ANAl.YTIIAI
92, ?- 10 (1979)
RICK HFMISTRY
REVIEW Size Determination
of Enzymes by Radiation
inactivation
E. S. KEMPNERANDWERNERSCHLEGEL of
Lahoruroric,s Narionul
In.vrirure National
Physicul
Biology
und
Received
From atomic physics it is known that the passage of radiation through matter is characterized by transfer of energy to the target material. For high energy radiation this occurs in discrete bursts called “primary ionizations. ” In each primary ionization, about Copyright All rights
‘F 1979 by Academic Pres. Inc. of reproductmn in any form rexrved.
and
und Digestive Mqvlund
Endocrinology. Diseases, 20014
66 eV of energy are transferred, corresponding to approximately 1500 kcal/mol. When X rays, y rays or high-energy electrons are used, ionizations occur randomly throughout the volume of the irradiated material. The probability of 0, 1,2, . . . n ionizations occurring in a unit volume is given by the Poisson formula e-*(x)” fJ(n) = ~ n! where x is a function of the amount of radiation exposure. If a biological material is irradiated, the same description applies. Exposure to ionizing radiation leads to a loss of function due to destruction of critical structures. The larger the structure, the more likely it will be “hit.” Because the energy deposition is so large, function is completely destroyed by a single hit; there are no partially damaged targets. Therefore, the only activity remaining after radiation exposure is due to units which have escaped ionization and these units are fully active. In the Poisson formula, the probability on no ionizations is P(0) = eps. It follows that the surviving biological activity, measured after exposure to different amounts of radiation, will decrease as a simple exponential function of dose,
THEORY
0003.2697/79/010002-09$02.00/O
Nutrition
July 21, 1978
The molecular size of an enzyme can be measured by a variety of physical techniques. Among these, ionizing radiation offers several unique advantages in that samples need not necessarily be purified and only minute amounts of enzyme are required. The method relies on measurement of the loss of biological activity with increasing radiation exposure. Since the diagnostic measurement is biological or biochemical activity, the technique describes a functional unit rather than the structural object that is determined by other methods. The target volume theory relating inactivation sensitivity to molecular weight (1) and its procedures (2), as well as applications of the technique, were widely published in the 1950s. Interest in the method waned when many of the results did not agree with those from other procedures. However, subsequent advances have vastly increased knowledge of protein structure. A reevaluation of the radiation studies now indicates the original validity of these results and reinforces the power of the technique. PHYSICAL
of
qf’Arthriris. MeraholiJm Institurc~s of Healrh. Bethesda.
A = AoeekD
where A is the activity measured after a radiation dose D. This exposure is given in 2
SIZE DETERMINATIONOF'ENZYMES
units of primary ionizations per cubic centimeter. The constant, k. is characteristic of each biological activity and has the units of volume (cubic centimeters). This is the radiation target or “sensitive volume” in which an ionization must occur in order to destroy the measured function. Since ionizations caused by such radiation occur at random throughout the volume of the sample, target volume is independent of the shape of the unit. Since so much energy is involved, radiation sensitivity is independent of chemical composition. This situation is entirely opposite to the effects of nonionizing radiation, e.g., ultraviolet light, where energy absorption and damage depends upon the presence of unique chemical sustituents, i.e., specific ring structures. disulfide bridges. or ligands. A direct measure of the target size, k, is obtained from the 37% dose. This is the specific radiation exposure, also known as the i’D37r” responsible for reducing the measured activity to 37% of that found in the unexposed controls: A = 0.37 A,, = A,,cJ-~~‘~;
In 0.37 = - 1 = -XD:,,
L =k. 037
3
mary ionization as 66 eV. This clarifies earlier confusion in the literature where values of 60 to 100 eV were quoted (1,2,4). Multiple radiation targets exist if the same function resides independently in several different structures. The inactivation is then described by an additive series of exponential terms. A = A II,e--klu + A 28 -kzLJ + . . . . For two targets of significantly different size, the survival curve can be resolved into two simple exponential functions from which both molecular weights can be calculated. By this means. the proportion of activity due to each unit is also revealed. This description constitutes the dirt~t action of radiation on matter, that is, the damage to functional units is caused entirely by primary ionizations in situ. In liquid solution so-called indirect effects can occur. For example, irradiation of water results in formation of H+. OH-, peroxide, and other radialytic products which, in the liquid state, can diffuse appreciable distances before reacting. Thus, inactivation of function can occur not only by the direct action of radiation, but also by secondary chemical inactivation. Empirically, enzymes are at least two orders of magnitude more sensitive to radiation in liquid solution than in the dry state. Target analysis is clearly not applicable under such conditions, and this is the most severe limitation of the method.
Calculation of the molecular weight from radiation inactivation involves two steps. First, the conventional unit of dose, the rad (defined as the deposition of 100 ergs of radiation energy/cubic centimeter of material), is converted to units of primary ionizaSOURCES OF RADIATION tions per cubic centimeter yielding the target volume. Then the density of protein and Any type of ionizing radiation capable of Avogadro’s number are used to convert to causing damage randomly throughout the molecular weight (1,2). The two steps were volume of a sample is adequate for the target implicitly combined by Kepner and Macey method. Under appropriate conditions, X (3). From a selection of radiation data they es- rays, gamma rays, or high-energy electrons tablished empirically that can fit the “random” requirement. It is necessary that the radiation penetrate fully 6.4 x 10” = molecular weight through the thickness of the sample. This is D:,, most easily seen in the case of electrons where the dose is given in rads. They also which have a defined range proportional to determined the energy requirement for a pri- their energy (1). For samples up to 1 mm
4
KEMPNEKANDSCHLEGEI
thick, 1 MeV electrons from a Van der Graaff accelerator, y rays from cobalt-60. or 1000 kVcp X rays are adequate. For l-cm thickness, at least 10 MeV electrons are needed, as can be produced by a linear electron accelerator or a betatron. A second criterion, the quantity of radiation, is related to the available dose rate and the acceptable time of exposure. Doses as high as 50 Mrad are sometimes needed for enzyme inactivation analysis. A large cobalt60 source of IO’ Ci can deliver IO4 radimin: total irradiation time would take several days. With a linear accelerator this could be decreased to several hours. Absorbed radiation energy ultimately appears as heat. Therefore. the temperature of the sample should be controlled during irradiation. This is often accomplished by holding the samples against a large metal block maintained at constant temperature. EVALUATION
OF THE METHOD
The theory of the direct action of radiation states that only ionizations within the target volume are significant. The nature of the surrounding environment, as long as it is dry, is not a factor. It follows that purifrcation of the enzyme is not required. This has been confirmed experimentally. Target analysis of enzymes has been successfully applied after irradiation of such divers dried cells as bacteria, yeast, and seeds (5-7). The radiation sensitivity of enzymes is the same in I%Y, as in cell extracts or purified preparations (7). Hence the procedure has potentially great advantages for membrane-bound systems and other complex or labile structures. A wide variety of enzymes has been studied with ionizing radiation. For the purposes of target theory analysis, all reports of irradiation in liquid solution have been deliberately omitted in our discussion. There remain more than 40 enzymes for which rddiation molecular weights have been determined. All were reported to show a single exponential inactivation curve. A number of
molecular weight values deduced from radiation experiments many years ago were considerably smaller than the values obtained contemporaneously by standard procedures. However, subsequent advances in conventional purification and molecular weight techniques led to a revision in these molecular weights to values which nearly matched those obtained by target analysis. A comprehensive literature review of target molecular weights suggests three categories. Table 1 lists enzymes for which there is good agreement between target and other molecular weight estimates. Also shown is the subunit structure which is now known; in general the latter were determined with sodium dodecyl sulfate gels or by sedimentation in guanidinium hydrochloride. A substantial number of enzymes remain for which radiation methods yielded apparently low molecular weights (Table 2). It is noteworthy that all ofthese values appear to correspond to the size of subunits. All enzymes for which radiation yields a value much larger than the standard value are listed in Table 3. There is only one entry. the single exception to the first two categories. In analyzing the data, several factors should be considered: (a) Choice of accepted molecular weight. The values were taken uncritically from the literature and some may be in doubt. especially in the case of membrane-bound enzymes. (b) Comparison of accepted and target values from different species. tc) Sample treatment and radiation techniques. Several procedures have been discussed repeatedly in the literature in relation to their effect on the target molecular weight. The presence or absence of oxygen during irradiation appears to be important only at low dose rates ( 19). There is usually no difficulty due to air with the radiation fluxes necessary for molecular weight determinations. Another factor, temperature dependence of radiation sensitivity, has been reported for
Glutamic Catalase
Lipoxygenase Starch phosphorylase Creatine kinase
Alkaline phosphatase (K+ stimulated) Deoxyribonuclease I Ribonuclease I
Lysozyme P-Galactosidase Hyaluronoglucuronidase Chymotrypsin
Trypsin Thrombin Pepsin A ATPase (Mg’+ mitochondria)
1.4.1.3 1.11.1.6
1.13.11.12 2.4.1.1 2.7.3.2
3.1.3.1
3.1.4.5 3.1.4.22
3.2.1.17 3.2.1.23 3.2.1.35 3.4.21.1
3.4.21.4 3.4.21.5 3.4.23.1 3.6.1.3
Tissue
semen
testicle
G. y rays; performed
Beef pancreas Human blood Pig stomach
Beef 9
Hen egg Escherichia
Bull
?
Calf intestine
Soy bean Potato Rabbit muscle
coli
FOR WHICH
Pseudomonas testosteroni Rhodospirillum rubrum Beef liver Beef liver
(’ A, o( particles; D, deuterons: E. electrons: b Dry-ice temperature. All other irradiations ” Lyophilized.
,
dehydrogenase
dehydrogenase
Succinic
1.3.99.1
dehydrogenase
name
Malate
Trivial
1.1.1.37
EC number
ENZYMES
1 GIVES
WHOLE
‘7
1 2 1 2-11
2
24 32 + 5 35 ?
14 116 14 23
14
69
54 108 44
2 2 2 2
57 58
60 + 27
36
Subunit (X lo-“)
THE
6 4
2
2
Number of chains
ANALYSIS
X, X rays. at room temperature
24 37 35 284
14 464 55 46
63 29
140
102 215 84
320 232
87
74
Molecular weight (X 10-S)
TARGET
TABLE
63 30 27 28 I5 370 75 50 48 28 34 35 39 280 280
300 230 110 120 210 73 68 140
80
73
Target size (X 10-3)
MOLECULE
bean
cell
blood Beef heart Rat liver
‘,
Human
‘,
Ox pancreas Beef pancreas ? Hen egg Eschrrichia coli Beef testicle ‘)
Potato Rabbit muscle Rabbit muscle Guinea pig kidney microsomes ?
Soy
Beef liver ? Beefred blood
M. lysodektikus membranes Bacillus subtilis
Source
Sample
Dry LYO LYO
Dry Dry
LYO Dry Dry Dry Dry Dry Dry
Dry Dry Dry LYO LYO Dry LYO
E D E E
E
E X E G E G.D.A D.F
E X E X G G G E
E
Dry Lyo’
Eb
Radiation”
Frozen
Condition
2 20,28 26.27 20,28 3,29 30
L
19.20 12.21 22 4 22 23.24 12.25 2.20
11.12 12.13 2 12.14 12.15 16.17 18 3,12
2.10
8.9
References
Alkaline phosphatase (no K+ stimulation) Glucose-6-phosphatase
Hexosediphosphatase a-Amylase
p-Fructofuranosidase
p-Glucoronidase Urease
ATPase (Ca”. sarcoplasmic reticulum) ATPase (Myosin) Cytochrome P-450
3.1.3.1
3.1.3.11 3.2.1.1
3.2.1.26
3.2.1.31 3.5.1.5
3.6.1.3
200
muscle
G. y rays: performed
Rabbit muscle Bovine adrenal
Rabbit
Rat liver Jack bean
Rat. human liver Rabbit var. Bacillus
154
Human red blood cell Calf intestine
GIVES
2 2 8
4 2 3 4
4 2 2 4
2
2
2
‘I 4
‘I
4 4 2 7
Number of chains
ANALYSIS
2
P. protons; X. X rays. at room temperature.
480 850 470
280 480 240 460
140 96 48 210
130
140
40 280
Horseradish Eel
heart
Bovine
Molecular weight (X lo-“)
TARGET
141 140 300 263
WHICH
Yeast Pig heart Cow’s milk Yeast
Tissue
FOR
‘I A, cy particles; D. deuterons: E. electrons; ’ Dry-ice temperature. All other irradiations ’ Lyophilized.
3.6.1.3
3.1.3.9
Peroxidase Acetylcholinesterase
1.11.1.7 3.1.1.7
name
Alcohol dehydrogenase Lactate dehydrogenase Xanthine oxidase Cytochrome c oxidase
Trivial
1.1.1.1 1.1.1.27 1.2.3.2 1.9.3.1
EC number
ENZYMES
TABLE
240 470 53
75 240 83 115
36 48 24 52
63
69
80
70
35 35 150 42,34.25.15. 15.12.11 ”
Subunit ( x 10-2)
A PARTIAL
Sample
‘! Muscle Mouse
230 57
Rabbit
87 190
liver
muscle
‘, Beef liver
liver
suhrilis
human
7 Bacillu.s
Rat.
Rat liver “purified” Horseradish Eel Eel Eel Eel Eel Red blood cell Red blood cell Guinea pig kidney
Yeast Rat, goldfish Cow’s milk Bacillus suhrilis
Source
WEIGHT
120 123 81
32 46
70
50 67 14 170 105 71 75 77 56 75 70
37 34 125 40
Target size (X lo-“)
MOLECULAR
-
LYO
Dry
Dry Dry
Dry Dry Dry
Dry
‘I
LY.0
LYO LYO
tyo
‘, 7
Frozen Frozen Dry Ly0” Dry Dry
Dry Dry Dry Dry
Condition
G
12.18.46
21.45
2.12.42 43.44 D E X
12.41 41 12.18
12.40 12.18
39
12.30 30 1x 3.12 35 35 36 36 3.37.38 36 3.12
6.12 12.31 32.33 2.34
References
E D G
E G
G
i E E
E E ‘7
Eh E” G E P
AD E A E
Radiation”
r
z
=;
?
zi tr
E
E 3 s
m
SIZE
DETERMINATION TABLE
ENZYMES
EC number 3.4.12.2
Trivial
Papain
Tissue Papaya
21
Number of chains I
seven enzymes: lysozyme (22). invertase (41). lactic dehydrogenase t48), hyaluronidase (49), catalase (50), ribonuclease (22. 52), and trypsin (53): all were irradiated in the dry state at different temperatures. Analyses of these temperature phenomena have been presented (225 1.5253) and led to calculation of activation energies for several unidentified processes. The experimental values given in the original studies indicate that the enzymes have different DZr7 values. However, we find that the radiation sensitivity at -100°C relative to that at +3o”C was similar for all. It is found that D:s,( - I OOT)
D:J + 30°C)
7
ENZYMES
3
FOR WHKH TARGCI ANALYSIS Gwts A MOLECULAR MUCH LAKG~R THAN I HE WHOI E MOI.KCILF Molecular weight ( x IO :‘)
name
OF
= 2.57 2 0.29.
Although the present analysis offers no insight into mechanism, the observed factor seems to be constant for a variety of enzymes. It can be used empirically in molecular weight calculations to adjust data obtained at - 100°C. Uncertainty in the precise value of this correction factor will increase the error in final molecular weight calculations by 10% over that due to experimental error. Finally. it appears that irradiation with X rays, y rays, or electrons allows determination of molecular weights in a straightforward manner. The use of deuterons, protons, (Y particles, and heavy ions is more complicated and interpretation is not always direct. With these reservations in mind, the data shown in Tables 1 and 2 allow a comparison between target molecular weight and the “accepted” value for the molecule or subunit. The average “error” for all the data is
WEIGHT
Subunit ( x IO-:‘:I
Target size ( x IO-“)
Sample Source
Condition
Radiation
References
21
287
Papaya
Lyophilized
X ray
20.47
14%, a remarkably low value considering the questions and caveats involved. Why radiation sometimes detects the whole molecule and sometimes a subunit is an intriguing question. In the simplest case, identification of a functional target with a single subunit means that the subunit is fully active and is sufficiently isolated from the rest of the molecule so that there is no transfer of ionization energy from other subunits (11). Although such isolation may exist in viva, it could be an artifact due to drying the sample. Nevertheless, the functional unit size, so determined, is correct. If the entire molecule is radiation sensitive. alternative explanations are possible. The simplest and most reasonable of these is that the structure of the entire molecule is required for enzymatic activity. An alternative explanation, albeit experimentally unsupported. is that the functional activity is restricted to one or more subunits but that the structure of the entire molecule is so compact or bound so tightly that the units are coupled, i.e., there is significant energy transfer between subunits; no evidence for this hypothesis exists at present. COMMENTS
This compilation of enzyme inactivation data indicates the enormous potential of the radiation technique. When enzymes were irradiated with electrons or y rays, target theory led to remarkably accurate values of total or subunit molecular weight. The size of simple proteins as well as more elaborate enzymes such as the ATPase of myosin (2 1) and the different enzymatic activities of xan-
8
KEMPNER
AND
thine oxidase (32) have been measured. It should be noted that the values given in the tables are those reported in the original study. There has been no “recalculation” from experimental values. The agreement with enzyme structures which were determined only recently inspires confidence in the method. The only radiation studies which have not been listed are those enzymes for which the size or subunits are not presently known. All of these are listed in Table 4 and may be viewed as further predictions of this technique. As with any method, there are limitations to this one. Primarily these have to do with irradiation conditions, including the need for a high dose rate of electrons or y rays. Exposure of lyophilized samples to radiation at room temperature leads directly to molecular size determination. However, lyophilized samples can be irradiated at low temperature, provided a simple temperature correction is made subsequently. PrelimiTABLE ENZYMES
WITH
UNKNOWN
MOLECULAR
WEIGHT
EC number
Trivial
1.3.99.1 2.3.1.6 3.6.1.3
Succinate dehydrogenase Choline acetyltransferase ATPase (Na-K)
4.6.1.1
Adenylate cyclase, F+ stimulated, glucagon stimulated Protoheme ferro-lyase NADH oxidase NADH fenicyanide reductase NADH dehydrogenase
4.99.1.1
” E, electrons. * Dry-ice temperature. c Lyophilized.
name
All other
SCHLEGEL
nary attempts have been made to see if target analysis was applicable to irradiation of enzymes frozen in solution. Both unchanged (3658) and reduced (3) radiation sensitivity were reported. Further studies of this approach offers the possibility of extending the method to enzymes previously excluded, i.e., those that could not be lyophilized. Many enzymes have been studied by radiation inactivation, a technique which spares the need for purification. Target theory analysis of these data has successfully predicted the size of functional units and has been shown to apply to simple and complex enzymes. This method has also been used to determine virus structure (2) and can be extended to any unit for which an activity can be measured quantitatively. It is the only method which directly relates molecular structure with function. Systems which have proved intractable to purification, e.g., membrane-bound and organellar enzymes, are logical candidates for the radiation method. A few such studies (3,18,55) indi4
OR SUBUNITS
WHICH
HAVE
BEEN
STUDIED
WITH
RADIATION
Target size (X 10-Z)
Source
Condition
Radiation
74 46 500 300 270 200 190
Rat liver Rat, human, horse Pig brain Human red blood cell ghosts Crayfish nerve Guinea pig kidney Guinea pig kidney
Frozen Lyo’ LYO LYO LYO LYO LYO
E Eb E E E E
30 54 55 3 3 3 3
160 389 286 77 40 70
Rat liver Rat liver Chicken red blood cells Rat liver Rat liver M. lysodektikus membranes
LYO LYO Dry Frozen Frozen Frozen
E E ED Eb ED Eb
56 56 57 30 30 8
irradiations
Sample
performed
at room
temperature
p*
References
SIZE
DETERMINATION
cate that they are amenable to analysis and may point to the next major achievement of this approach.
OF ENZYMES
24. 25.
REFERENCES 26. I. Lea, D. E. (1955) Actions of Radiations on Living Cells, 2nd ed. Cambridge Univ. Press. Cambridge. England. 2. Pollard, E. C., Guild, W. R.. Hutchinson. F., and Setlow. R. B. (1955) in Progress in Biophysics (Butler. J. A. V., and Randall. J. T.. cds.). Vol. 5, pp. 72-108. Pergamon. Long Island City. N.Y. 3. Kepner, G. R.. and Macey, R. I. (1963) Biwhim. Bioptzy.~. Ac,tu 163, 188-203. 4. Marshall, M.. Holt. P. D.. and Gibson, J. A. B. (1970) In/. J. Rodicd. Bicd. 18, 139- 146. 5. Powell, W. F., and Pollard, E. C. (1955) Rdicr/. Rrs. 2, 109- 118. 6. Hutchinson. F.. Preston. A.. and Vogel. B. (1957) Rndicrr. Kc.\. 7, 465-472. 7. Jagger, J., and Wilson. D. (1955) Radiur. Rex. 3, l27- 134. 8. Ostrovskii, D. N.. Tsfasman. I. M., and Gel’man, N. S. (1969) Biokhimiyu 34. 993-999. 9. You. K. S., and Kaplan. N. 0. ( 1975j.1. Bcrc~rc~riol. 123, 704-716. IO. Davis, K. A.. Hatefi. Y.. Crawford. I. P.. and Baltscheffsky. H. (1977) Arch. Biwhcrn. BiophJs. 180, 459-464. 1 I. Blum. E.. and Alper, I. (1971) Biochrm. J. 122, 677-680. 12. Damell, D. W.. and Klotz. I. M. (1975) Birwhirn. BicJphys. Ac,icr 166, 65 l-682. 13. Norman. A.. and Ginoza. W. (1958) Rdiut. Rcs. 9, 77-83. 14. Budnitskaya, E. V.. Borisova. I. G.. and Pasinsky. A. G. (1956) Biokhimi~u 21, 702-708 15. Phillips, G. 0.. and Grifflths, W. (1965) Rotlitrt. Rcs. 26. 363-377. 16. Friedberg. F. and Hayden. G. A. (1966) Rtrdicrt. Rcs. 28, 717-725. 17. Mani. R. S., and Kay. M. (1976) Bioc,hirrl Biophys. Ac,fcr 453, 391-399. 18. Shikita. M.. and Hitano-Sato. F. (1973)F,FBS 1x1/. 36, 187-189. 19. Butler, J. A. V.. and Robins. A. B. (196:!) Rodiut. Rt’.\. 17, 63-73. 20. Dixon. M.. and Webb. E. C. (1964) Enzymes, 2nd ed., Academic Press, New York. 21. Lea. D., Smith, K. M., Holmes. B., and Markham, R. (1944) Pnrusilcdo~y 36, 1 lo- 118. 21. Fluke, D. J. (1966) Radiut. Rec. 28, 677-693. 23. Pollard. E. (1959) in Biophysical Science-A
27.
28. 29. 30. 31. 32.
33. 34. 35. 36. 37. 38. 39.
40. 41. 42. 43.
44.
45. 46. 47. 48.
9
Study Program (Oncley. J. L.. ed.). pp. 2731281. Wiley. New York. Fowler, A. V.. and Zabin. I. (1977) Proc,. Nuti. ,4c,rrd. 5c.i. USA 74. l507-1510. Setlow. R., and Doyle. B. (1955) RudW/. Res. 2, 15-25. Aronson. D. L.. and Preiss. J. W. (1962) Ratliut. RCA. 16, 138-143. Fenton. J. W.. II. Fasco, M. J.. Stackrow. A. B.. Aronson. D. L., Young. A. M.. and Finlayson, .I. S. (1977) /. Rio/. Chrm. 252, 3587-3598. Pollard, E.. Buzzell. A.. Jeffreys, C., and Forro. I?., Jr. ( 195l)Arc.h. Bioc hrm. Biophy.s. 33.9-21. Penefsky, H. S., and Warner, R. C. ( 1965)J. BirJl. Chrm. 240, 4694-4702. Kagawa. Y. (1967) Birwhim. BiophJs. Ac,tu 131, 586-588. Fluke. D. J.. and Hochachka. P. W. ( 1965) Rrrdiclt. Rzs. 26. 395-402. Fluke. D. J. (1959) in Proceedings of the First National Biophysics Conference, pp. 161-168. Yale Univ. Press. New Haven. Conn. Nagler. L. G.. and Vartanyan. L. S. (1976) Bit)chirtr. Bictphxs. Acicr 427, 78-90. Phan. S. H.. and Mahler. H. R. (1976) ./. Biol. Chcm. 251, 257-269. Serlin, I., and Fluke. D. J. (1956) J. Biol. Chum. 2123, 727-736. Levinson. S. R.. and Ellory. J. C. (1974) Biochrnr. J’. 137, 123-125. Grossman, H.. and Lieflander. M. (1975) Hoppe!ic~~lrr.\ 2. Ph~siol. C‘hrm. 356, 663-669. Ott, P.. Jenny, B.. and Brodbeck. U. (1975) Eur. Y. Bioc hem. 57, 469-480. Collipp. P. J.. Carsten. A., Chen, S. Y.. Thomas, J.. and Maddiah. V. T. ( 1974) Biochc,m. Med. 10, 312-319. Griffin. L. (1962) Masters Thesis. Duke University, Durham. N. C. [quoted in (31)]. Pollard, E.. Powell, W. F.. and Reaume. S. H. (1952) Proc,. Not. Acud. Sc,i. USA 38, 173- 180. Setlow. R. B. (1952) Arch. Birx.hem. Biophy.s. 36. 328-335. Vegh. K., Spiegler. P.. Chamberlain. C.. and Mommaerts. W. F. H. M. (1968) Bic>c,hinr. BicJphy.v. Ac~cr 163, 266-268. LeMaire, M.. Jorgensen. K. E.. Roigaard-Petersen, H.. and Moller. J. V. (1976) Bicwhemistt-> 15, 5805-5812. Lowey. S.. Slayter. H. S., Weeds. A. G.. and Elaker. H. I 1969) J. A-lr~l. Bkd. 42, l-29. Haugen. D. A., and Coon. M. J. (1976) J. Biot. C‘hrm. 251, l817- 1827. Giovannozzi-Sermanni. G.. and Di Marco. G. (1965) Rodicrf. Rcs. 26, 63-68. Fluke, D. J. (1972) Rndicrt. Rrs. 51, 56-71.
10
KEMPNER
AND
49. Vollmer. R. T.. and Fluke, D. J. (19671 Rtrdinl. Ra.\. 31, 867-875. SO. Setlow, R.. and Doyle. B. (1953) AK/I. Bitwhcw. Biophys. 46, 46-52. 51. Augenstein, L. G.. Brustad. T.. and Mason, R. (1964)m Advances in Radiation Biology (Augenstein. L. G.. Mason. R.. and Quastler. H.. eds.). Vol. I. pp. 227-266. Academic Press. New York. 52. Gunther. W., and Jung. H. (1967) Z. Nnfu$~rsclz. 22, 313-320. 53. Brustad. T. (1964) irr Biological Effects of Neutron and Proton Irradiations, Vol. II. pp. 404-410. International Atomic Energy Agency. Vienna.
SCHLEGEL 54. Banns, H. E.. and Stephens-Newsham. L. ( 1974) .I. Pk~.sid. (Lontlm~ 242. 16P-18P.
G.
55. Nakao, M.. Nagano. K.. Nakao, T., Mizuno. N., Tashima. Y., Fujita. M.. Maeda, H.. and Matsudaira, H. r 1967) Bicx.hc~n~. Bic~ph~.t. Kc~.\. ~‘rvnmu/~. 29. 588-592. 56. Houslay. M.. Ellory. J.. Smith. G.. Hesketh, T., Stein. J.. Warren. G., and Metcalfe. J. C. (1977) Bicrhirn. Birqhys. AC,IN 467. 208-219. 57. Tanaka. S.. Nagahama, Yoneyama. Y. (1976) 1071. 58. Kagawa,
S., Takeshita, J. Biochem.
Y. r 19691 J. Biochem.
M.. and 80, lO67-
65, 925-934
92, ?- 10 (1979)
RICK HFMISTRY
REVIEW Size Determination
of Enzymes by Radiation
inactivation
E. S. KEMPNERANDWERNERSCHLEGEL of
Lahoruroric,s Narionul
In.vrirure National
Physicul
Biology
und
Received
From atomic physics it is known that the passage of radiation through matter is characterized by transfer of energy to the target material. For high energy radiation this occurs in discrete bursts called “primary ionizations. ” In each primary ionization, about Copyright All rights
‘F 1979 by Academic Pres. Inc. of reproductmn in any form rexrved.
and
und Digestive Mqvlund
Endocrinology. Diseases, 20014
66 eV of energy are transferred, corresponding to approximately 1500 kcal/mol. When X rays, y rays or high-energy electrons are used, ionizations occur randomly throughout the volume of the irradiated material. The probability of 0, 1,2, . . . n ionizations occurring in a unit volume is given by the Poisson formula e-*(x)” fJ(n) = ~ n! where x is a function of the amount of radiation exposure. If a biological material is irradiated, the same description applies. Exposure to ionizing radiation leads to a loss of function due to destruction of critical structures. The larger the structure, the more likely it will be “hit.” Because the energy deposition is so large, function is completely destroyed by a single hit; there are no partially damaged targets. Therefore, the only activity remaining after radiation exposure is due to units which have escaped ionization and these units are fully active. In the Poisson formula, the probability on no ionizations is P(0) = eps. It follows that the surviving biological activity, measured after exposure to different amounts of radiation, will decrease as a simple exponential function of dose,
THEORY
0003.2697/79/010002-09$02.00/O
Nutrition
July 21, 1978
The molecular size of an enzyme can be measured by a variety of physical techniques. Among these, ionizing radiation offers several unique advantages in that samples need not necessarily be purified and only minute amounts of enzyme are required. The method relies on measurement of the loss of biological activity with increasing radiation exposure. Since the diagnostic measurement is biological or biochemical activity, the technique describes a functional unit rather than the structural object that is determined by other methods. The target volume theory relating inactivation sensitivity to molecular weight (1) and its procedures (2), as well as applications of the technique, were widely published in the 1950s. Interest in the method waned when many of the results did not agree with those from other procedures. However, subsequent advances have vastly increased knowledge of protein structure. A reevaluation of the radiation studies now indicates the original validity of these results and reinforces the power of the technique. PHYSICAL
of
qf’Arthriris. MeraholiJm Institurc~s of Healrh. Bethesda.
A = AoeekD
where A is the activity measured after a radiation dose D. This exposure is given in 2
SIZE DETERMINATIONOF'ENZYMES
units of primary ionizations per cubic centimeter. The constant, k. is characteristic of each biological activity and has the units of volume (cubic centimeters). This is the radiation target or “sensitive volume” in which an ionization must occur in order to destroy the measured function. Since ionizations caused by such radiation occur at random throughout the volume of the sample, target volume is independent of the shape of the unit. Since so much energy is involved, radiation sensitivity is independent of chemical composition. This situation is entirely opposite to the effects of nonionizing radiation, e.g., ultraviolet light, where energy absorption and damage depends upon the presence of unique chemical sustituents, i.e., specific ring structures. disulfide bridges. or ligands. A direct measure of the target size, k, is obtained from the 37% dose. This is the specific radiation exposure, also known as the i’D37r” responsible for reducing the measured activity to 37% of that found in the unexposed controls: A = 0.37 A,, = A,,cJ-~~‘~;
In 0.37 = - 1 = -XD:,,
L =k. 037
3
mary ionization as 66 eV. This clarifies earlier confusion in the literature where values of 60 to 100 eV were quoted (1,2,4). Multiple radiation targets exist if the same function resides independently in several different structures. The inactivation is then described by an additive series of exponential terms. A = A II,e--klu + A 28 -kzLJ + . . . . For two targets of significantly different size, the survival curve can be resolved into two simple exponential functions from which both molecular weights can be calculated. By this means. the proportion of activity due to each unit is also revealed. This description constitutes the dirt~t action of radiation on matter, that is, the damage to functional units is caused entirely by primary ionizations in situ. In liquid solution so-called indirect effects can occur. For example, irradiation of water results in formation of H+. OH-, peroxide, and other radialytic products which, in the liquid state, can diffuse appreciable distances before reacting. Thus, inactivation of function can occur not only by the direct action of radiation, but also by secondary chemical inactivation. Empirically, enzymes are at least two orders of magnitude more sensitive to radiation in liquid solution than in the dry state. Target analysis is clearly not applicable under such conditions, and this is the most severe limitation of the method.
Calculation of the molecular weight from radiation inactivation involves two steps. First, the conventional unit of dose, the rad (defined as the deposition of 100 ergs of radiation energy/cubic centimeter of material), is converted to units of primary ionizaSOURCES OF RADIATION tions per cubic centimeter yielding the target volume. Then the density of protein and Any type of ionizing radiation capable of Avogadro’s number are used to convert to causing damage randomly throughout the molecular weight (1,2). The two steps were volume of a sample is adequate for the target implicitly combined by Kepner and Macey method. Under appropriate conditions, X (3). From a selection of radiation data they es- rays, gamma rays, or high-energy electrons tablished empirically that can fit the “random” requirement. It is necessary that the radiation penetrate fully 6.4 x 10” = molecular weight through the thickness of the sample. This is D:,, most easily seen in the case of electrons where the dose is given in rads. They also which have a defined range proportional to determined the energy requirement for a pri- their energy (1). For samples up to 1 mm
4
KEMPNEKANDSCHLEGEI
thick, 1 MeV electrons from a Van der Graaff accelerator, y rays from cobalt-60. or 1000 kVcp X rays are adequate. For l-cm thickness, at least 10 MeV electrons are needed, as can be produced by a linear electron accelerator or a betatron. A second criterion, the quantity of radiation, is related to the available dose rate and the acceptable time of exposure. Doses as high as 50 Mrad are sometimes needed for enzyme inactivation analysis. A large cobalt60 source of IO’ Ci can deliver IO4 radimin: total irradiation time would take several days. With a linear accelerator this could be decreased to several hours. Absorbed radiation energy ultimately appears as heat. Therefore. the temperature of the sample should be controlled during irradiation. This is often accomplished by holding the samples against a large metal block maintained at constant temperature. EVALUATION
OF THE METHOD
The theory of the direct action of radiation states that only ionizations within the target volume are significant. The nature of the surrounding environment, as long as it is dry, is not a factor. It follows that purifrcation of the enzyme is not required. This has been confirmed experimentally. Target analysis of enzymes has been successfully applied after irradiation of such divers dried cells as bacteria, yeast, and seeds (5-7). The radiation sensitivity of enzymes is the same in I%Y, as in cell extracts or purified preparations (7). Hence the procedure has potentially great advantages for membrane-bound systems and other complex or labile structures. A wide variety of enzymes has been studied with ionizing radiation. For the purposes of target theory analysis, all reports of irradiation in liquid solution have been deliberately omitted in our discussion. There remain more than 40 enzymes for which rddiation molecular weights have been determined. All were reported to show a single exponential inactivation curve. A number of
molecular weight values deduced from radiation experiments many years ago were considerably smaller than the values obtained contemporaneously by standard procedures. However, subsequent advances in conventional purification and molecular weight techniques led to a revision in these molecular weights to values which nearly matched those obtained by target analysis. A comprehensive literature review of target molecular weights suggests three categories. Table 1 lists enzymes for which there is good agreement between target and other molecular weight estimates. Also shown is the subunit structure which is now known; in general the latter were determined with sodium dodecyl sulfate gels or by sedimentation in guanidinium hydrochloride. A substantial number of enzymes remain for which radiation methods yielded apparently low molecular weights (Table 2). It is noteworthy that all ofthese values appear to correspond to the size of subunits. All enzymes for which radiation yields a value much larger than the standard value are listed in Table 3. There is only one entry. the single exception to the first two categories. In analyzing the data, several factors should be considered: (a) Choice of accepted molecular weight. The values were taken uncritically from the literature and some may be in doubt. especially in the case of membrane-bound enzymes. (b) Comparison of accepted and target values from different species. tc) Sample treatment and radiation techniques. Several procedures have been discussed repeatedly in the literature in relation to their effect on the target molecular weight. The presence or absence of oxygen during irradiation appears to be important only at low dose rates ( 19). There is usually no difficulty due to air with the radiation fluxes necessary for molecular weight determinations. Another factor, temperature dependence of radiation sensitivity, has been reported for
Glutamic Catalase
Lipoxygenase Starch phosphorylase Creatine kinase
Alkaline phosphatase (K+ stimulated) Deoxyribonuclease I Ribonuclease I
Lysozyme P-Galactosidase Hyaluronoglucuronidase Chymotrypsin
Trypsin Thrombin Pepsin A ATPase (Mg’+ mitochondria)
1.4.1.3 1.11.1.6
1.13.11.12 2.4.1.1 2.7.3.2
3.1.3.1
3.1.4.5 3.1.4.22
3.2.1.17 3.2.1.23 3.2.1.35 3.4.21.1
3.4.21.4 3.4.21.5 3.4.23.1 3.6.1.3
Tissue
semen
testicle
G. y rays; performed
Beef pancreas Human blood Pig stomach
Beef 9
Hen egg Escherichia
Bull
?
Calf intestine
Soy bean Potato Rabbit muscle
coli
FOR WHICH
Pseudomonas testosteroni Rhodospirillum rubrum Beef liver Beef liver
(’ A, o( particles; D, deuterons: E. electrons: b Dry-ice temperature. All other irradiations ” Lyophilized.
,
dehydrogenase
dehydrogenase
Succinic
1.3.99.1
dehydrogenase
name
Malate
Trivial
1.1.1.37
EC number
ENZYMES
1 GIVES
WHOLE
‘7
1 2 1 2-11
2
24 32 + 5 35 ?
14 116 14 23
14
69
54 108 44
2 2 2 2
57 58
60 + 27
36
Subunit (X lo-“)
THE
6 4
2
2
Number of chains
ANALYSIS
X, X rays. at room temperature
24 37 35 284
14 464 55 46
63 29
140
102 215 84
320 232
87
74
Molecular weight (X 10-S)
TARGET
TABLE
63 30 27 28 I5 370 75 50 48 28 34 35 39 280 280
300 230 110 120 210 73 68 140
80
73
Target size (X 10-3)
MOLECULE
bean
cell
blood Beef heart Rat liver
‘,
Human
‘,
Ox pancreas Beef pancreas ? Hen egg Eschrrichia coli Beef testicle ‘)
Potato Rabbit muscle Rabbit muscle Guinea pig kidney microsomes ?
Soy
Beef liver ? Beefred blood
M. lysodektikus membranes Bacillus subtilis
Source
Sample
Dry LYO LYO
Dry Dry
LYO Dry Dry Dry Dry Dry Dry
Dry Dry Dry LYO LYO Dry LYO
E D E E
E
E X E G E G.D.A D.F
E X E X G G G E
E
Dry Lyo’
Eb
Radiation”
Frozen
Condition
2 20,28 26.27 20,28 3,29 30
L
19.20 12.21 22 4 22 23.24 12.25 2.20
11.12 12.13 2 12.14 12.15 16.17 18 3,12
2.10
8.9
References
Alkaline phosphatase (no K+ stimulation) Glucose-6-phosphatase
Hexosediphosphatase a-Amylase
p-Fructofuranosidase
p-Glucoronidase Urease
ATPase (Ca”. sarcoplasmic reticulum) ATPase (Myosin) Cytochrome P-450
3.1.3.1
3.1.3.11 3.2.1.1
3.2.1.26
3.2.1.31 3.5.1.5
3.6.1.3
200
muscle
G. y rays: performed
Rabbit muscle Bovine adrenal
Rabbit
Rat liver Jack bean
Rat. human liver Rabbit var. Bacillus
154
Human red blood cell Calf intestine
GIVES
2 2 8
4 2 3 4
4 2 2 4
2
2
2
‘I 4
‘I
4 4 2 7
Number of chains
ANALYSIS
2
P. protons; X. X rays. at room temperature.
480 850 470
280 480 240 460
140 96 48 210
130
140
40 280
Horseradish Eel
heart
Bovine
Molecular weight (X lo-“)
TARGET
141 140 300 263
WHICH
Yeast Pig heart Cow’s milk Yeast
Tissue
FOR
‘I A, cy particles; D. deuterons: E. electrons; ’ Dry-ice temperature. All other irradiations ’ Lyophilized.
3.6.1.3
3.1.3.9
Peroxidase Acetylcholinesterase
1.11.1.7 3.1.1.7
name
Alcohol dehydrogenase Lactate dehydrogenase Xanthine oxidase Cytochrome c oxidase
Trivial
1.1.1.1 1.1.1.27 1.2.3.2 1.9.3.1
EC number
ENZYMES
TABLE
240 470 53
75 240 83 115
36 48 24 52
63
69
80
70
35 35 150 42,34.25.15. 15.12.11 ”
Subunit ( x 10-2)
A PARTIAL
Sample
‘! Muscle Mouse
230 57
Rabbit
87 190
liver
muscle
‘, Beef liver
liver
suhrilis
human
7 Bacillu.s
Rat.
Rat liver “purified” Horseradish Eel Eel Eel Eel Eel Red blood cell Red blood cell Guinea pig kidney
Yeast Rat, goldfish Cow’s milk Bacillus suhrilis
Source
WEIGHT
120 123 81
32 46
70
50 67 14 170 105 71 75 77 56 75 70
37 34 125 40
Target size (X lo-“)
MOLECULAR
-
LYO
Dry
Dry Dry
Dry Dry Dry
Dry
‘I
LY.0
LYO LYO
tyo
‘, 7
Frozen Frozen Dry Ly0” Dry Dry
Dry Dry Dry Dry
Condition
G
12.18.46
21.45
2.12.42 43.44 D E X
12.41 41 12.18
12.40 12.18
39
12.30 30 1x 3.12 35 35 36 36 3.37.38 36 3.12
6.12 12.31 32.33 2.34
References
E D G
E G
G
i E E
E E ‘7
Eh E” G E P
AD E A E
Radiation”
r
z
=;
?
zi tr
E
E 3 s
m
SIZE
DETERMINATION TABLE
ENZYMES
EC number 3.4.12.2
Trivial
Papain
Tissue Papaya
21
Number of chains I
seven enzymes: lysozyme (22). invertase (41). lactic dehydrogenase t48), hyaluronidase (49), catalase (50), ribonuclease (22. 52), and trypsin (53): all were irradiated in the dry state at different temperatures. Analyses of these temperature phenomena have been presented (225 1.5253) and led to calculation of activation energies for several unidentified processes. The experimental values given in the original studies indicate that the enzymes have different DZr7 values. However, we find that the radiation sensitivity at -100°C relative to that at +3o”C was similar for all. It is found that D:s,( - I OOT)
D:J + 30°C)
7
ENZYMES
3
FOR WHKH TARGCI ANALYSIS Gwts A MOLECULAR MUCH LAKG~R THAN I HE WHOI E MOI.KCILF Molecular weight ( x IO :‘)
name
OF
= 2.57 2 0.29.
Although the present analysis offers no insight into mechanism, the observed factor seems to be constant for a variety of enzymes. It can be used empirically in molecular weight calculations to adjust data obtained at - 100°C. Uncertainty in the precise value of this correction factor will increase the error in final molecular weight calculations by 10% over that due to experimental error. Finally. it appears that irradiation with X rays, y rays, or electrons allows determination of molecular weights in a straightforward manner. The use of deuterons, protons, (Y particles, and heavy ions is more complicated and interpretation is not always direct. With these reservations in mind, the data shown in Tables 1 and 2 allow a comparison between target molecular weight and the “accepted” value for the molecule or subunit. The average “error” for all the data is
WEIGHT
Subunit ( x IO-:‘:I
Target size ( x IO-“)
Sample Source
Condition
Radiation
References
21
287
Papaya
Lyophilized
X ray
20.47
14%, a remarkably low value considering the questions and caveats involved. Why radiation sometimes detects the whole molecule and sometimes a subunit is an intriguing question. In the simplest case, identification of a functional target with a single subunit means that the subunit is fully active and is sufficiently isolated from the rest of the molecule so that there is no transfer of ionization energy from other subunits (11). Although such isolation may exist in viva, it could be an artifact due to drying the sample. Nevertheless, the functional unit size, so determined, is correct. If the entire molecule is radiation sensitive. alternative explanations are possible. The simplest and most reasonable of these is that the structure of the entire molecule is required for enzymatic activity. An alternative explanation, albeit experimentally unsupported. is that the functional activity is restricted to one or more subunits but that the structure of the entire molecule is so compact or bound so tightly that the units are coupled, i.e., there is significant energy transfer between subunits; no evidence for this hypothesis exists at present. COMMENTS
This compilation of enzyme inactivation data indicates the enormous potential of the radiation technique. When enzymes were irradiated with electrons or y rays, target theory led to remarkably accurate values of total or subunit molecular weight. The size of simple proteins as well as more elaborate enzymes such as the ATPase of myosin (2 1) and the different enzymatic activities of xan-
8
KEMPNER
AND
thine oxidase (32) have been measured. It should be noted that the values given in the tables are those reported in the original study. There has been no “recalculation” from experimental values. The agreement with enzyme structures which were determined only recently inspires confidence in the method. The only radiation studies which have not been listed are those enzymes for which the size or subunits are not presently known. All of these are listed in Table 4 and may be viewed as further predictions of this technique. As with any method, there are limitations to this one. Primarily these have to do with irradiation conditions, including the need for a high dose rate of electrons or y rays. Exposure of lyophilized samples to radiation at room temperature leads directly to molecular size determination. However, lyophilized samples can be irradiated at low temperature, provided a simple temperature correction is made subsequently. PrelimiTABLE ENZYMES
WITH
UNKNOWN
MOLECULAR
WEIGHT
EC number
Trivial
1.3.99.1 2.3.1.6 3.6.1.3
Succinate dehydrogenase Choline acetyltransferase ATPase (Na-K)
4.6.1.1
Adenylate cyclase, F+ stimulated, glucagon stimulated Protoheme ferro-lyase NADH oxidase NADH fenicyanide reductase NADH dehydrogenase
4.99.1.1
” E, electrons. * Dry-ice temperature. c Lyophilized.
name
All other
SCHLEGEL
nary attempts have been made to see if target analysis was applicable to irradiation of enzymes frozen in solution. Both unchanged (3658) and reduced (3) radiation sensitivity were reported. Further studies of this approach offers the possibility of extending the method to enzymes previously excluded, i.e., those that could not be lyophilized. Many enzymes have been studied by radiation inactivation, a technique which spares the need for purification. Target theory analysis of these data has successfully predicted the size of functional units and has been shown to apply to simple and complex enzymes. This method has also been used to determine virus structure (2) and can be extended to any unit for which an activity can be measured quantitatively. It is the only method which directly relates molecular structure with function. Systems which have proved intractable to purification, e.g., membrane-bound and organellar enzymes, are logical candidates for the radiation method. A few such studies (3,18,55) indi4
OR SUBUNITS
WHICH
HAVE
BEEN
STUDIED
WITH
RADIATION
Target size (X 10-Z)
Source
Condition
Radiation
74 46 500 300 270 200 190
Rat liver Rat, human, horse Pig brain Human red blood cell ghosts Crayfish nerve Guinea pig kidney Guinea pig kidney
Frozen Lyo’ LYO LYO LYO LYO LYO
E Eb E E E E
30 54 55 3 3 3 3
160 389 286 77 40 70
Rat liver Rat liver Chicken red blood cells Rat liver Rat liver M. lysodektikus membranes
LYO LYO Dry Frozen Frozen Frozen
E E ED Eb ED Eb
56 56 57 30 30 8
irradiations
Sample
performed
at room
temperature
p*
References
SIZE
DETERMINATION
cate that they are amenable to analysis and may point to the next major achievement of this approach.
OF ENZYMES
24. 25.
REFERENCES 26. I. Lea, D. E. (1955) Actions of Radiations on Living Cells, 2nd ed. Cambridge Univ. Press. Cambridge. England. 2. Pollard, E. C., Guild, W. R.. Hutchinson. F., and Setlow. R. B. (1955) in Progress in Biophysics (Butler. J. A. V., and Randall. J. T.. cds.). Vol. 5, pp. 72-108. Pergamon. Long Island City. N.Y. 3. Kepner, G. R.. and Macey, R. I. (1963) Biwhim. Bioptzy.~. Ac,tu 163, 188-203. 4. Marshall, M.. Holt. P. D.. and Gibson, J. A. B. (1970) In/. J. Rodicd. Bicd. 18, 139- 146. 5. Powell, W. F., and Pollard, E. C. (1955) Rdicr/. Rrs. 2, 109- 118. 6. Hutchinson. F.. Preston. A.. and Vogel. B. (1957) Rndicrr. Kc.\. 7, 465-472. 7. Jagger, J., and Wilson. D. (1955) Radiur. Rex. 3, l27- 134. 8. Ostrovskii, D. N.. Tsfasman. I. M., and Gel’man, N. S. (1969) Biokhimiyu 34. 993-999. 9. You. K. S., and Kaplan. N. 0. ( 1975j.1. Bcrc~rc~riol. 123, 704-716. IO. Davis, K. A.. Hatefi. Y.. Crawford. I. P.. and Baltscheffsky. H. (1977) Arch. Biwhcrn. BiophJs. 180, 459-464. 1 I. Blum. E.. and Alper, I. (1971) Biochrm. J. 122, 677-680. 12. Damell, D. W.. and Klotz. I. M. (1975) Birwhirn. BicJphys. Ac,icr 166, 65 l-682. 13. Norman. A.. and Ginoza. W. (1958) Rdiut. Rcs. 9, 77-83. 14. Budnitskaya, E. V.. Borisova. I. G.. and Pasinsky. A. G. (1956) Biokhimi~u 21, 702-708 15. Phillips, G. 0.. and Grifflths, W. (1965) Rotlitrt. Rcs. 26. 363-377. 16. Friedberg. F. and Hayden. G. A. (1966) Rtrdicrt. Rcs. 28, 717-725. 17. Mani. R. S., and Kay. M. (1976) Bioc,hirrl Biophys. Ac,fcr 453, 391-399. 18. Shikita. M.. and Hitano-Sato. F. (1973)F,FBS 1x1/. 36, 187-189. 19. Butler, J. A. V.. and Robins. A. B. (196:!) Rodiut. Rt’.\. 17, 63-73. 20. Dixon. M.. and Webb. E. C. (1964) Enzymes, 2nd ed., Academic Press, New York. 21. Lea. D., Smith, K. M., Holmes. B., and Markham, R. (1944) Pnrusilcdo~y 36, 1 lo- 118. 21. Fluke, D. J. (1966) Radiut. Rec. 28, 677-693. 23. Pollard. E. (1959) in Biophysical Science-A
27.
28. 29. 30. 31. 32.
33. 34. 35. 36. 37. 38. 39.
40. 41. 42. 43.
44.
45. 46. 47. 48.
9
Study Program (Oncley. J. L.. ed.). pp. 2731281. Wiley. New York. Fowler, A. V.. and Zabin. I. (1977) Proc,. Nuti. ,4c,rrd. 5c.i. USA 74. l507-1510. Setlow. R., and Doyle. B. (1955) RudW/. Res. 2, 15-25. Aronson. D. L.. and Preiss. J. W. (1962) Ratliut. RCA. 16, 138-143. Fenton. J. W.. II. Fasco, M. J.. Stackrow. A. B.. Aronson. D. L., Young. A. M.. and Finlayson, .I. S. (1977) /. Rio/. Chrm. 252, 3587-3598. Pollard, E.. Buzzell. A.. Jeffreys, C., and Forro. I?., Jr. ( 195l)Arc.h. Bioc hrm. Biophy.s. 33.9-21. Penefsky, H. S., and Warner, R. C. ( 1965)J. BirJl. Chrm. 240, 4694-4702. Kagawa. Y. (1967) Birwhim. BiophJs. Ac,tu 131, 586-588. Fluke. D. J.. and Hochachka. P. W. ( 1965) Rrrdiclt. Rzs. 26. 395-402. Fluke. D. J. (1959) in Proceedings of the First National Biophysics Conference, pp. 161-168. Yale Univ. Press. New Haven. Conn. Nagler. L. G.. and Vartanyan. L. S. (1976) Bit)chirtr. Bictphxs. Acicr 427, 78-90. Phan. S. H.. and Mahler. H. R. (1976) ./. Biol. Chcm. 251, 257-269. Serlin, I., and Fluke. D. J. (1956) J. Biol. Chum. 2123, 727-736. Levinson. S. R.. and Ellory. J. C. (1974) Biochrnr. J’. 137, 123-125. Grossman, H.. and Lieflander. M. (1975) Hoppe!ic~~lrr.\ 2. Ph~siol. C‘hrm. 356, 663-669. Ott, P.. Jenny, B.. and Brodbeck. U. (1975) Eur. Y. Bioc hem. 57, 469-480. Collipp. P. J.. Carsten. A., Chen, S. Y.. Thomas, J.. and Maddiah. V. T. ( 1974) Biochc,m. Med. 10, 312-319. Griffin. L. (1962) Masters Thesis. Duke University, Durham. N. C. [quoted in (31)]. Pollard, E.. Powell, W. F.. and Reaume. S. H. (1952) Proc,. Not. Acud. Sc,i. USA 38, 173- 180. Setlow. R. B. (1952) Arch. Birx.hem. Biophy.s. 36. 328-335. Vegh. K., Spiegler. P.. Chamberlain. C.. and Mommaerts. W. F. H. M. (1968) Bic>c,hinr. BicJphy.v. Ac~cr 163, 266-268. LeMaire, M.. Jorgensen. K. E.. Roigaard-Petersen, H.. and Moller. J. V. (1976) Bicwhemistt-> 15, 5805-5812. Lowey. S.. Slayter. H. S., Weeds. A. G.. and Elaker. H. I 1969) J. A-lr~l. Bkd. 42, l-29. Haugen. D. A., and Coon. M. J. (1976) J. Biot. C‘hrm. 251, l817- 1827. Giovannozzi-Sermanni. G.. and Di Marco. G. (1965) Rodicrf. Rcs. 26, 63-68. Fluke, D. J. (1972) Rndicrt. Rrs. 51, 56-71.
10
KEMPNER
AND
49. Vollmer. R. T.. and Fluke, D. J. (19671 Rtrdinl. Ra.\. 31, 867-875. SO. Setlow, R.. and Doyle. B. (1953) AK/I. Bitwhcw. Biophys. 46, 46-52. 51. Augenstein, L. G.. Brustad. T.. and Mason, R. (1964)m Advances in Radiation Biology (Augenstein. L. G.. Mason. R.. and Quastler. H.. eds.). Vol. I. pp. 227-266. Academic Press. New York. 52. Gunther. W., and Jung. H. (1967) Z. Nnfu$~rsclz. 22, 313-320. 53. Brustad. T. (1964) irr Biological Effects of Neutron and Proton Irradiations, Vol. II. pp. 404-410. International Atomic Energy Agency. Vienna.
SCHLEGEL 54. Banns, H. E.. and Stephens-Newsham. L. ( 1974) .I. Pk~.sid. (Lontlm~ 242. 16P-18P.
G.
55. Nakao, M.. Nagano. K.. Nakao, T., Mizuno. N., Tashima. Y., Fujita. M.. Maeda, H.. and Matsudaira, H. r 1967) Bicx.hc~n~. Bic~ph~.t. Kc~.\. ~‘rvnmu/~. 29. 588-592. 56. Houslay. M.. Ellory. J.. Smith. G.. Hesketh, T., Stein. J.. Warren. G., and Metcalfe. J. C. (1977) Bicrhirn. Birqhys. AC,IN 467. 208-219. 57. Tanaka. S.. Nagahama, Yoneyama. Y. (1976) 1071. 58. Kagawa,
S., Takeshita, J. Biochem.
Y. r 19691 J. Biochem.
M.. and 80, lO67-
65, 925-934
