 
fifth were nearly colorless. 
Second Series.-The second series of globin preparations differed 
from the first in two respects. The original solution of hemoglobin, 
obtained from the laked corpuscular mass, was treated with alu- 
mina cream according to the method of Marshall and Welker (10) ; 
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A. Hunter and H. Borsook 509 
and the number of reorystallizations was reduced to two. The 
series included, therefore, only three specimens. Of these the 
first and second were rather pigmented; the third was practically 
white. 
The nitrogen content of the globin preparations, determined 
by the method of Kjeldahl, varied between 16.76 and 16.93 per 
cent. The average found was 16.86, which agrees with the 
value 16.89 reported by Schulz (11). The variations, such as 
they were, were quite irregular, and in themselves gave no evi- 
dence of increasing purity in successive fractions. 
Determinations of Tryptophane and Tyrosine. 
1 gm. lots of the eight preparations of globin, each of which had 
been dried for 48 hours in vacua over sulfuric acid, were hydrolyzed 
with baryta, and assayed for tryptophane and tyrosine by the 
method of Folin and Looney (6). Each determination was 
carried out on duplicate samples of the product of hydrolysis, the 
volume taken4 to 5 cc.-being always such as would yield 
color depths approximately equal to those of the standards. 
The results are reported in Table I, in which is shown also for 
each case the indicated molecular ratio of tyrosine to tryptophane. 
TABLE I. 
Source of globin. 
Molecular ratio 
Tyrosine 
Tryptophane’ Trypto- 
phane. 
I 
First crystallization 
Second “ 
First series. Third “ 
Fourth “ 
t Fifth 
‘I 
‘I 
“ 
Third “ 
........ 1.93 
........ 1.99 
........ 2.51 
........ 2.49 
........ 2.48 
........ 2.56 
........ 2.61 
........ 2.62 
-I 
oer cent 
3.23 1.9 
3.57 2.0 
3.97 1.8 
3.96 1.8 
4.13 1.9 
4.10 1.8 
4.64 2.0 
4.63 2.0 
Discussion of the Tryptophane and Tyrosine Determinations. 
It is at once apparent from the data that no diminution of 
tryptophane content is effected by successive attempts at purifica- 
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510 Nitrogen Distribution in Globin 
tion. Whether the increase observed in. the earlier samples of 
each series, and the higher maximum shown by the second, have 
a real significance is not quite certain. The differences seem, 
especially in the first series, to be rather beyond the experimental 
error of the method; but the fact that they are paralleled by nearly 
proportional changes in tyrosine content makes them difficult to 
explain by simple contamination of the Crst fractions with at any 
rate the serum proteins. Serum globulin (ox) contains, according 
to Folin and Looney, 6.7 per cent of tyrosine and 2.28 per cent of 
tryptophane. A highly purified specimen of serum albumin 
(horse), to which we applied Folin and Looney’s procedure, 
yielded values of 4.63 per cent for tyrosine and 1.79 per cent for 
tryptophane. The gradual removal of serum proteins would 
therefore be expected to raise the tryptophane content of the 
preparations, but to depress their tyrosine content. Actually 
the proportion of each amino-acid is seen to increase regularly 
to a maximum, while the molecular ratio remains nearly con- 
stant. Possibly an admixture of stroma proteins, regarding the 
composition of which we know next to nothing, may be respon- 
sible for the low tyrosine content of the early fractions. 
Taking the analytical results at their face value, it would seem 
that to obtain a globin of constant composition by simple recrys- 
tallization of oxyhemoglobin, three crystallizations at least are 
necessary; whereas, when a preliminary treatment with alumina 
cream is employed, two will sufEce. This is a confirmation of 
the claims made (10) for the efficacy of alumina cream in purify- 
ing oxyhemoglobin from other colloids. 
There is, it must be admitted, a discrepancy between the two 
series in respect to the ultimate values, especially for tyrosine, 
to which they rise. While we cannot at present offer an adequate 
explanation, we feel that the second series deserves the greater 
confidence, and would therefore put the tryptophane yield of 
pure globin at 2.61 per cent and its tyrosine at 4.63. This would 
attribute to each amino-acid the same proportion-2.12 per cent 
-of the total globin nitrogen. 
At these values we get a molecular ratio of tryptophane to 
tyrosine that is exactly 1:2. On the assumption that this im- 
plies the presence in the globin molecule of two tryptophane and 
four tyrosine radicles, we may then calculate the molecular 
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A. Hunter and H. Borsook 511 
weight of globin to be from 15,630 to 15,640. This agrees sur- 
prisingly with the 15,274 deduced by Osborne from the sulfur 
content (12). 
The tryptophane value of 2.6 per cent, yielded by the method 
of Folin and Looney, is identical with that which we formerly 
obtained (2) by the method of Fiirth and Nobel; but the latter 
in the hands of Kiyotaki (7) gave much higher figures-from 3.1 
to 4.0, with an average of ‘3.6. A higher value by the Fiirth- 
Nobel procedure is the general rule (6), and it may be that the 
identity of our two results is the chance outcome of the conditions 
under which the earlier determinations were made. Kiyotaki 
shows, what our own experience had already suggested, that the 
result of a Fiirth-Nobel determination is greatly dependent upon 
the concentration of the protein analyzed. We suspect that it 
is affected by still other factors. We have observed, for instance, 
that the Fiirth-Nobel procedure gives a far deeper and at the 
same time redder color when applied to gIycyl-trypt,ophane than 
when applied to an equivalent quantity of free tryptophane;l 
and that the few instances where the Folin-Looney result is not 
decidedly lower than the other-casein (6), serum albumin 
(1.8 per cent by Folin and Looney, 1.4 by Fiirth and Nobel (7)), 
and globin (if the comparison is limited to our own determina- 
tions)-include only proteins that are free from glycocoll. These 
observations are too limited in number to be decisive; but they 
certainly suggest that the Voisenet color reaction is affected, both 
as to hue and as to intensity, by the mode of combination of 
tryptophane in the protein molecule. This suspicion is not 
calculated to increase one’s confidence in any method of which 
it forms the basis. 
The tyrosine content of globin was determined by Kiyotaki 
in two ways; (1) by a bromine titration, and (2) by a coIorimetric 
application of the diazo reaction. Neither method gave very 
consistent results, the percentage found varied from 2.2 to 5.5. 
The figure 3.5 to 4.0 is proposed as a probable approximation. 
From his data Kiyotaki calculates that if globin has a molec- 
ular weight of about 15,000, it should contain 3 molecules each 
‘Proteins also give a redder color than pure tryptophane, a fact already 
commented upon by Liischer (Liischer, E., Biochem. J., 1922, xvi, 556)) 
who produces, moreover, fairly conclusive evidence that values obtained 
by the Ftirth-Nobel method are 30 to 60 per cent too high. 
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512 Nitrogen Distribution in Globin 
of tyrosine and of tryptophane. We feel that the greater con- 
sistency of our own results, together with the pains taken to 
prove the purity of our globin, make our estimate the more 
probably correct one. 
General Distribution of Nitrogen in Globin. 
The preparation of globin used for the Van Slyke analysis 
had been obtained from oxyhemoglobin, twice crystallized after 
treatment with alumina cream. It had a light brown color, and 
included, therefore, doubtless a trace of hematin. Two portions 
were separately hydrolyzed with 20 per cent hydrochloric acid, 
the one (designated A in Table II) by heating in a bath of boiling 
water for 48 hours, the other (B) by boiling over a free flame for 
30 hours. Table II shows the results obtained with aliquot 
portions of each hydrolysate. The original analytical data have 
been corrected for the solubility of the phosphotungstates of 
the bases. No correction was applied for cystine, for like Van 
Slyke in his analysis of hemoglobin (13) we found, even in the 
much larger quantities with which we were dealing, no evidence 
of the presence in the base fraction of any appreciable quantity 
of that amino-acid; the solution absorbed only a trace of bromine 
in the method of Okuda (14), and a one-fift,h aliquot, ignited 
with the reagent of Denis, yielded less than 1 mg. of BaS04. 
TABLE II. 
Distribution of Nitrogen in Globin. 
Nitrogen. 
Peroentlrge of 
total nitrogen. 
Calm&ted 
rd& of 
A B A 
I I 
B 
AVX- 
age. 
globin. 
Total N taken .......... 
Ammonia. .............. 
Humin .................. 
Arginine ................ 
Histidine. ............... 
Lysine. ................. 
Total bases ............. 
“ filtrate ............ 
Amino N of fi1trat.e ..... 
Non-amino N of filtrate 
?w. mg. per cent per cent per cent 
. 375.4 457.0 
. . . 19.3 25.6 5.14 5.60 5.37 
. . 7.0 8.8 1.86 1.93, 1.90 
30.3 36.4 8.07 7.97 8.0 
. . 47.1 55.6 12.55 12.8 12.7 
. . . . 42.4 49.5 11.3 10.8 11.1 
. . . . 119.9 144.5 31.9 31.6 31.8 
. 225.2 277.0 60.0 60.6 60.3 
. . . . . 213.8 259.9 57.0 56.9 57.0 
. . . . . . 11.4 17.1 3.0 3.7 3.3 
5.35 
1.5 
7.85 
13.0 
11.1 
32.0 
61.2 
58.2 
3.0 
Total N recovered.. . . . . . . . . .I 371.41 455.9; 99.0 / 99.8 1 99.4 1 (100.0) 
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A. Hunter and H. Borsook 513 
Discussion of Nitrogen Distribution. 
Considering the somewhat different conditions of hydrolysis 
under which the two analyses were conducted the agreement 
between their results is almost surprisingly good. It may be 
added that two other analyses, carried through upon entirely 
different preparations of globin, gave figures differing only within 
the recognized limits of the method from those presented. Our 
data stand, moreover, in excellent accord with those obtained 
by Van Slyke (13) in an analysis of ox hemoglobin. This is 
shown by the figures in the last column of Table II, where Van 
Slyke’s results have been recalculated for globin upon the as- 
sumptions that hematin accounts for 2.1 per cent of the total 
nitrogen of hemoglobin, and that all this appears in the humin 
fraction. 
The proportion of histidine found may be compared with the 
estimate of Hanke and Koessler (15), whose calorimetric method 
indicated the presence in horse hemoglobin of 8.8 per cent his- 
tidine, corresponding to 13.8 per cent of the total globin nitrogen. 
It is perhaps of some interest to attempt from the analytical 
data, an estimate of the number of basic radicles in the globin 
molecule. This is made possible by the knowledge already 
gained that 2.12 per cent of t$he total nitrogen corresponds to 
four monoamino-acid (tyrosine) radicles. One amino group, 
therefore, corresponds to 0.53 per cent of the total globin nitrogen. 
Dividing the percentages of arginine, histidine,and lysine nitro- 
gen by 4, 3, and 2, respectively, and each quotient by 0.53, wc 
find t,hat globin yields 3.8 molecules of arginine, 8.0 of histidine, 
and 10.4 of lysine; or, taking the integers to which these numbers 
approximate, 4, 8, and 10 molecuIes of arginine, histidine, and 
lysine, respectively. The exact nitrogen percenbages to be expect.ed 
from such a composition (assuming always the precise correct- 
ness of our tyrosine determination) are 8.48 for arginine, 12.72 
for histidine, and 10.60 for lysine. 
A similar calculation based upon the percentage of filtrate 
amino nitro ,en indicates the presence of 108 monoamino-acid 
f radicles in g obin. 
While such estimates have no claim to precision, they probably 
approximate pretty closely to the actual composition of the 
protein. In the case of the bases they can hardly differ by more 
than unity from the true number. 
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514 Nitrogen Distribution in Globin 
SOMMARY. 
Analytical data are presented which suggest that the globin 
molecule yields upon hydrolysis 2 molecules of tryptophane, 4 
each of tyrosine and arginine, 8 of histidine, 10 of lysine, and 
approximately 100 other amino-acid molecules, including dicar- 
boxylic acids. 
BIBLIOGRAPHY. 
1. Furth, O., and Lieben, F., Biochem. Z., 1920, cix, 124. 
2. Hunter, A., and Borsook, H., Tr. Roy. Sot. Canada, 1922, xvi, sect. v, 79. 
3. Ftirth, O., and Nobel, E., Biochem. Z., 1920, cix, 103. 
4. Abderhalden, E., Z. physiol. Chem., 1902-03, xxxvii, 484. 
5. Strauss, E., and Griitzner, R., Z. physiol. Chem., 1920-21, cxii, 169. 
6. Folin, O., and Looney, J. M., J.,BioZ. Chem., 1922, li, 421. 
7. Kiyotaki, U., Biochem. Z., 1922-23, cxxxiv, 322. 
8. Zinoffsky, O., Z. physiol. Chem., 1886, x, 23. 
9. Robertson, T. B., The physical chemistry of the proteins, New York, 
London, Bombay, Calcutta, and Madras, 1920. 
10. Marshall, J., and Weiker, W. H., J. Am. Chem. Sot., 1913, xxxv, 820. 
Welker, W. H., and Williamson, C. S., J. Biol. Chem., 1920, xli, 75. 
Howard, F. H., J. Biol. Chem., 1920, xli, 537. 
11. Schulz, F. N., Z. physiol. Chem., 1898, xxiv, 449. 
12. Cited from Mathews, A. P., Physiological chemistry, New York, 
3rd edition, 1920, 139. 
13. Van Slyke, D. D., J. Biol. Chem., 1911-12, x, 52. 
14. Okuda, Y., J. CoZZege Agric. Imp. Unit!. Tokyo, 1919, vii, 69. 
15. Hanke, M. T., andKoessler, K. K., J. BioZ. Chem., 1920, xliii, 527. 
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Nitrogen distribution by globin

NITROGEN DISTRIBUTION IN GLOBIN. 
BY ANDREW HUNTER AND HENRY BORSOOK. 
(From the Department of Biochemistry, University of Toronto, 
Toronto, Canada.) 
(Received for publication, July 2, 1923.) 
In endeavoring to isolate any single component from a naturally 
occurring mixture of proteins, one is almost certain to be embar- 
rassed by the lack of some safe yet convenient criterion which 
shall guarantee the completeness of the separation. The writers 
lately experienced this difficulty in attempting to obtain prepa- 
rations of globin which should be demonstrably uncdntaminated 
by the other proteins of the blood. They had barely encountered 
the problem when its solution seemed to be offered. Fiirth and 
Lieben (1) reported in 1920 the entire absence of the tryptophane 
radicle from the molecule of hemoglobin. Here appeared to be 
the desired criterion. The other blood proteins are known to be 
fairly rich in tryptophane, so that the problem apparently resolved 
itself into one of purifying hemoglobin, or its globin component, 
until it gave a negative glyoxylic reaction. 
We have already feported elsewhere the failure of the expecta- 
tions thus raised (2). It has been found that not even five recrys- 
tallizations of hemoglobin made any appreciable difference in 
the intensity of the glyoxylic reaction as given either by the pig- 
ment itself or by the globin prepared from it. Not only is this 
so, but the method of tryptophane estimation based by Fiirth and 
Nobel (3) upon the Voisenet reaction-the very method the use 
of which had led IGirth and Lieben to conclude that hemoglobin 
is free from tryptophane-indicated in our hands the presence 
in globin of 2.6 per cent of that amino-acid. This proportion was 
not in the least diminished by successive purifications. We were 
forced to the conclusion-quite in accord with earlier belief 
(4,5)-that tryptophane is an integral part of the globin molecule; 
and could only suppose that Fiirth and Lieben had been misled 
by the use of globin solutions altogether too dilute to give the 
Voisenet reaction. 
507 
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508 Nitrogen Distribution in Globin 
This and other experiences with the tryptophane method of 
Fiirth and Nobel led us to doubt seriously the reliability of quan- 
titative data obtained by its application. When, therefore, just 
as we completed our work with it, Folin and Looney (6) described 
another and apparently better method of determination, a method 
based upon a different color reaction and capable moreover of 
convenient combination with a quantitative procedure for tyrosine, 
it seemed to us worth while to review the problem again. With 
the aid of this newer method we have now determined the trypto- 
phane and tyrosine content of two series of globin preparations, 
and have, we believe, settled fairly decisively the proportion of 
these amino-acids yielded by the pure protein. We have also 
taken occasion to determine by the method of Van Slyke the 
general distribution of nitrogen in the globin molecule. 
Meanwhile, our original conclusion has been independently con- 
firmed in Fiirth’s own laboratory. Kiyotaki (7), still using the 
method of Fiirth and Nobel, but observing certain precautions 
neglected by Fiirth and Lieben, estimates the tryptophane content 
of globin at the rather high figure of 3.6 per cent. He has also 
determined the tyrosine content, which he places at 3.5 to 4 per 
cent. Since in each determination his result as well as his method 
is different from ours, the presentation of our data would still 
seem to be worth while. 
Globin Preparations. 
First Series.-A large quantity of crystalline oxyhemoglobin 
was obtained from horse blood by the method of Zinoffsky (8). 
The preparation was recrystallized four times. Of each of the 
fist four crops of crystals a portion was reserved. From each 
reserved portion, and from the whole of the fifth and final crop, 
globin was separated by a slight modification of the method of 
Robertson (9). Five successive specimens of globin, of presumably 
increasing purity, were thus obtained. The first and fourth were 
somewhat contaminated with hematin; but the second, third, and 
fifth were nearly colorless. 
Second Series.-The second series of globin preparations differed 
from the first in two respects. The original solution of hemoglobin, 
obtained from the laked corpuscular mass, was treated with alu- 
mina cream according to the method of Marshall and Welker (10) ; 
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A. Hunter and H. Borsook 509 
and the number of reorystallizations was reduced to two. The 
series included, therefore, only three specimens. Of these the 
first and second were rather pigmented; the third was practically 
white. 
The nitrogen content of the globin preparations, determined 
by the method of Kjeldahl, varied between 16.76 and 16.93 per 
cent. The average found was 16.86, which agrees with the 
value 16.89 reported by Schulz (11). The variations, such as 
they were, were quite irregular, and in themselves gave no evi- 
dence of increasing purity in successive fractions. 
Determinations of Tryptophane and Tyrosine. 
1 gm. lots of the eight preparations of globin, each of which had 
been dried for 48 hours in vacua over sulfuric acid, were hydrolyzed 
with baryta, and assayed for tryptophane and tyrosine by the 
method of Folin and Looney (6). Each determination was 
carried out on duplicate samples of the product of hydrolysis, the 
volume taken4 to 5 cc.-being always such as would yield 
color depths approximately equal to those of the standards. 
The results are reported in Table I, in which is shown also for 
each case the indicated molecular ratio of tyrosine to tryptophane. 
TABLE I. 
Source of globin. 
Molecular ratio 
Tyrosine 
Tryptophane’ Trypto- 
phane. 
I 
First crystallization 
Second “ 
First series. Third “ 
Fourth “ 
t Fifth 
‘I 
‘I 
“ 
Third “ 
........ 1.93 
........ 1.99 
........ 2.51 
........ 2.49 
........ 2.48 
........ 2.56 
........ 2.61 
........ 2.62 
-I 
oer cent 
3.23 1.9 
3.57 2.0 
3.97 1.8 
3.96 1.8 
4.13 1.9 
4.10 1.8 
4.64 2.0 
4.63 2.0 
Discussion of the Tryptophane and Tyrosine Determinations. 
It is at once apparent from the data that no diminution of 
tryptophane content is effected by successive attempts at purifica- 
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510 Nitrogen Distribution in Globin 
tion. Whether the increase observed in. the earlier samples of 
each series, and the higher maximum shown by the second, have 
a real significance is not quite certain. The differences seem, 
especially in the first series, to be rather beyond the experimental 
error of the method; but the fact that they are paralleled by nearly 
proportional changes in tyrosine content makes them difficult to 
explain by simple contamination of the Crst fractions with at any 
rate the serum proteins. Serum globulin (ox) contains, according 
to Folin and Looney, 6.7 per cent of tyrosine and 2.28 per cent of 
tryptophane. A highly purified specimen of serum albumin 
(horse), to which we applied Folin and Looney’s procedure, 
yielded values of 4.63 per cent for tyrosine and 1.79 per cent for 
tryptophane. The gradual removal of serum proteins would 
therefore be expected to raise the tryptophane content of the 
preparations, but to depress their tyrosine content. Actually 
the proportion of each amino-acid is seen to increase regularly 
to a maximum, while the molecular ratio remains nearly con- 
stant. Possibly an admixture of stroma proteins, regarding the 
composition of which we know next to nothing, may be respon- 
sible for the low tyrosine content of the early fractions. 
Taking the analytical results at their face value, it would seem 
that to obtain a globin of constant composition by simple recrys- 
tallization of oxyhemoglobin, three crystallizations at least are 
necessary; whereas, when a preliminary treatment with alumina 
cream is employed, two will sufEce. This is a confirmation of 
the claims made (10) for the efficacy of alumina cream in purify- 
ing oxyhemoglobin from other colloids. 
There is, it must be admitted, a discrepancy between the two 
series in respect to the ultimate values, especially for tyrosine, 
to which they rise. While we cannot at present offer an adequate 
explanation, we feel that the second series deserves the greater 
confidence, and would therefore put the tryptophane yield of 
pure globin at 2.61 per cent and its tyrosine at 4.63. This would 
attribute to each amino-acid the same proportion-2.12 per cent 
-of the total globin nitrogen. 
At these values we get a molecular ratio of tryptophane to 
tyrosine that is exactly 1:2. On the assumption that this im- 
plies the presence in the globin molecule of two tryptophane and 
four tyrosine radicles, we may then calculate the molecular 
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A. Hunter and H. Borsook 511 
weight of globin to be from 15,630 to 15,640. This agrees sur- 
prisingly with the 15,274 deduced by Osborne from the sulfur 
content (12). 
The tryptophane value of 2.6 per cent, yielded by the method 
of Folin and Looney, is identical with that which we formerly 
obtained (2) by the method of Fiirth and Nobel; but the latter 
in the hands of Kiyotaki (7) gave much higher figures-from 3.1 
to 4.0, with an average of ‘3.6. A higher value by the Fiirth- 
Nobel procedure is the general rule (6), and it may be that the 
identity of our two results is the chance outcome of the conditions 
under which the earlier determinations were made. Kiyotaki 
shows, what our own experience had already suggested, that the 
result of a Fiirth-Nobel determination is greatly dependent upon 
the concentration of the protein analyzed. We suspect that it 
is affected by still other factors. We have observed, for instance, 
that the Fiirth-Nobel procedure gives a far deeper and at the 
same time redder color when applied to gIycyl-trypt,ophane than 
when applied to an equivalent quantity of free tryptophane;l 
and that the few instances where the Folin-Looney result is not 
decidedly lower than the other-casein (6), serum albumin 
(1.8 per cent by Folin and Looney, 1.4 by Fiirth and Nobel (7)), 
and globin (if the comparison is limited to our own determina- 
tions)-include only proteins that are free from glycocoll. These 
observations are too limited in number to be decisive; but they 
certainly suggest that the Voisenet color reaction is affected, both 
as to hue and as to intensity, by the mode of combination of 
tryptophane in the protein molecule. This suspicion is not 
calculated to increase one’s confidence in any method of which 
it forms the basis. 
The tyrosine content of globin was determined by Kiyotaki 
in two ways; (1) by a bromine titration, and (2) by a coIorimetric 
application of the diazo reaction. Neither method gave very 
consistent results, the percentage found varied from 2.2 to 5.5. 
The figure 3.5 to 4.0 is proposed as a probable approximation. 
From his data Kiyotaki calculates that if globin has a molec- 
ular weight of about 15,000, it should contain 3 molecules each 
‘Proteins also give a redder color than pure tryptophane, a fact already 
commented upon by Liischer (Liischer, E., Biochem. J., 1922, xvi, 556)) 
who produces, moreover, fairly conclusive evidence that values obtained 
by the Ftirth-Nobel method are 30 to 60 per cent too high. 
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512 Nitrogen Distribution in Globin 
of tyrosine and of tryptophane. We feel that the greater con- 
sistency of our own results, together with the pains taken to 
prove the purity of our globin, make our estimate the more 
probably correct one. 
General Distribution of Nitrogen in Globin. 
The preparation of globin used for the Van Slyke analysis 
had been obtained from oxyhemoglobin, twice crystallized after 
treatment with alumina cream. It had a light brown color, and 
included, therefore, doubtless a trace of hematin. Two portions 
were separately hydrolyzed with 20 per cent hydrochloric acid, 
the one (designated A in Table II) by heating in a bath of boiling 
water for 48 hours, the other (B) by boiling over a free flame for 
30 hours. Table II shows the results obtained with aliquot 
portions of each hydrolysate. The original analytical data have 
been corrected for the solubility of the phosphotungstates of 
the bases. No correction was applied for cystine, for like Van 
Slyke in his analysis of hemoglobin (13) we found, even in the 
much larger quantities with which we were dealing, no evidence 
of the presence in the base fraction of any appreciable quantity 
of that amino-acid; the solution absorbed only a trace of bromine 
in the method of Okuda (14), and a one-fift,h aliquot, ignited 
with the reagent of Denis, yielded less than 1 mg. of BaS04. 
TABLE II. 
Distribution of Nitrogen in Globin. 
Nitrogen. 
Peroentlrge of 
total nitrogen. 
Calm&ted 
rd& of 
A B A 
I I 
B 
AVX- 
age. 
globin. 
Total N taken .......... 
Ammonia. .............. 
Humin .................. 
Arginine ................ 
Histidine. ............... 
Lysine. ................. 
Total bases ............. 
“ filtrate ............ 
Amino N of fi1trat.e ..... 
Non-amino N of filtrate 
?w. mg. per cent per cent per cent 
. 375.4 457.0 
. . . 19.3 25.6 5.14 5.60 5.37 
. . 7.0 8.8 1.86 1.93, 1.90 
30.3 36.4 8.07 7.97 8.0 
. . 47.1 55.6 12.55 12.8 12.7 
. . . . 42.4 49.5 11.3 10.8 11.1 
. . . . 119.9 144.5 31.9 31.6 31.8 
. 225.2 277.0 60.0 60.6 60.3 
. . . . . 213.8 259.9 57.0 56.9 57.0 
. . . . . . 11.4 17.1 3.0 3.7 3.3 
5.35 
1.5 
7.85 
13.0 
11.1 
32.0 
61.2 
58.2 
3.0 
Total N recovered.. . . . . . . . . .I 371.41 455.9; 99.0 / 99.8 1 99.4 1 (100.0) 
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A. Hunter and H. Borsook 513 
Discussion of Nitrogen Distribution. 
Considering the somewhat different conditions of hydrolysis 
under which the two analyses were conducted the agreement 
between their results is almost surprisingly good. It may be 
added that two other analyses, carried through upon entirely 
different preparations of globin, gave figures differing only within 
the recognized limits of the method from those presented. Our 
data stand, moreover, in excellent accord with those obtained 
by Van Slyke (13) in an analysis of ox hemoglobin. This is 
shown by the figures in the last column of Table II, where Van 
Slyke’s results have been recalculated for globin upon the as- 
sumptions that hematin accounts for 2.1 per cent of the total 
nitrogen of hemoglobin, and that all this appears in the humin 
fraction. 
The proportion of histidine found may be compared with the 
estimate of Hanke and Koessler (15), whose calorimetric method 
indicated the presence in horse hemoglobin of 8.8 per cent his- 
tidine, corresponding to 13.8 per cent of the total globin nitrogen. 
It is perhaps of some interest to attempt from the analytical 
data, an estimate of the number of basic radicles in the globin 
molecule. This is made possible by the knowledge already 
gained that 2.12 per cent of t$he total nitrogen corresponds to 
four monoamino-acid (tyrosine) radicles. One amino group, 
therefore, corresponds to 0.53 per cent of the total globin nitrogen. 
Dividing the percentages of arginine, histidine,and lysine nitro- 
gen by 4, 3, and 2, respectively, and each quotient by 0.53, wc 
find t,hat globin yields 3.8 molecules of arginine, 8.0 of histidine, 
and 10.4 of lysine; or, taking the integers to which these numbers 
approximate, 4, 8, and 10 molecuIes of arginine, histidine, and 
lysine, respectively. The exact nitrogen percenbages to be expect.ed 
from such a composition (assuming always the precise correct- 
ness of our tyrosine determination) are 8.48 for arginine, 12.72 
for histidine, and 10.60 for lysine. 
A similar calculation based upon the percentage of filtrate 
amino nitro ,en indicates the presence of 108 monoamino-acid 
f radicles in g obin. 
While such estimates have no claim to precision, they probably 
approximate pretty closely to the actual composition of the 
protein. In the case of the bases they can hardly differ by more 
than unity from the true number. 
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514 Nitrogen Distribution in Globin 
SOMMARY. 
Analytical data are presented which suggest that the globin 
molecule yields upon hydrolysis 2 molecules of tryptophane, 4 
each of tyrosine and arginine, 8 of histidine, 10 of lysine, and 
approximately 100 other amino-acid molecules, including dicar- 
boxylic acids. 
BIBLIOGRAPHY. 
1. Furth, O., and Lieben, F., Biochem. Z., 1920, cix, 124. 
2. Hunter, A., and Borsook, H., Tr. Roy. Sot. Canada, 1922, xvi, sect. v, 79. 
3. Ftirth, O., and Nobel, E., Biochem. Z., 1920, cix, 103. 
4. Abderhalden, E., Z. physiol. Chem., 1902-03, xxxvii, 484. 
5. Strauss, E., and Griitzner, R., Z. physiol. Chem., 1920-21, cxii, 169. 
6. Folin, O., and Looney, J. M., J.,BioZ. Chem., 1922, li, 421. 
7. Kiyotaki, U., Biochem. Z., 1922-23, cxxxiv, 322. 
8. Zinoffsky, O., Z. physiol. Chem., 1886, x, 23. 
9. Robertson, T. B., The physical chemistry of the proteins, New York, 
London, Bombay, Calcutta, and Madras, 1920. 
10. Marshall, J., and Weiker, W. H., J. Am. Chem. Sot., 1913, xxxv, 820. 
Welker, W. H., and Williamson, C. S., J. Biol. Chem., 1920, xli, 75. 
Howard, F. H., J. Biol. Chem., 1920, xli, 537. 
11. Schulz, F. N., Z. physiol. Chem., 1898, xxiv, 449. 
12. Cited from Mathews, A. P., Physiological chemistry, New York, 
3rd edition, 1920, 139. 
13. Van Slyke, D. D., J. Biol. Chem., 1911-12, x, 52. 
14. Okuda, Y., J. CoZZege Agric. Imp. Unit!. Tokyo, 1919, vii, 69. 
15. Hanke, M. T., andKoessler, K. K., J. BioZ. Chem., 1920, xliii, 527. 
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Nitrogen distribution by globin

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