THE PHOTOCHEMICAL SPECTRUM OF THE PASTEUR ENZYME IN RETINA* The aerobic ...

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THE PHOTOCHEMICAL SPECTRUM OF THE PASTEUR
ENZYME IN RETINA*
BY KURT G. STERN AND JOSEPH L. MELNICKT
WITH THE COLLABORATION OF DELAFIELD DUBOIS
(From the Laboratories of Physiological Chemistry and of Physiology, Yale
University School of Medicine, New Haven)
(Received for publication, January 7, 1941)
The aerobic glycolysis of mammalian tissues is increased by
carbon monoxide. This effect is reversible and photosensitive
(2, 3). A primary CO inhibition of respiration is not the cause,
since the respiratory rate remains unaltered under these conditions
(3). The hypothesis that the Pasteur reaction, i.e. the check of
fermentation by oxygen, is mediated by a specific catalyst con-
taining heavy metal is strengthened by the selective inhibition
of the effect by ethyl isocyanide (4). The term Pasteur enzyme
is herewith proposed for this thermolabile (5), negative catalyst.
In order to determine the chemical constitution of the enzyme, its
spectrum has been studied by Warburg’s photochemical method
(6). This method utilizes the affinity of ferrous iron for carbon
monoxide and the reversible photodissociation of iron-carbonyl
complexes. Since only that fraction of incident light which is
absorbed can exert a chemical effect, the efficiency of monochro-
matic radiation of constant intensity will be proportional to the
degree of absorption of light of a given wave-length by the FeCO
complex. When continuous illumination is used and the photo-
chemical effects of the various wave-lengths are referred to one
wave-length chosen arbitrarily as the reference standard, the
reihtive spectrum of the catalyst is obtained. This yields the
* This work was aided by a grant from the Jane Coffin Childs Memorial
Fund for Medical Research. A preliminary report has been published (1).
Some of the data were presented before the Thirty-fourth annual meeting
of the American Society of Biological Chemists (1940).
t Finney-Howell Research Foundation Fellow, 193941.
301
This is an Open Access article under the CC BY license.
Spectrum of Pasteur Enzyme
pattern and the positions of the absorption maxima. The absolute
spectrum in terms of extinction coefficients referred to 1 gm. atom
of iron is determined with the aid of intermittent illumination.
The photochemical efficiency spectrum has been shown to be
identical with the absorption spectrum of iron-carbonyl complexes
(7). The present paper deals with the relative spectrum of the
Pasteur enzyme in the region from 405 to 655 mp.
The arrangement of the experiments is briefly the following.
Rat retinas are suspended in a medium containing bicarbonate and
glucose; they are then equilibrated in the dark with a gas mixture
containing CO, 02, and COZ. With the inhibition of the Pasteur
effect by CO, the already considerable aerobic glycolysis of this
tissue is further increased to values approaching the level of an-
aerobic glycolysis. Each molecule of lactic acid formed by the
tissue liberates 1 molecule of CO2 from the bicarbonate of the
medium. The resulting increase in pressure is measured with the
aid of a differential manometer. Upon illumination with mono-
chromatic light of known, high intensity, the rate of lactic acid
production and of the subsequent COZ formation is decreased to
an extent depending on the intensity and the wave-length of the
radiation employed. The photochemical efficiency of each wave-
length is compared with the effect of the reference wave-length
436 mp on the same retinas.
EXPERIMENTAL
InjIuence of CO on Metabolism of Retina Tissue
Laser’s experiments on the effect of CO on tissue metabolism (3)
were conducted at 38’; Warburg and Negelein (S), however, had
already demonstrated a marked effect of light on the glycolysis
of CO-treated rat retinas at 25”. In view of the finding of Kubo-
witz and Haas (9) that the light sensitivity of the CO compound
of the respiratory enzyme is increased upon lowering the tempera-
ture, the present photochemical experiments were carried out at
26.6’. This made it necessary to repeat the experiments of Laser
at this temperature. With Warburg’s two vessel method and
Summerson’s modification of the Dixon-Keilin method, it was
found that at 26.6”, just as at 38”, the respiration is not affected by
replacing a gas mixture of 95 per cent O2 and 5 per cent CO2
by one of 10 per cent 02, 5 per cent COZ, and 85 per cent CO.
K. G. Stern and J. L. Melnick 303
However, the glycolyxis approaches the anaerobic level in the
presence of the CO. In the absence of 02, CO has no stimulat-
ing effect on glycolysis. The data arc summarized in Table I.
Photochemical Apparatus and Procedure
The arrangement of the photochemical apparatus is schemati-
cally represented in Fig. 1. It follows essentially the design of that
used by Warburg and his associates (9, 11).
The glycolysis is measured with the aid of a differential man-
ometer with cylindrical vessels of the form sketched. Four to
TABLE 1
Selective Inhibition of Pasteur Reaction in Retina by CO
1 At 26.6” (present experiments)
I
Atmosphere Two vessel Dixon-Keilin- (LaserA t 3(38,” 10))
method of Sll~l~hWLXl
Warburg
I
QG QG QC
95% N 2, 5% co2 .................. 24* 30* I 88
95% co, 5% “ .................. 25*
85yo “ 10% og, 5% co2 .......... 22 31.5 79
85yo N,, 10% “ 5% “ .......... 1G 18 70
05% 02, 5% coz .................. 11 12 45
Qo, Qoz Qo2
85% CO, 10% 02, 5% COz .......... 9 9 31
85% N,, 10% “ 5% “ .......... 9 11 31
(35% 02, 5% co2 .................. 10 9.5 31
* These values were obtained with simple Warburg-Barcroft manometers
at the same time when the other determinations in the same series were
performed.
six freshly removed rat retinas are placed in one vessel containing
2 cc. of glucose-Ringer-bicarbonate solution. The Ringer’s so-
lution contains 96 cc. of 0.9 per cent NaCl, 2 cc. of 1.22 per cent
CaCL, 2 cc. of 1.15 per cent KCl, 20 cc. of 1.3 per cent NaHC03
(through which COZ has been previously passed), and 240 mg.
of glucose. A control experiment in which the glucose concentra-
tion was increased S-fold (600 mg. per 100 cc. of Ringer’s solution)
yielded the same results as with the smaller glucose concentration,
which indicates that during the photochemical experiments the
enzyme systems concerned were saturated with substrate. The
304 Spectrum of Pasteur Enzyme
first experimental reading is taken 20 to 25 minutes after the gas
flow has been stopped, and the pressure has been equilibrated
against the atmosphere. The compensation vessel contains 2 cc.
of the Ringer’s solution. The volume of each vessel is 20.2 cc.,
so that the ratio of gas volume to fluid volume is large. Under
these conditions the respiratory exchange produces no appreciable
pressure changes; the positive pressures recorded result, therefore,
IIOV. 60N
PHOTO-CHEMICAL APPARATUS
FIG. 1. Schematic diagram of the photochemical apparatus (not drawn
to scale).
from lactic acid formation and the subsequent liberation of carbon
dioxide from the bicarbonate-containing medium.
The cross-section of the manometer capillary is 0.5 sq. mm. ;
the capillary is filled with isocaproic acid (11,160 mm. = 760
mm. of Hg) or with Brodie’s fluid (10,000 mm. = 760 mm. of
Hg). To facilitate the filling of the capillary a stop-cock is in-
serted at the base of the U-shaped capillary tube.
The manometer is rotated around its vertical axis by means of
K. G. Stern and J. L. Melnick 305
a motor in connection with an eccentric disk, with the result that
the vessels make excursions of 1 to 2 mm. amplitude about a
fixed point in the thermostatically controlled bath. The usual
rate of rotation is 350 revolutions per minute. The pressure
changes taking place are automatically recorded during the ex-
periment by photographing the levels of the manometer fluid at
regular intervals with a camera fitted with a specially corrected
long focus lens of 5 cm. aperture df = 82 cm.). The magnification
ratio is 0.8. The photographic recording process is controlled
by a clockwork in conjunction with relays and a solenoid switch.
When the minute hand of the clock makes contact with metal
posts which can be spaced at distances corresponding to intervals
of 1 to 10 minutes, an electromagnet becomes energized, and the
following sequence of events takes place. (1) The shutter of the
camera opens. (2) A gas-filled fluorescent sign tube mounted on
the back of the manometer is lighted for a fraction of a second by
means of a 110 volt D.C. impulse sent through a transformer (9000
volts; 0.045 ampere). (3) The shutter is closed. (4) The break-
ing of the direct current connection through the transformer
causes the sign tube to flash again. (5) The film in the camera
is advanced by a motor to receive the next image. (6) The motor
is stopped automatically. The flash of the sign tube caused by
the “make” charge during step (2) is rapid enough and of suffi-
cient intensity to record an unblurred image of the levels of the
manometer fluid on the film (Eastman positive, 35 mm.) without
stopping the motion of the manometer. The second flash (step
(4)) is not recorded because the shutter has been closed in the
meantime. The films are later read either with the aid of a
Bausch and Lomb measuring magnifier or by projection of the
film on a graduated screen.
Isolation of Monochromatic Radiation
The light sources used include a high pressure mercury arc
lamp (General Electric, Type H-3), an electrical sodium lamp
(General Electric Vapor Lamp Company, sodium Labarc), and
a high intensity carbon arc lamp modeled after that of Kubowitz
and Haas (9). The arc is operated on 220 volts D.C. and 50 to 75
amperes. It is stabilized by an air draft produced by an oil burner
fan, a pair of electromagnets on either side of the arc, and by
Spectrum of Pasteur Enzyme
mechanically rotating the water-cooled anode during the experi-
ment. The light emission is varied by means of anode carbons,
the cores of which are filled with various salts (Ca, Sr, Li, Mg, Cu).’
The cathode carbons were usually copper-coated. A diagram of
the lamp will be found in the paper of Kubowitz and Haas (9).
During the experimental light periods the carbon arc is regulated
by advancing the cathode and the rotating anode by hand. By
reflecting, with an inclined clear glass plate inserted in the light
path, a small part of the light upon a Weston photronic cell con-
nected to a galvanometer, it is possible to keep the light intensity
constant by maintaining a constant deflection of this galvanometer
through manual control of the electrodes. This is facilitated by
projecting an enlarged image of the burning electrodes on a screen.
With the aid of Dr. L. H. Ott, an attempt was also made to utilize
high voltage sparks between magnesium and tungsten electrodes
in nitrogen and carbon dioxide atmospheres respectively. How-
ever, the equipment available to us yielded radiation of insufficient
intensity. The light coming from the various sources is rendered
parallel by suitable biconvex glass lenses made by Bausch and
Lomb. The light filters employed include polished, colored glasses
(Corning and Schott filter glasses), and solutions of dyestuffs
and inorganic salts contained in plane-parallel cemented glass
cells. The mirrors are first surface silver mirrors on optical flats
(Bausch and Lomb). All optical parts are mounted on riders on
optical benches (Fuess). The manometer vessels are immersed
in a closely controlled thermostat equipped with glass windows.
The light enters the vessel containing the tissue from below with
the aid of an adjustable mirror placed in the tank. The illumina-
tion is uniformly spread over an area larger than that covered by
the vessel during its excursions. The intensity of the light in
gm. calories per sq. cm. per minute is measured before and after
each experiment by means of a carefully shielded, calibrated Moll
thermopile (Kipp and Zonen) in conjunction with a Leeds and
Northrup mirror galvanometer (sensitivity, 0.1 microvolt = 10
mm. deflection). By connecting a 1.5 volt battery in series with a
500,000 ohm resistance, and then placing this circuit in parallel
1 The special carbon electrodes used in this work were made by the
Siemens-Plania works which had furnished the carbons for Warburg’s work.
*A number of the dyes used were kindly given to us by the National
Aniline and Chemical Company, Inc.
K. G. Stern and J. L. Melnick
with a 1 ohm resistance in the thermopile-galvanometer circuit, one
may check the sensitivity of the galvanometer during the energy
measurements. Because of zero drifts of the galvanometer it is
necessary to admit an opposing small voltage (of the order of 1OV
volt) which is controlled by a potentiometer as illustrated in Fig. 1.
The intensity of the light beam is measured before it enters
the thermostat. The loss due to reflection and absorption by the
windows and water is estimated with the aid of a photoelectric
cell (W&on) which may be held in the bath in place of the experi-
mental vessel. A record of the spectral purity of the radiation
is obtained with a Hilger glass spectrograph. The photometry
of the spectrograms enables one to determine the spectral purity
of the light. The wave-lengths are checked by photographing
the mercury spectrum in juxtaposition with the spectrum of the
experimental radiation. The absence of infra-red light is ascer-
tained by photographing the spectrum of the filtered radiation
on infra-red-sensitive plates (Eastman No. III-L). Table II
lists the wave-lenths of light employed in the present study, to-
gether with the source of light, the filters necessary for their isola-
tion, and the intensity of the light beam before entering the ther-
mostat.
Determination of Photochemical E$iciency Ratios (Px/&)
The method of charting photochemical absorption spectra has
been described by Warburg (6). In the present case the photo-
chemical effect consists in the relief of the CO inhibition of the
Pasteur reaction; that is to say, glycolysis in the presence of 85
per cent CO and 10 per cent 02, in the dark, is enhanced, and the
illumination of the system tends to diminish the rate of glycolysis.
When the intensities of two wave-lengths of light which will
produce the same photochemical effect are known, the relative
light absorption coefficient as referred to a standard wave-length
may be calculated, for the ratio of the absorption coefficients is
the reciprocal of the ratio of the light intensities times the wave-
lengths.
al/L% = iPXllilX1
This equation is based on fundamental quantum relationships and
has been experimentally verified for the case of FeCO complexes
by Warburg and Negelein (7).
An additional factor must be considered in the case of retina,
308 Spectrum of Pasteur Enzyme
TABLE II
Isolation of Monochromatic Light Used in Photochemical I 9eriments
Filters ntemity X 104
m. Cal&e per
mr . cm. pm min.
405 !&ercury lamI Noviol shade 0,2.7 mm., Corning 4.2
No. 306; red-purple ultra, 3.8
mm., Corning No. 597; light
blue-green, 2.0 mm., Corning
No. 428
407 Strontium- Water, 5 cm.; ammoniacal copper 3.0
carbon sulfate (25 gm. CuS04+5HsO +
1000 cc. H20 + 55 cc. 25%
NH*OH), 2.5 cm.; noviol shade
0, 2.7 mm., Corning No. 306;
red-purple ultra, 3.8 mm.,
Corning No. 597
436 Mercury iamr Extra light aklo, 2.3 mm., Corn- 1.54.0
ing No. 395; noviol shade A, 2.5
mm., Corning No. 038; violet,
2.0 mm., Corning No. 511; am
maniacal copper sulfate (as 407
mL)
42%459* Zalcium- Water, 5 cm.; copper sulfate, t 1.5
carbon lo’%, 2.5 cm. ; extra light aklo,
2.3mm., Corning No. 395; vio-
let, 2.0 mm., Corning No. 511;
noviol shade A, 2.5 mm., Corn-
ing No. 038
Clopper-car- Water, 5 cm. ; ammoniacal copper 1.1
bon sulfate (as 407 mp); copper sul-
fate, lo%, 2.5 cm.; noviol shade
A, 2.5 mm., Corning No. 038;
violet, 2.0 mm., Corning No.
511; dark blue-green, 3.9 mm.,
Corning No. 430
431462s Strontium- Same as 430470 rnr from copper- 1.0
carbon carbon
45711 Magnesium- Water, 5 cm.; ammoniacal copper 2.3
carbon sulfate (as 407 mp); noviol
shade A, 2.5 mm., Corning No.
038; dark blue-green, 3.9 mm.,
Corning No. 430; blue-purple
ultra, 3.1 mm., Corning No.
585; extra light aklo, 2.3 mm.,
Corning No. 395
K. G. Stern and J. L. Melnick 309
TABLE II-Conlinued
wsv&!ngtt Light *oure* Filters ‘ntensity x 10’
rm. calae per
WJ ,. cm. per min.
460 Lithium-car- Water, 5 cm. ; ammoniacal copper 5.8
bon sulfate (as 407 mp); noviol.
shade A, 2.5 mm., Corning No.
038; extra light aklo, 2.3 mm.,
Corning No. 395
487, 4971 Strontium- Water, 5 cm.; Guinea green B** 4.3
carbon (18 mg. in 100 cc.), 2.5 cm.; ex-
tra light aklo, 2.3 mm., Corning
No. 395; H. R. lantern blue, 2.8
mm., Corning No. 554; noviol
shade C, 4.5 mm., Corning No.
338
494 Magnesium- Water, 5 cm.; Guinea green (as 3.8
carbon 487 mr); light blue-green, 2.0
mm., Corning No. 428; H. R.
lantern blue, 2.8 mm., Corning
No. 554; noviol shade C, 4.5
mm., Corning No. 338
517 I‘ I‘ Water, 5 cm.; copper sulfate, 4.6
lo’& 2.5 cm. ; Guinea green (as
487 mp); noviol shade D, 2.0
mm., Corning No. 338D
515,522t. Copper-car- Same as 517 mp + extra light 7.5
bon aklo, 2.3 mm., Corning No. 395
525 Strontium- Water, 5 cm.; Guinea green (as 10.9
carbon 487 mp); tartrazine** (64 mg.
+ 100 cc. water), 2.5 cm.; light
blue-green, 2.0 mm., Corning
No. 428
546 Mercury lamI Extra light aklo, 2.3 mm., Corn- 13.6
ing No. 395; didymium, 5.0
mm., Corning No. 512; H. R.
illusion pink, 4.6 mm., Corning
No. 592; yellow shade yellow,
2.0 mm., Corning No. 351; cop-
per sulfate, lo%, 2.5 cm.
553 Magnesium- Water, 5 cm.; copper sulfate, 5.1
carbon 24%, 2.5 cm.; H. R. yellow
shade yellow, 2.0 mm., Corning
No. 351; didymium, 14.0 mm.,
Jena No. BG-11
310 Spectrum of Pasteur Enzyme
‘FABLE II --C’ontinued
Wave-length Filters ntensity X 104
m. cnlori.9 per
mr
I. cm. per min.
560 Calcium-car- Water, 5 cm.; copper sulfate, 7.1
bon lo%, 2.5 cm.; H. R. yellow
shade yellow, 2.0 mm., Corn-
ing No. 351; didymium, 14.0
mm., Jena No. BG-11; green,
2.0 mm., Jena No. VG-3
578 Mercury lam1 Extra light aklo, 2.3 mm., Corn- 3.6
ing No. 395; H. R. red shade
yellow, 2.0 mm., Corning No.
348; copper sulfate, 1070, 2.5
cm.
582 Strontium- Water, 5 cm.; copper sulfate, 2.8
carbon 240/o, 2.5 cm. ; H. R. red shade
yellow, 2.0 mm., Corning No.
348
589 Sodium lamp Ferrous sulfate,$$ 20% in 10% 5.4
H2S04, 2.5 cm.; H. R. red shade
yellow, 2.0 mm., Corning No.
348
597 Strontium- Water, 5 cm. ; copper sulfate, 7.1
carbon lo%, 2.5 cm.; H. R. lighthouse
red, 3.2 mm., Corning No. 246
“ IL
640-655 Water, 5 cm.; ferrous sulfate (as 7.6
589 mr); copper sulfate, lo%,
2.5 cm. ; H. R. signal red, 2.1
mm., Corning No. 243
640-650 Calcium-car- Same as 640--655 rnp from stron- 9.5
bon tium-carbon
The thickness of the impregnated anode carbons was 20 mm. except in
the instance of the lithium-carbon which was 12 mm. The cathode carbons
were copper-coated and, for the most part, copper-filled. Their thickness
varied from 8 mm. to 14 mm., 8 mm. being the size usually employed.
These latter electrodes were kindly supplied by the National Carbon Com-
pany, Inc., Cleveland, Ohio.
* The following lines are included: 428, 429, 430, 431, 432, 436, 443, 453,
458, and 459 mp.
t CuSOa .5H20.
$ The multiple line spectrum and the strong background emission in this
region produce a fairly continuous spectrum between these wave-lengths.
8 Lines at 431, 433, 434, 436, 444, 453, 457, and 462 m.q and rather pro-
nounced background emission.