Biological Effects of Nonionizing Radiation - American Chemical Society

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3 Dielectric

Properties

of

Water

in

Myoglobin

Solution Ε. H. GRANT, N. R. V. NIGHTINGALE, and R. J. SHEPPARD Physics Department, Queen Elizabeth College, London, W8 7AH, U.K.

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S. R. GOUGH Division of Chemistry, National Research Council of Canada, Ottawa, Ontario, Canada, K1A OR9 Biological material has a high aqueous content and it is acknowledged that the physical properties of the water are different from those of pure liquid water. This has given rise to the respective terms "bound" water and "free water" although, of course, the terms are purely relative. The actual properties of the water in biological solution are however subject to controversy and there is still considerable disagreement about its nature. One method of investigation is to determine the dielectric properties of an appropriate biological material over a frequency range from around 100MHz or lower right up to high microwave frequencies, and in the present paper some recent measurements on myoglobin solutions are described. The determinations, which were made at 20 C on solutions of concentration 200mg/ml, over a frequency range 0.6 - 15000MHz represent the first stage of a comprehensive programme to measure the dielectric properties of water in solution. Owing to the difficulty of measuring accurately the conductivity at the low frequency end of this range only the values were used in the analysis. Over the frequency range 0.6 to 100MHz bridge apparatus was used; the methods having been previously described (1,2). At frequencies between 250 15000MHz the permittivity determinations were made using coaxial line apparatus. Between 250 - 4000MHz methods were used which involve a coaxial line cell having a sliding inner conductor with a protruding probe which detects the electric field strength as a function of distance within the liquid sample. The cell construction and the appropriate techniques of measurement have been described previously(3-6). In the overlapping range 1500-15000MHz the results were obtained using a coaxial line cell terminated by a movable short circuiting plunger(7). o

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Illinger; Biological Effects of Nonionizing Radiation ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

58

BIOLOGICAL EFFECTS OF NONIONIZING RADIATION

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The s o l u t i o n s were prepared at a c o n c e n t r a t i o n of 2 0 0 m g / m l by d i s s o l v i n g sperm w h a l e m y o g l o b i n ( M i l e s L a b o r a t o r i e s (PTY) Limited) i n d e - i o n i s e d water. The s a m p l e s were e l e c t r o d i a l y s e d before measurement to reduce the i o n i c c o n d u c t i v i t y . F u l l d e t a i l s of the s o l u t i o n preparation procedure have appeared p r e v i o u s l y (8). The p e r m i t t i v i t y measurements were obtained at 20 C u s i n g sample c e l l s t h e r m o s t a t i c a l l y c o n t r o l l e d to - 0.1 Q The d e t e r m i n a t i o n s were made at 45 frequencies and the mean v a l u e of € at e a c h frequency i s shown i n Figure 1. Interpretation of d i e l e c t r i c data on m y o g l o b i n s o l u t i o n In order to a n a l y s e the r e s u l t s i n Figure 1 it i s n e c e s s a r y to make v a r i o u s a s s u m p t i o n s about the nature of the p r o c e s s e s i n v o l v e d at a m o l e c u l a r l e v e l . B a s i c a l l y there are two t y p e s of p o l a r m o l e c u l e present - m y o g l o b i n and water - and t h e r e ­ fore a s i m p l e approach would be to represent the information by two d i s p e r s i o n r e g i o n s . S i n c e , h o w e v e r , the water m o l e c u l e s adjacent to the m a c r o m o l e c u l e s have d i e l e c t r i c properties different from t h o s e i n the bulk of the f l u i d (J-, 9) a more r e a l i s t i c interpretation i s to c o n s i d e r two separate d i s p e r s i o n r e g i o n s for the water i . e . three d i s p e r s i o n s i n t o t a l . Further refinements would be p o s s i b l e by s u b d i v i d i n g the water into more than two c a t e g o r i e s s i n c e it i s an o v e r s i m p l i f i e d p i c t u r e to c o n s i d e r the water m o l e c u l e s as either r i g i d l y bound to the m y o g l o b i n on the one h a n d , or i n an i d e n t i c a l state to those i n pure water on the other. From work c a r r i e d out p r e v i o u s l y on m y o g l o b i n (8) at frequencies w i t h i n the d i s p e r s i o n r e g i o n of the m y o g l o b i n m o l e c u l e it i s k n o w n that t h i s d i s p e r s i o n may not be c h a r a c t e r i s e d by a D e b y e d i s p e r s i o n , and therefore the present data were fitted to three r e l a x a t i o n regions c o n s i s t i n g of a C o l e - C o l e d i s p e r s i o n to represent the m y o g l o b i n f o l l o w e d by two D e b y e d i s p e r s i o n s to a c c o u n t for the w a t e r . The C o l e - C o l e spread parameter for the m y o g l o b i n was found to be 0 . 1 5 - 0 . 0 3 ; the v a l u e s of the r e l a x a t i o n time and d i e l e c t r i c i n c r e m e n t are shown i n T a b l e I. Table I. D i e l e c t r i c increments and r e l a x a t i o n frequencies for the m y o g l o b i n s o l u t i o n (Temperatuie 20 C ; C o n c e n t r a t i o n 2 0 0 m g / m l ; Errors are the 95% c o n f i d e n c e i n t e r v a l s ) Dispersion

Dielectric increment (Δ) 34 - 5% 3 . 6 - 11% 59.0 - 0.4%

Relaxation f r e q u e n c y / M H z ($· ) Λ

2.2

583 -

14274 -

7% 22%

1.6%

Illinger; Biological Effects of Nonionizing Radiation ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

Illinger; Biological Effects of Nonionizing Radiation ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

ε

1

30^

5(Η

7(Η

90Η

Figure 1.

10

Frequency/Μ Η ζ

100

δ

1000

···%·· ν

10000

Relative permittivity of an aqueous solution of myoglobin at 20°C, concentration 200 mg/mL

1

V

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5

vo

ο

δ· 2'

>

Η W Η

2

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60

BIOLOGICAL EFFECTS OF NONIONIZING RADIATION

Attempts made to fit two d i s p e r s i o n s o n l y f a i l e d on a c c o u n t of the u n a c c e p t a b l y h i g h root mean square d e v i a t i o n . In an attempt to fit four d i s p e r s i o n s it was found that the parameters were h i g h l y c o r r e l a t e d w h i c h means that although the p o s s i b i l i t y of four d i s p e r s i o n s i s not p r e c l u d e d there i s not s u f f i c i e n t e v i d e n c e from the present experiments to demonstrate t h e i r existence. The v a l u e s of the parameters i n d i c a t e d for the β d i s p e r s i o n (myoglobin) and S d i s p e r s i o n (water i m m e d i a t e l y adjacent to the myoglobin) are i n good agreement w i t h those c o n c l u d e d p r e v i o u s l y from work c a r r i e d out at frequencies between 0 . 2 1 3 0 0 M H z (8,10.). For the JT d i s p e r s i o n , however it i s found (Table I) that the r e l a x a t i o n frequency i s s i g n i f i c a n t l y l e s s than the literature v a l u e for pure water of 1 6 . 7 - 0 . 1 G H z w h i c h has been v e r i f i e d i n our laboratory (7) u s i n g the same apparatus as that employed for the present m y o g l o b i n s t u d i e s . Moreover t h i s c o n c l u s i o n i s not affected by the model used to represent the d a t a . For e x a m p l e , i f the S and y d i s p e r s i o n s are e a c h represented by a C o l e - C o l e d i s t r i b u t i o n the mean v a l u e s of a l l the parameters i n Table I are unchanged to w i t h i n e x p e r i m e n t a l error and the c o n f i d e n c e i n t e r v a l s of the v a l u e s of the C o l e - C o l e spread parameter overlap- zero i n both c a s e s . The v a l u e of i n t h i s c a s e i s now 14. 2 - 0 . 3 G H z as compared w i t h 1 4 . 3 - 0 . 2 G H z i n Table I and 1 6 . 7 - 0 . 1 G H z for pure w a t e r . M o l e c u l a r interpretation of m y o g l o b i n s o l u t i o n data Our r e s u l t s imply that v e r y l i t t l e of the bulk water component of the s o l u t i o n has d i e l e c t r i c properties the same as those of pure water. O n m o l e c u l a r grounds t h i s appears to be a r e a s o n a b l e c o n c l u s i o n . For a 20% s o l u t i o n of m y o g l o b i n the m o l e c u l e s w o u l d on average be separated by a d i s t a n c e of around one m o l e c u l a r r a d i u s ( - - 1 . 5 n m ) . This d i s t a n c e c o u l d be o c c u p i e d by about five water m o l e c u l e s and thus no water m o l e c u l e would on average be further a w a y from a m a c r o m o l e c u l e than the t h i r d s h e l l . This l o w e r i n g i n r e l a x a t i o n frequency of the bulk water component of a b i o l o g i c a l s o l u t i o n from that of pure water i s an e s t a b l i s h e d phenomenon and was o b s e r v e d i n the e a r l y 1950's by C o o k (_Π,_12) and by H a g g i s , H a s t e d and Buchanan (13) a l t h o u g h the rigour of the c o n c l u s i o n s was hampered by the p r e s e n c e of measurements at a few frequencies o n l y . It i s w e l l k n o w n that o r g a n i c s o l u t e s in g e n e r a l modify the properties of bulk water. For i n s t a n c e an a d d i t i o n of 20% by weight of tetrohydrofuran to water i n c r e a s e s the water d i e l e c t r i c r e l a x a t i o n time by o v e r 50% as a r e s u l t of s t r u c t u r a l m o d i f i c a t i o n of the hydrogen-bonded network

Illinger; Biological Effects of Nonionizing Radiation ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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3.

GRANT ET AL.

Water in Myoglobin Solution

61

(14). It i s not s u r p r i s i n g that b i o l o g i c a l m a c r o m o l e c u l e s impose a s i m i l a r s t a b i l i z i n g effect. A similar proposal concerning the i n c r e a s i n g of the water r e l a x a t i o n time w a s made r e c e n t l y by M a s s z i , Szuarto and G a f (15) from c o n d u c t i v i t y measurements made o n m u s c l e t i s s u e but i n t h i s c a s e the h i g h e s t frequency of measurement w a s 4 G H z w h i c h i s too l o w to p r o v i d e unambiguous e v i d e n c e i n r e s p e c t of the c h a r a c t e r i s t i c s of the dispersion. Other work on m u s c l e t i s s u e c a r r i e d out by F o s t e r , Schepps a n d Schwan (16) at 1 C came to the o p p o s i t e c o n c l u s i o n , n a m e l y , that the bulk water r e l a x a t i o n frequency w a s not s i g n i f i c a n t l y different from that of pure water at that temperature. T h i s i s at v a r i a n c e w i t h the present work a l t h o u g h the d i f f e r e n c e s i n the temperature and i n the m a t e r i a l b e i n g s t u d i e d may be at l e a s t part of the e x p l a n a t i o n of the apparent discrepancy. The amount of t i g h t l y bound water (water o f hydration) present i n the m y o g l o b i n s o l u t i o n c a n be c a l c u l a t e d from the a m p l i t u d e of the S d i s p e r s i o n u s i n g a method p r e v i o u s l y d e s c r i b e d (1_,10) . From the v a l u e of 3 . 6 for t h i s parameter (Table I) a v a l u e of h y d r a t i o n of 0 . 1 5 - 0 . 0 2 unit m a s s of water per unit mass of m y o g l o b i n i s o b t a i n e d . C o n s i d e r e d as a v o l u m e fraction of the t o t a l water content t h i s w o u l d amount to about 4%, w h i c h compares w e l l w i t h the figure of 5% r e c e n t l y proposed for m u s c l e fibres by F o s t e r , Schepps and S c h w a n (16). Acknowledgements W e w o u l d l i k e to thank the U n i t e d States Office of N a v a l R e s e a r c h ( P h y s i o l o g y Program Grant N o . N 0 0 0 1 4 - 7 7 - G - 0 0 7 5 ) for supporting NRVN and the N a t i o n a l R e s e a r c h C o u n c i l of C a n a d a for the p r o v i s i o n of s a b b a t i c a l l e a v e for S R G .

1.

2. 3. 4. 5. 6. 7. 8.

Literature Cited Grant,E.H: Sheppard,R.J; South,G.P: "Dielectric behaviour of biological molecules in solution"; Oxford University Press, 1976. Essex,C.G; South,G.P; Sheppard,R. J; Grant,E.H: J.Phys. Ε 1975, 8, 385 Grant, E . H ; Keefe S.E. Rev. Sci. Instrum 1968, 39, 1800 Sheppard,R.J; J.Phys.D 1972, 5, 1576 Sheppard,R.J; Grant,E.H: J. Phys. Ε 1972, 5, 1208 Szwarnowski,S; Sheppard,R. J. J.Phys. Ε 1979, 12, 937 Nightingale, Ν.R.V: Szwarnowski, S; Sheppard,R. J; Grant,E.H; Submitted to J.Phys.Ε South,G.P; Grant,E.H; Proc.R.Soc.Lond.A. 1972, 328,371

Illinger; Biological Effects of Nonionizing Radiation ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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9. Pennock,B.E; Schwan, H.P; J.Phys. Chem 1969,73, 2600 10. Grant,E.H; Mitton, B.G.R; South,G.P; Sheppard,R. J. Biochem J. 1974, 139, 375 11. Cook,H.F. Brit. J.Appl. Phys. 1951, 2, 295 12. Cook,H.F. Brit. J.Appl. Phys. 1952, 3, 249 13. Haggis,G.H; Hasted,J.B; Buchanan, T.J. J. Chem. Phys. 1952, 20, 1452 14. Gough,S.R. J.Soln. Chem. 1978, 8, 371 15. Masszi, G; Szverto,A; Graf, P. Biophys. Acad. Sci. Hung. 1976, 129 16. Foster, K.R; Schepps, J.L; Schwan, H.P. Biophys. J. 1980, 29, 271 RECEIVED

February 3, 1981.

Illinger; Biological Effects of Nonionizing Radiation ACS Symposium Series; American Chemical Society: Washington, DC, 1981.