Experiments previously reported (1) have shown that the eggs of the Pacific marine worm, Urechis caupo, contain a reversible oxidation-reduction pigment. The pigment, called urechrome, is autoxidizable and changes color from red to yellow on oxidation. It is soluble in water (reduced form insoluble below pH 5) and in acidified methanol, but insoluble in ether, acetone, chloroform, and neutral alcohol. Evidence for participation of the pigment in cellular respiration has been previously given

Baumberger, J. Percy

Horowitz, N. H.

English

Caltech Authors - Main

STUDIES ON THE RESPIRATORY PIGMENT OF 
URECHIS EGGS 
BY N. H. HOROWITZ* AND J. PERCY BAUMBERGER 
(Prom the Schaol of Biological Sciences, Stanford University, California) 
(Received for publication, June 9, 1941) 
Experiments previously reported (1) have shown that the eggs 
of the Pacific marine worm, Urechis caupo, contain a reversible 
oxidation-reduction pigment. The pigment., called urechrome, 
is autoxidixable and changes color from red to yellow on oxidation. 
It is soluble in water (reduced form insoluble below pH 5) and in 
acidified methanol, but insoluble in ether, acetone, chloroform, 
and neutral alcohol. Evidence for participat’ion of the pigment 
in cellular respiration has been previously given. 
Because of its biochemical interest and possible embryological 
significance, a more extensive study of the substance has been 
made, the results of which are reported below. 
Occurrence and Preparation-Urechrome occurs chiefly in the 
eggs of the worm. None has been found in the muscle, viscera, 
egg sacs, or sperm. Traces are found in the centrifuged blood 
elements of the female, probably in association with the oocytes, 
which mature in the blood of this species. 
To prepare the pigment, the egg sacs are dissected from the 
animal and dropped into sea water, care being taken to prevent 
contamination by blood. The eggs are released by puncturing 
the sacs and the empty sacs are removed. After the eggs have 
been washed several times, they are centrifuged down and 
transferred to a Soxhlet apparatus where they are extracted first 
with acetone and then with ether until yellow pigments no longer 
come over. The urechrome, which remains behind, is extracted 
by shaking with 0.1 N HCl-methanol at 40’. The solution is 
* National Rescarch Council Fellow in t.hc Natural Sciences, 1939%4(!. 
Present address, California Instit,ute of Technology, Pasadena. 
407 
Respiratory Pigment of Urechis 
filtered and then concentrated in vacua to about one-fourth of the 
original volume. It is now brought to pH 2 (glass electrode) with 
1 N NaOH, precipitating reduced urechrome. After standing at 
0” for several hours, the precipitate is centrifuged down, redis- 
solved in HCl-methanol, and reprecipitated with 4 to 5 volumes of 
ether. The ether precipitation is repeated, after which the pig- 
ment is collected in a centrifuge tube, washed twice with absolute 
methanol, and twice with distilled w-ater. It is dried in vacua 
over calcium chloride. The reduced pigment dries in dark violet, 
glistening flakes. It is non-crystalline. 10 gm. of dehydrated, 
defatted eggs yield about 40 to 50 mg. of urechrome. An addi- 
tional 15 to 20 mg. can be recovered from the supernatant of the 
NaOH precipitation by reducing the oxidized pigment, which has 
remained in solution, with a small crystal of stannous chloride and 
proceeding as with the first precipitate. It has been found that 
reduced urechrome can also be precipitat,ed from HCI-methanol 
with a small amount of pyridine, apparently as the result of salt 
format,ion. 
Oxidation-i&Auction I’otential-------‘l’l-le oxidation-reduction poten- 
tial has been studied polarographically (2-4), checked by po- 
tentiometric titration. The advantage of the polarographic 
method consists in its being particularly adaptable to small quanti- 
ties in which the ratios of oxidant to reduct.ant and the correspond- 
ing values of E, are determinable without the addition of a second 
system. The method as used by us is less accurate than the po- 
t.entiometric titration, the error being ~5 millivolts. 
The instrument used was the Heyrovsky-Kejedly type with a 
dropping mercury electrode and a saturated calomel reference 
half-cell. The set-up m-as essentially that described by Miiller 
and Baumberger (2). The temperature was 25” f I”.’ A 
saturated solution of pigment (1 to 3 mg. per cc.) in phosphate or 
borate buffer of the desired pH was deoxygenated by a stream of 
02-free nitrogen. A small quantity of platinized asbestos was 
added and the gas changed to hydrogen. At various stages of 
reduction (judged roughly by the color of the solution) the hydro- 
1 Owing to a typographical error, the trmperature of the erperimcnt,s 
was previously rcportcd RS 250” (1). 
N. H. Horowitz and J. P. Baumberger 409 
gen was replaced by nitrogen and a current-voltage curve was 
photographically recorded (Fig. 1). After complete reduction 
had been attained, the process could be reversed by introducing 
successive small amounts of air into the solution until complete 
oxidation was reached. From the polarographic curves so ob- 
tained, Eh and the relative heights of the cathodic and anodic 
waves can be measured, from which E’o, the ratio [Ox] : [Red], and 
the number of electrons involved in the reaction can be calculated. 
FIG. 1. Polarogram of urechrome. Phosphate buffer, pH 6.26. The 
horizontal line indicates “galvanometer zero,” above which a dropping Hg 
electrode is the cathode and below which it is the anode. A short vertical 
line is drawn through the half waves, intersecting “galvanometer zero” 
at E’O. The ratios [Ox]: [Red] are as follows: Curve I, 4:3; Curve II, 7:lS; 
Curves III and IV, practically zero. 
Details of the calculations are given in the paper of Miiller and 
Baumberger (2). The potentials were readily reproducible, and 
were not noticeably affected by variations in the total concen- 
tration of dye. 
The change in E’, with pH was investigated from pH 5 to 10. 
These are the practical limits of pH for the substance, since below 
pH 5 the reduced form is insoluble and above pH 10 the pigment is 
unstable. E’o decreases by 59 millivolts per unit increase in pH, 
within the limits of experimental error (Table I). 
The reduction of urechrome involves only 1 electron, as can 
410 Respiratory Pigment of Urechis 
be seen in Table I, where calculated values of Eh for 1 and 2 
electron processes are compared with the observed potentials. 
With some preparations a second polarographic wave was observed 
at a potential several hundred millivolts negative to the main wave. 
The height and slope of this wave were extremely variable, in 
some cases being pronounced, in others imperceptible. The 
TABLE I 
Relation oj’ E’, to QH and Evaluation o,f n, jrom Polarographic Data 
PH E’Q 
5.31 0.287 
6.26 0.228 
7.39 0.163 
7.70 0.143* 
8.62 O.OSQ* 
9.15 0.057 
9.97 0.007 
[ox] 
[Red1 
4:3 0.236 0.232 0.239 
7:lS 0.203 0.215 0.206 
3:l 0.192 0.178 0.191 
9~7 0.170 0.167 0.172 
5:36 0.112 0.137 0.106 
2:25 0.097 0.130 0.091 
1:23 0.081 0.122 0.077 
7:3 0.079 0.068 0.077 
19:13 0.067 0.062 0.066 
21:16 0.064 0.061 0.063 
2:ll 0.013 0.035 0.011 
8:47 0.011 0.034 0.011 
1:23 -0.025 0.016 -0.020 
7:l 0.058 0.033 0.056 
Mean deviation from observed 0.003 0.019 
volt 
Eh 
Calculated 
TL=Z 
volt 
Observed 
volt 
* Potentiometric titration 
“half wave” potential did not vary in any regular manner with 
pH, nor was it ever observed to fall below the “galvanometer 
zero” line, i.e. in the region corresponding to a completely reduced 
system, even after prolonged bubbling of hydrogen. We believe 
the wave to be due to an unknown impurity or decomposition 
product. 
As a check on the polarographically determined potentials a few 
potentiometric titrations with a platinum electrode were carried 
N. H. Horowitz and J. P. Baumberger 411 
out. The reducing agent was NazSz04, the oxidizing agent fer- 
ricyanide. The atmosphere was nitrogen. The results on the 
whole are in close agreement with the polarographically deter- 
mined potentials, although the curves tend to be somewhat 
steeper t,han the theoretical, with drifting potentials at the ex- 
tremes of the curve. This is attributed to poor poising of the 
system. A titration curve at pH 8.62 is shown in Fig. 2. The 
agreement between the polarographic and titrimetric measure- 
I I I I 1 t I I I          
20 40 60 80 100 
% OXIDATION 
FIG. 2. Titration of reduced urcchrome with ferricyanide, 
The curves are the theoretical for vz = 1 and n = 2. The circles 
experimental values. 
~1-1 8.62. 
represent 
ments is in accord with the findings of previous studies on electro- 
active reversible systems. 
We conchIde that urechromc is a reversible oxidation-reduction 
system conforming, within the experimental limits, to the equation 
EJ, = 0.599 + 7 In g$ + y In [H+] 
where the formulation of the reaction is as follows: 
Oxidant + IS+ + e S rcductant 
Absorption Xpectrum-Solutions of the pigment exhibit no 
specific absorption in the visible region. Aqueous solutions of the 
412 Respiratory Pigment of Urechis 
oxidized form show general absorption of wave-lengths below 
540 mp, whereas the reduced form absorbs generally below 620 
ml.c. Reduced urechrome dissolved in acid methanol shows a 
band in the near ultraviolet at 388 rnp. The oxidized form has 
not been investigated in this region. 
Reduced urechrome forms a deep violet solution in concen- 
trated sulfuric acid. This solution exhibits two marked absorp- 
tion bands: a broad band centering at 553 rnp and a narrow band at 
688 rnp. The bands resemble those of hematoporphyrin an- 
hydride, formed from hemoglobin in concentrated sulfuric acid, 
which shows a broad band at 553 rnp and a narrow one at 613 rnp. 
It was therefore attempted to make a pyridine hemochromogen 
derivative of urechrome by reduction in alkaline solution in the 
presence of pyridine. The resulting solution showed a strong band 
at 548 rnp and a weak band at 515 rnp, typical of pyridine hemo- 
chromogen. It should be noted that the spectrum of pyridine 
hemochromogen cannot have resulted from contamination of the 
preparation with hemoglobin, since no hemoglobin spectrum was 
observed in untreated solutions, nor is the spectrum obtained by 
sulfuric acid treatment that of hematoporphyrin. 
Molecular Weight-Since the degree of purity of the pigment 
was not known, the molecular weight was approximated from 
diffusion measurements. A Northrop and Anson type of diffusion 
cell (5, 6) of 7.29 cc. capacity was used. The membrane constant, 
K, determined by standardization against 2 M NaCl, was 0.470. 
The temperature of the thermostat was 20’ f 0.05”. In order 
to obtain a high concent,ration of urechrome in the diffusion cell, 
acidified methanol was the solvent used in the dye experiments. 
Relative concentrations on the two sides of t,he membrane were 
measured with an Evelyn photoelectric calorimeter. 
Three determinations of t’he diffusion constant of urechrome 
gave L&O , methnnol = 0.363, 0.366, and 0.377 sy. cm. per day; aver- 
age, 0.368. Assigning t,he values 0.0062 to the viscosity of the 
solvent and 1.3 to t.hc density of the pigment, one arrives at an 
approximat,e molecular weight of 1700. The method of calcula- 
tion is given by Northrop and Anson (5). Since the calculation 
is based on the assumptions of a spherical molecule and the 
absence of solvation and ionizat,ion effects, the strict validity of 
which is in most cases douht’ful, this value is to be taken as an 
approximation only. 
N. H. Horowitz and J. P. Baumberger 413 
The molecular weight from diffusion data is in fair agreement 
with t*hat calculated from hydrogenation experiments. Ure- 
chrome is rapidly and reversibly reduced by hydrogen gas in the 
presence of finely divided plabinum. From the potentiometric 
data it is known that the reduction involves I hydrogen atom per 
molecule of pigment. By carrying out the reaction in Warburg 
manometers, the uptake of hydrogen can be measured and the 
equivalent weight of pigment calculated. It was found that 
8.18 c.mm. of Hz are taken up per mg. of pigment,. The weight 
of pigment equivalent to 1 gm. at.om of hydrogen is then 
$ x 22,400 
= 1370 
8.18 
DISCUSYIOK 
Crechrome is an autoxidizable pigment of relatively high poten- 
tial. Evidence for its participation in the respimtion of the egg 
cell has been presented in a previous paper. Kecause of t,he 
difficulty of obtaining sufficient quantities of the suhstancc, fur- 
ther chemical studies of t#he molecule have not been possible. The 
available data, however, suggest that it may be related to the 
heme pigmenbs. Such a hypothesis is consistent \?;it,h the observed 
valence change of 1 on reduction, with the absorption spectra of 
the derivatives studied, and with the solubility properties of the 
pigment. That it is not a typical heme pigment,. however, is 
evidenced by the absence of characteristic absorption bands in 
solut.ions of the untreated dye, as well as by the molecular weight, 
which appears to be too high for that of a free metalloporphyrin 
and too low for that of a chromoprotein. The fact, that the 
pigment is not cxtract,ed from the cell by aqueous solTents, 
alt.hough it is soluble in t,hem, suggests t.hat, as oht,ained in extracts, 
it ma.y represent a fragment of a larger molcculn. This would 
explain the molecular weight and possibly other properties of the 
substance. 
Concerning the biological significance of urechrome, the out- 
standing fact is that it has been found only in the eggs. Its poten- 
tial, which is in the range of the cytochromes, and its autoxidizabil- 
ity indicate that it intermediates between oxygen and the reducing 
systems of the cell. It is notewort,hy that spectroscopic examina- 
tions made by us of both living eggs and extracts have failed to 
414 Respiratory Pigment of Urechis 
reveal the absorption bands of cytochrome. The studies of 
Brachet (7) and of Ball and Meyerhof (8) on the eggs of other 
species have given similar results. As is pointed out by the 
latter authors, this does not constitute proof that the cytochromes 
are not present, since they may occur in quantities too small for 
detection by ordinary methods. They suggest the possibility 
that a hemin other t.han cytochrome may be involved in the respi- 
ration of the egg. The present work constitutes evidence that 
such may be the case in the eggs of Urechis. On the basis of 
microscopic examinations, Baumberger and Michaelis (9) have 
concluded that pigmented granules present in these eggs are 
derived from the hematin of the blood. It appears likely that 
these granules are to be identified with urechrome, although this 
would be difficult to prove. It has been observed (10) that 
during the embryonic development of the egg red granules become 
localized in the ciliated regions of the embryo, suggesting a rela- 
tionship with the ciliary metabolism. 
SUMMARY 
1. Urechrome, the red pigment of the eggs of Urecizis CCLU~O, is a 
reversible oxidation-reduction system. At pH 7 E’, = +0.186 
volt; ‘iz = 1. 
2. Solutions of the untreated pigment show no specific absorp- 
tion in the visible region. In concentrat,ed sulfuric acid bands 
appear at 553 and 688 rnp. Reduction in alkaline pyridine solu- 
tion brings out the spectrum of a pyridine hemochromogen. 
3. The approximate molecular weight determined from diffusion 
experiments is 1700. 
4. It is suggested that the pigment is related to the hemins. 
5. ‘1’1~ possible physiological rBle of urechrornc is discussed. 
One of us (N. H. H.) desires to express his gratitude to l’rofessol 
D. M. Whitaker and Professor C. B. van Niel of Stanford Uni- 
versity and the Hopkins Marine Station for their generous aid 
during his residence there. 
BIBLIOGRAPHY 
1. Horowitz, N. II., Z’roc. Nat. Acad. SC., 26, 161 (1940). 
2. Miiller, 0. I-I., and Baumbergcr, J. P., TT. Electrrochem. Sot., 71, 169, 
181 (1937). 
N. H. Horowitz and J. P. Baumberger 415 
3. Kolthoff, I. M., and Lingane, J. J., Chem. Rev., 24, 1 (1939). 
4. Mtiller, 0. H., Chem. Rev., 24, 95 (1939); Cold Spring Harbor symposia 
on quantitative biology, Cold Spring Harbor, 7, 59 (1939). 
5. Northrop, J. H., and Anson, M. L., J. Gen. Physiol., 12, 543 (1929). 
6. Anson, M. L., and Northrop, J. H., J. Gen. Physiol., 20, 575 (1937). 
7. Brachet, J., Arch. biol., 46, 611 (1934). 
8. Ball, E. G., and Meyerhof, B., J. BioZ. Chem., 134, 483 (1940). 
‘3. Baumberger, J. I’., and bIichaclis, I~., Biol. Bull., 61, 417 (1931). 
10. IIorowitz, N. II., Thesis, California Institute of Technology (193Q). 


Studies on the respiratory pigment of Urechis eggs

Cold Spring Harbor symposia on quantitative biology,

http://authors.library.caltech.edu/11854/1/HORjbc41b.pdf

Studies on the respiratory pigment of Urechis eggs

Abstract

Similar works

Full text

Available Versions

Caltech Authors - Main

Caltech Authors - Main