International Journal of Radiation Biology and Related

Studies in Physics, Chemistry and Medicine

ISSN: 0020-7616 (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/irab19

The Inactivation of SH Enzymes by x-rays

Rolf Lange, Alexander Pihl & Lorentz Eldjarn

To cite this article: Rolf Lange, Alexander Pihl & Lorentz Eldjarn (1959) The Inactivation of SH
Enzymes by x-rays, International Journal of Radiation Biology and Related Studies in Physics,
Chemistry and Medicine, 1:1, 73-79, DOI: 10.1080/09553005914550111

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 The inactivation of SH e n z y m e s by x-rayst
ROLF LANGE~, ALEXANDERPII-IL and LORENTZELDJARN
Norsk Hydro's Institute for Cancer Research,
The Norwegian Radium Hospital, Oslo, Norway

(Received 31 December 1958)

The susceptibility of SH-enzymes to the indirect effect of ionizing

radiation has been re-investigated. The claims of previous authors that
these enzymes are particularly radiosensitive could not be confirmed. Dilute
solutions of crystalline glyceraldehyde-3-phosphate dehydrogenase and yeast
alcohol dehydrogenase were found to be inactivated by x-rays, with ionic
yields of about 0'02. In contrast to previous reports no reactivation was
obtained by the addition of reduced glutathione after the irradiation.
The discrepancy between the present results and those of earlier investi-
gators can in part be explained by the fact that the earlier estimations of ionic
yields were based on molecular weights for the enzymes which differ by a
factor of about 2 from those currently accepted. The present data seem to be
consistent with a number of radiochemical and enzymologicalfindings.

1. INTRODUCTION
The findings of Barron and Dickman (1949), Barron et al. (1949, 1954),
Barton and Johnson (1954) that SH-enzymes, in contrast to enzymes not requiring
free SH-groups for activity, are exceptionally susceptible to the indirect action of
ionizing radiation has been extensively cited, and these papers initiated a great
interest in the role of SH-enzymes in the mechanism of radiation damage.
Barron et al. (1949, 1954) and Barron and Johnson (1954) reported that
crystalline phosphoglyceraldehyde dehydrogenase (GAPDH) and alcohol
dehydrogenase (ADH) were inactivated by x-rays in dilute aqueous solution
with an ionic yield of about 1. G A P D H could be protected by the presence of
reduced glutathione (GSH), and it was reported that the activity of the irradiated
enzyme could be partially restored by the addition of G SH after the irradiation.
The above findings seemed reasonable, since it was found that small molecular
thiols were oxidized to the corresponding disulphides with an ionic yield of 3-4
(Barron and Flood 1950). It was assumed that, in the case of the SH-enzymes,
the predominant part of the radiation lesions involved the oxidation of the
SH-groups to SS-groups, which were thought to be radiochemically stable.
However, recent studies of proteins and enzymes irradiated in solution have
demonstrated that in addition to the extensive radiochemical reactions of
the sulphur atoms, radiochemical changes occur at a number of other chemical
groupings as well (Drake et al. 1957, Okada and Gehrmann 1957, Barron et al.
1955). Furthermore, disulphides are also highly susceptible to the indirect
action of ionizing radiation (Shapiro and Eldjarn 1955). In view of these facts
it seemed improbable to the present authors that any enzyme could be inactivated
J" Supported by Landsforeningen mot Kreft, Oslo, Norway.
Fellow of Landsforeningen mot Kreft, Oslo, Norway.
74 R. Lange et al.

with an overall yield of 1, since this implies that one enzyme molecule should be
inactivated per ion pair produced in solution. Such a finding appears particularly
unlikely in the case of G A P D H and ADH, since these enzymes are known to
possess a considerable number of free SH-groups. A re-investigation of the
inactivation of SH-enzymes by ionizing radiation was therefore found desirable.
A preliminary note on this work has appeared elsewhere (Pihl et al. 1958).

2. EXPERIMENTAL
2.1. Materials
For the experiments, conductivity water obtained by double distillation
from glass equipment and passage through a mixed-bed ion-exchange column
was used. All solutions were equilibrated with air.
The buffer constituents were of the highest analytical grade. Diphospho-
pyridine nucleotide (DPN) and D-3-phosphoglyceraldehyde (GAP) were
obtained from Boehringer & Soehne G.m.b.H., Mannheim-Waldhof. The
D P N (98-100 per cent pure) was 85 per cent enzymatically active, due to the
admixture of the inactive ~-isomer. The phosphoglyceraldehyde was obtained
in solution together with dihydroxyacetone. The aldehyde concentration, as
titrated by thiazolidine formation with a'~S-cysteamine, was found to be 0.018 M.
Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle was prepared
according to Velick (1955), and recrystallized five times. Usually, all solutions
used for preparation of the enzyme contained 0.002 M disodium ethylenediamine-
tetraacetate (EDTA).
Crystalline alcohol dehydrogenase was prepared from baker's yeast according
to Racker (1955).

2.2. Methods
Since the results set out in this paper disagree strongly with those of previous
reports, the experimental technique is described in some detail.

2.3. Enzymatic measurements

The crystalline G A P D H suspension was centrifuged at approximately
10.000 x G for 20 minutes at + 3°c. The supernatant was carefully removed,
and the crystals were dissolved in 0-007 M EDTA-free phosphate buffer, pH 7.4.
The enzyme concentration of this stock solution was determined by measuring
the optical density at 2.760~ (Velick 1955). For the irradiation experiments a
solution containing 70/zg G A P D H per ml (5 x 10-7M) was made by dilution
with the phosphate buffer.
The enzymatic activity was determined by measuring in a Zeiss spectro-
photometer the increment in optical density at 3.400 A. The quartz cells (light
path 1 cm) contained: 2-60ml 0"03 M pyrophosphate buffer, pIJ 8.45;
0.10ml DPN, 1 . 0 2 x l 0 - ~ M ; 0.10ml 0 . 4 M arsenate; 0.10ml GAP,
8.01× 10 -a M; and 0-10 ml of the enzyme solution (5 × 10 -7 M). The
reaction was started by adding the enzyme with rapid mixing of the reactants.
The increment in optical density during the first minute was taken as a measure
of the enzyme activity.
 The inactivation of S H enzymes by x-rays 75

In the alcohol dehydrogenase experiments the suspension of enzyme crystals

was centrifuged at 10.000 × G for 20 minutes at 0°c. The crystals were dissolved
in 0-01 M phosphate buffer ( E D T A free) at pH 7.4, and the enzyme concentration
was determined spectrophotometrically according to Warburg and Christian
(1942). For the irradiation experiments a solution containing 45 tzg A D H per ml
(3 x 10 -7 M) was made by dilution of the stock solution with the buffer. The
measurement of the enzymatic activity was carried out essentially as described
by Barron and Johnson (1954). To the quartz cells were added : 2-20 ml water ;
0.50 ml 0-1 M phosphate buffer, pH 8"86; 0"10 ml 0-033 M D P N ; and 0.10 ml
of the enzyme solution. The reaction was started by adding 0.10 ml of
absolute ethanol. After rapid mixing, the optical density at 3400 & was measured
every 15 seconds. The increment in optical density between the 15- and 45-second
readings, multiplied by two, was taken as the enzyme activity per minute (Racker
1955).
All measurements of enzyme activity were carried out at 22°c. Under the
above conditions the enzymes were found to be the rate-limiting factors, and the
activity was proportional to the enzyme concentration.

2.4. Irradiation conditions

The irradiation was carried out with conventional x-rays (' Stabilipan',
Siemens), using irradiation factors of 200 key, 4 mA and 0.5 mm Cu-filter. Three
millilitres of the solutions to be irradiated were pipetted into a 10 ml Erlenmeyer
flask, which was placed directly on the Cu-filter. The samples were irradiated
intermittently at a dose-rate of 234r per minute, each exposure lasting for
2.5 minutes (585 r). Between irradiations the enzymatic activity was rapidly
measured on aliquots, while the rest of the irradiated solution was kept in the
ice-box. Usually five to six doses were given. The activity of the irradiated
solutions was expressed as a percentage of that of the unirradiated controls,
which were treated identically. Since the controls showed no decline in activity
during the experimental procedure, it was found unnecessary to provide cooling
during the short exposures.
The dosimetry was carried out with a Victoreen chamber as well as with the
ferrous-ferric system (Baarli and Borge 1957), under conditions identical to
those used in the actual experiments.

3. RESULTSAND DISCUSSION
In the figure typical examples of the data obtained are demonstrated. Clearly,
the degree of inactivation of both enzymes is proportional to the radiation dose.
The curves correspond to ionic yields of 0.023 and 0.020 for G A P D H and ADH,
respectively.
In table 1 the ionic yields obtained in a series of experiments with G A P D H
and A D H are summarized. Each value given was calculated from an inactivation
curve like those demonstrated in the figure. It appears that the data, which stand
in striking contrast to those reported by Barron et al. (1949) and Barron and
Johnson (1954), are remarkably uniform, and the yields of inactivation are almost
equal for the two enzymes. The present yields fall in the same range as those
previously reported for various non-SH-enzymes (Dale 1942, Forssberg 1947,
Dale et al. 1949, Barron et al. 1949, Barron and Dickman 1949).
76 R. L a n g e et al.

100
~ 'o I '' l I I

C-
O 80
0

fl_ 60
¢-

>, 4 0
>

o 20
<f

, I m I ~ I ~ ' I

1170 2340 3510 4680

Dose in r
Inactivation of glyceraldehyde-3-phosphate dehydrogenase ( G A P D H ) and alcohol
dehydrogenase (ADH) by x-rays. For conditions, see table 2.

Enzyme Number of Average

Specific
Enzyme preparation Treatment doses ionic
activityt
number tested yield{

1 3-8x 105 0.025

3.8x 105 0.020
2 3.8x 105 0.023
3 3-8× 105 0.023
4 Crystallization No. 1 3"2 x 105 0"023
4 . . . . 2 3"5 x 108 0"023
GAPDH 4 ~ ~ 3 3"8× 10 ~ 0'023
4 ,, ,, 4 3-8× 105 0.023
4 ~ ~ 5 3.8x 105 0.023
EDTA-free
preparation 3"8 x 105 0.027
Dialysis for 20 hrs. 3'8 x 105 0"046
G S H (10 2 M) added
after irradiation 3"8 x 10 5 0"023

1 . 8 1 x l0 s 0.020
ADH Ageing 7.60 × 104 0"020
4.65 x 104 0-020
r
t Calculated according to Velick (1955) and Racker (1955) for G A P D H and A D H
respectively.
1 r is assumed to give 3 x 10 -9 M ion-pairs in solution.
Table 1. T h e inactivation of glyceraldehyde-3-phosphate dehydrogenase ( G A P D H ) and
alcohol dehydrogenase (ADH) by x-rays.
 The inactivation of S H enzymes by x-rays 77

In the case of G A P D H a number of different enzyme preparations gave

almost the same yield. In one case the E D T A had been omitted from all solutions
used during the preparation of the enzyme ; the observed yield was only slightly
increased. Upon extensive dialysis of the enzyme in the cold room the ionic yield
increased by a factor of 2. The data demonstrate that the difference between the

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

Barron et al. Barron et al. Present

(1949) (1954) experiments

Enzyme conc. 70/zg/ml 16.7/zg/ml (?) 70/zg/ml

Irradiation Buffer Phosphate, 0"033 M Buffer or water Phosphate, 0'0667 M
conditions pH 7'0 ? 7.4
Temperature 0°c 0°c + 3°c

Buffer Pyrophosphate Pyrophosphate, Pyrophosphate,

1 0 -2 M 2"6 x 10 -2 M
pH 8.5 ? 8"4
Assay DPN 1 . 6 7 x 1 0 -8 M 1"67 x 1 0 -5 M 3"4 x 10 -4 M
conditions Arsenate 4 x 1 0 7M 2"0 x 10 -4 M 1"3 x 10 .2 M
GAP 4 x 10 -8 M 4 x 10 -5 M 2"64 x 10 .4 M
Enzyme conc. 7/zg/3 ml 5/zg/3 ml 7/zg/3 ml
Enzyme M.W. 70000 137000

Ionic yield
(M.W. = 137000) 0"35 0"08 (?) 0"023

Alcohol dehydrogenase (ADH)

Barron and Johnson Present

(1954) experiments

Specific activity of ? 1"81 x 105 to

enzymes 4.65 x 104
Irradiation Enzyme 43"8/zg/ml 45"0/zg/ml
conditions Buffer Phosphate, 0"01 M Phosphate, 0'01 M
pH 7-6 (?) 7.4
Temperature 0Oc 0°c

Buffer Pyrophosphate, 0"01 M Pyrophosphate, 0'017 M

pH 8'86 8"86
Assay DPN 4"4 x 10 3 M 1"1 x 10 -3 M
conditions Ethanol 0'56 M 0.56 M
Enzyme 4"38 ~g/3 ml 4"5 pg/3 ml
Enzyme M.W. 70000 150000

Ionic yield
(M.W. 150000) 0'51 0"02

Table 2. Present experimental conditions compared with those of previous authors.

78 R. Lange et al.

present results and those of Barron et al. cannot be accounted for by the presence
of contaminating protective substances. With ADH, ageing of the enzyme
with a consequent decrease in the specific activity did not alter the ionic yield,
in spite of the fact that this procedure decreases the number of free titratable
SH-groups (Wallenfels and Sund 1957).
The reason for the discrepancy between the present results and those of
previous authors is not obvious. In the ADH experiments the experimental
conditions of Barton et al. were closely reproduced (table 2), the main difference
being that in our experiments the D P N concentration during the assay was ~ of
that used by Barron et al. Under our assay conditions the activity was proportional
to the concentration of the apoenzyme. It seems unlikely that the discrepancy
can be explained by different specific activity of the enzyme preparations studied ;
since we observed (table 1) that the ionic yield of inactivation is independent
within fairly wide limits of the specific activity of the ADH enzyme. The data
of Barton and Johnson (1954) do not permit a re-calculation of the specific
activity of their enzyme preparation. In the present calculation of the ionic
yields a molecular weight of 150 000 (Racker 1955) was used, whereas Barton et al.
used a molecular weight of 70 000. Consequently, if their data are re-calculated
on the basis of the more recent value, their ionic yield will be reduced to about 0.5.
In the case of GAPDH a detailed comparison of the present results with those
of Barron et al. is difficult; since the experimental procedure given differs
considerably in two reports presenting the same data (apparently a single
experiment only) (table 2). In this case Barron et al. also used a molecular
weight (70 000) which differs by a factor of about 2 from the one currently accepted.
If these data are re-calculated on the basis of the molecular weight 137000
(Fox and Dandliker 1956), ionic yields of 0.35 and 0"08 respectively are found.
As pointed out above, the x-ray-induced inactivation of SH-enzymes was
ascribed by Barron et al. (1949) to a radiochemical oxidation of SH- to S S-groups.
This view was supported by the finding that x-ray-inactivated GAPDH could
be partially reactivated by the addition of reduced glutathione after irradiation.
In the present experiments G SH treatment after the exposure consistently failed
to reactivate the enzyme.
In recent years a considerable body of knowledge of the radiochemistry of
proteins in solution has accumulated (Barron et al. 1955, Drake et al. 1957,
Okada and Gehrmann 1957, Jayko and Garrison 1958). Drake et al. found
that on irradiation of insulin in dilute aqueous solution at different pH values
a number of different amino acids were destroyed. Cystine was most extensively
degraded; but tyrosine, phenylalanine, proline, and histidine were also found
to be very radiosensitive. It is thus clear that, although SH- and SS-groups
are particularly vulnerable to the attack of free radicals, a number of other
chemical groupings in proteins will suffer radiation damage.
Even if the predominant part of the radiation effects in the case of ADH and
G A P D H involve the sulphydryl groups, a relatively low overall ionic yield of
inactivation would be expected; since it is known that GAPDH possesses
8-15 free SH-groups (Boyer and Segal 1954), whereas ADH possesses 4.5-36
SH-groups, the number of which is linearly related to the enzymatic activity
(Wallenfels and Sund 1957). An overall yield of inactivation of 1 would seem
to require a chain mechanism which is difficult to visualize in the case of protein
SH-groups, the reactivity of which is strongly restricted by sterical factors.
 The inactivation of S H enzymes by x-rays 79

On a fait de nouvelles recherches sur la susceptibilit6 des enzymes SH aux effets

indirects des radiations ionisantes. Ces recherches n'ont pas confirm~ les affirmations
d'autres savants que ces enzymes soient particuli~rement radiosensibles. On a trouv6
qu'une solution dilute de glyc6rald~hyde-3-phosphate d~hydrog6n6 cristallin ou d'aleool
d6hydrog6n~ de levure fur inactivis~e par des rayons x avec un rendement ionique d'environ
0"02. Contrairement aux rapports existants, on n'a trouv~ aucune r6activation en ajoutant
de la glutathione apr~s l'irradiation.
La diff6rence entre les r6sultats de ces derni~res recherches et ceux des reeherehes
ant~rieures s'explique en partie par le fait que les calculs d~j~ publi~s ont ~t6 bas6s sur un
poids mol6culaire pour les enzymes qui diff~re par un facteur d'environ 2 des poids actuelle-
ment accept~s.
Le r6sultat auquel on est arriv6 dans ces recherches s'accorde, paratt-il, avec les r6sultats
de recherches radio-chimiques et enzymologiques.

Man hat eine erneute Untersuchung der Empfiinglichkeit der SH-Enzyme einem
indirekten Effekt der ionizierender Bestrahlung gegentiber unternommen.
Die Behauptung frtiherer Forscher, dass diese Enzyme besonders radio empfindlich
sein sollten, wurde nicht best~itigt. Eine verdiinnte L6sung yon kristallierter Glyceral-
dehyde-3-Phosphate Dehydrogenase und Alkohol Dehydrogenase aus Hefe wurde, nach
unseren Untersuchungen, dutch eine R6ntgen-Bestrahlung mit einem Ionen-Ausbeute
yon 0.02 inaktiviert.
Im Gegensatz zu friiheren Berichten hat keine Reaktivierung durch einen Zusats yon
reduzierter Glutathione Each der Bestrahlung stattgefunden.
Die Abweichungen zwischen dem Resultat yon diesen Untersuchungen und den
Resultaten die friihere Forscher erreicht haben, lassen sich zum Teil dadurch erld~iren,
dass die friiheren Berechnungen yon dem Ionen-Ausbeute auf den Molekiilgewichten der
Enzyme ruhen, und diese Molekfilgewichte zeigen eine Abweichung yon den heute angenom-
menen Molekfilgewichten, und zwar im VerhMtnis 1 : 2 . Diese Daten zeigen auch
~bereinstimmung mit vielen radiochemischen und enzymologischen Untersuchungen.

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