1178

Specialia

b l u e - s t a i n e d p r o t e i n b a n d s . T h i s also a p p l i e d t o t h e zones of t h e p r o t e i n f r a c t i o n s s e p a r a t e d i n cellulose a c e t a t e . E s t e r a s e b a n d s 7, 13, 15 a n d 16 were l o c a t e d in t h e prea l b u m i n fraction, while B a n d s 10, 11 a n d 12 m o s t l y in t h e ~t-globulin fraction, a n d B a n d s 2, 3, 4, 5 a n d 8 in t h e x~g l o b u l i n fraction. B a n d 6 and, p a r t l y , B a n d s 2 , 4 a n d 8 were in t h e fl-globulin f r a c t i o n a n d B a n d i m a i n l y in t h e y-globulin fraction. I n a c o n c e n t r a t i o n of 10 -SM, w h i c h was f o u n d to b e effective, eserine i n h i b i t e d t h e a c t i v i t y in B a n d s 2, 3, 4, 5, 10, 11 a n d 12; w i t h lower c o n c e n t r a t i o n s of eserine B a n d s 3 a n d 12 were o n l y p a r t i a l l y i n h i b i t e d . I n a c o n c e n t r a t i o n of 10-4M, i s o - O M P A i n h i b i t e d ]3ands 2, 3 a n d 11, p r e s u m a b l y non-specific cholinesterases. I n t h e c o n c e n t r a t i o n of

10-SM o n l y t h e b a n d 11 w a s s e n s i t i v e t o t h e i n h i b i t o r . E v e n in t h e h i g h e s t c o n c e n t r a t i o n s used (10-SM) 284 C 51 failed t o cause a n y i n h i b i t i o n of t h e esterase b a n d s ; t h i s suggests t h a t a c e t y l c h o l i n e s t e r a s e is e n t i r e l y a b s e n t f r o m t h e r a t serum. With the two-dimensional step-gradient acrylamide electrophoresis, i t was possibie t o c o r r e l a t e t h e i n d i v i d u a l s e r u m p r o t e i n s a n d esterase t o t h e c~-, fi- a n d y - g l o b u l i n b a n d s in t h e s a m e w a y as was p r e v i o u s l y e s t a b l i s h e d b y SMITHIES I~, u s i n g t h e less d i s c r i m i n a t i n g p a p e r a n d s t a r c h s y s t e m . O n e - d i m e n s i o n a l gel e l e c t r o p h o r e s i s g a v e v e r y l i m i t e d i n f o r m a t i o n of t h e d i s t r i b u t i o n of esterases in t h e classical s e r u m p r o t e i n fractions. T h e o b s e r v a t i o n t h a t t h e esterase b a n d s d i d n o t a l w a y s c o r r e s p o n d t o t h e areas of s t a i n a b l e p r o t e i n is in a g r e e m e n t w i t h earlier o b s e r v a t i o n s b y HUNTER et al. 7 on s e p a r a t i o n s w i t h p a p e r a n d s t a r c h .

Zusammen/assung. I s o e n z y m e v o n S e r u m e s t e r a s e n wurd e n z u e r s t m i t Z e l l u l o s e a z e t a t u n d d a n n m i t e i n e m Gradientgel yon Polyakrylamid mit zweidimensionaler Elekt r o p h o r e s e b e t d e r R a t t e u n t e r s u c h t . E i n n e u e s B a n d , das eine s t a r k e A l b u m i n e s t e r a s e a k t i v i t ~ i t a u f w e i s t u n d u n e m p f i n d l i c h gegen 1 0 - 3 M P h y s o s t i g m i n ist, w u r d e s o m i t gef u n d e n . V o n 17 E s t e r a s e b ~ n d e r n w a r e n 7 e m p f i n d l i c h auf P h y s o s t i g m i n , u n d 3 w u r d e n a u c h d u r c h I s o - O M P A geh e m m t , w i i h r e n d 284-C-51 gar k e i n e n E f f e k t zeigte.

+

c~2~

EXPERIENTIA 29/9

I1

r ~ 8

O. ER]4NK6 !3, R. L. HUNTER 14 a n d A. I. I(OSKIMIES IS

~ 6

Department o/ Anatomy, University o/Helsinki, Siltavuorenpenger 20, Helsinki 17 (Finland), 26 March 1973.

I

I

I

I

I

I

I

I

Fig. 3. Diagrammatic presentation of the esterase bands found. Tile same numbering is used in the text. The eorres'ponding serum protein fractions are marked at the bottom of the two-dimensional separation.

is o. SMITHIES,Adv. Protein Chem. ld, 65 (1959). 1s Supported by a grant from the Sigrid Jus61ius Foundation, Finland. 1~ On sabbathieal leave from the.Department of Human Anatomy, School of Medicine, University of California, Davis (California 95616, USA).

Mercury Binding to Human Haemoglobin M e r c u r y solutions, b o t h as i n o r g a n i c m e r c u r y salts a n d as organic m e r c u r y complexes, h a v e f o u n d wide a p p l i c a t i o n i n t h e e s t i m a t i o n of s u l p h y d r y l g r o u p s in p r o t e i n s 1-6. T h e s e m e t h o d s g e n e r a l l y use e i t h e r a n a m p e r o m e t r i c b a c k - t i t r a t i o n of m e r c u r y a f t e r r e a c t i o n w i t h t h e - S H g r o u p s or a s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n . F o r t h e s e s y s t e m s t h e o r g a n i c m e r c u r y clerivatives, s u c h as 'pm e r c u r i b e n z o a t e ' h a v e p r o v e d m o r e successful t h e n i n o r g a n i c m e r c u r y w h i c h suffers f r o m t h e d i s a d v a n t a g e of its d i v a l e n c y since i t c a n r e a c t w i t h e i t h e r one - S H g r o u p to f o r m R S H g X or t w o t o f o r m (RS)2Hg 1,~. T h i s h a s lead t o conflict c o n c e r n i n g t h e n u m b e r of a v a i l a b l e sulphydryl groups present. T h e degree of d e n a t u r a t i o n of t h e p r o t e i n , especially w i t h h a e m o g l o b i n , m a r k e d l y affects t h e e s t i m a t e d n u m b e r of t h i o l groups. S t r i c t a d h e r e n c e t o t h e c o n t r o l of "pH a n d c o m p o s i t i o n is essential a n d e v e n a m m o n i a b a s e d b u f f e r s are r e p o r t e d to yield e r r o n e o u s r e s u l t s d u e to t h e f o r m a t i o n of m e t a l l i c a m m i n e s s. H o w e v e r , t h e i n t r o d u c t i o n of tris buffers h a s s u b s t a n t i a i l y i m p r o v e d t h e confidence p l a c e d in e x p e r i m e n t a l r e s u l t s 7. T h e w o r k d e s c r i b e d here offers a n a l t e r n a t i v e a p p r o a c h t~o t h e s t u d y of h a e m o g l o b i n s u l p h y d r y l g r o u p s a n d t h e i r

reaction with mercury using a technique analogous to t h a t p r e v i o u s l y r e p o r t e d in c o n n e c t i o n w i t h t h e i n t e r a c t i o n of lead w i t h h a e m o g l o b i n s . Method. W h o l e b l o o d was collected b y v e n e p u n c t u r e , t h e cells s e p a r a t e d a n d w a s h e d 3 t i m e s w i t h physiological saline. T h e e r y t h r o c y t e s were t h e n h a e m o l y s e d b y r e p e a t e d freezing a n d t h a w i n g a n d t h e s t r o m a r e m o v e d b y c e n t r i f u g a t i o n 9. T h e s u p e r n a t a n t was r e m o v e d , d i l u t e d • 2 w i t h tris-maleic acid buffer, p H 7.0 a n d s a m p l e s t a k e n for

1 A. C. ALLISON and R. CECIL, Biochem. J. 69, 27 (1958). R. CECIL and J. R. MCPHEE, Adv. Prot. Chem. 74, 255 (1959). s V. M. INGRAM, Biochem. J. 59, 653 (1955). 4 F. A. HOMMES,J. SANTEMA-DRINKWAARDand T. H. J. HUISMAI% Biochim. biophys. Acts 20, 564 (1956). 5 W. STRICKS and I. M. KOLTHOFF,J. Am. chem. Soe. 75, 5673 (1953). s W. STRICXSand I. M. KOLTI~OFF,J. Am. chem. Soe. 72, 1952 (1950). 7 R.E. BENESCH,H. A. LARDY and R. ]~ENESCH,J. biol. Chem. 216, 663 (1955). 8 D. BARLTROP and A. M. SMITH, Experientia 28, 76 (1972). 9 D. BARLTROPand A. M. SMITH, Experientia 27, 92 (1971).

15.9.1973

Specialia

t h e d e t e r m i n a t i o n , of h a e m o g l o b i n 10 a n d t o t a l p r o t e i n 11. M e r c u r y s o l u t i o n s of c o n c e n t r a t i o n s v a r y i n g f r o m 5 t o 500 p p m were p r e p a r e d f r o m A R g r a d e m e r c u r i c c h l o r i d e dissoIved in tris-maleic acid b u f f e r p t l 7.0. T h e s e s o l u t i o n s were l a b e l l e d b y t h e a d d i t i o n of H g r 2 0 3 s o l u t i o n t o give a specific a c t i v i t y of 10 v.Ci/tlg Hg. A l i q u o t s of t h e h a e m o g l o b i n a n d m e r c u r y s o l u t i o n s were m i x e d in sealed d i a l y s i s t u b i n g b a g s (Visking) a n d c e n t r i f u g e d for 2 h a t 2,500 g in a t e m p e r a t u r e c o n t r o l l e d c e n t r i f u g e a t 25~ s. I n i t i a l studies, o m i t t i n g h a e m o g l o b i n , s h o w e d m a r k e d b i n d i n g of m e r c u r y t o t h e bag. P r e l i m i n a r y t r e a t m e n t of t h e b a g s w i t h successive w a s h e s of 0.1 N HC1 0.1. N s o d i u m h y d r o x i d e s o l u t i o n a n d f i n a l l y s e v e r a l c h a n g e s of b o i l i n g distilled w a t e r was n e c e s s a r y t o r e d u c e t h e m e r c u r y b i n d ing t o a n a c c e p t a b l e a n d c o n s i s t e n t level. A f t e r c e n t r i f u g a t i o n ; a l i q u o t s of t h e u l t r a f i l t r a t e a n d t h e r e s i d u a l h a e m o l y s a t e were o b t a i n e d a n d t h e m e r c u r y c o n t e n t d e t e r m i n e d b y m e a s u r e m e n t of t h e 7 - e m i s s i 0 n of Hg-203 u s i n g a H e w l e t t - P a c k a r d auto-y-spectromeCer. T h e m e r c u r y b o u n d t o t h e h a e m o g l o b i n was d e t e r m i n e d b y difference. Findings. T h e r e s u l t s were s u b j e c t e d t o a n a l y s i s b y t h e m e t h o d of sCATCHARD 12 a n d are g i v e n in t h e Figure. T h e i n t e r c e p t o n t h e abscissa i n d i c a t e s t h a t t h e h a e m o globin m o l e c u l e h a s a m e a n v a l u e of 2.5 sites a v a i l a b l e for mercury binding. From the intercept on the ordinate the u n c o r r e c t e d a s s o c i a t i o n c o n s t a n t (Ku) for t h e m e r c u r y h a e m o g l o b i n c o m p l e x was c a l c u l a t e d a n d g a v e a v a l u e for log Ku = 6.65. I o d o a c e t a t e (10-2M) b l o c k e d t h e b i n d i n g

1179

of m e r c u r y to t h e h a e m o g l o b i n molecule t h u s c o n f i r m i n g t h e a d d i t i o n of t h e m e t a l t o t h e a v M l a b l e - S H groups. T h e s t a b i l i t y c o n s t a n t n e e d s t o be c o r r e c t e d for r e d u c t i o n in c o n c e n t r a t i o n of free m e t a l b y tile c o m p e t i n g t~is a n d c h l o r i d e c o m p l e x e s ~ , ~*. T h e c o r r e c t e d v a l u e ~s s u c h t h a t log K = 19.6. T h i s v a l u e agrees well w i t h t h a t o b t a i n e d b y o t h e r w o r k e r s ~4. Discussion. T h e w o r k d e s c r i b e d offers a n a l t e r n a t i v e a p p r o a c h t o t h e s t u d y of m e a l b i n d i n g in biological s y s t e m s . I t h a s t h e a d v a n t a g e of s i m p l i c i t y a n d r a p i d i t y c o m p a r e d w i t h t h e dialysis t e c h n i q u e a n d does n o t suffer f r o m t h e p r o b l e m of electrode c o n t a m i n a t i o n e x p e r i e n c e d in p o l a r o g r a p h y . T h e m e t h o d c a n b e a d a p t e d t o t h e s t u d y of o t h e r p r o t e i n s a n d m e t a l s a n d a l t h o u g h a r a d i o a c t i v e t r a c e r d e t e r m i n a t i o n was i n v o l v e d in t h i s s t u d y , conv e n t i o n a l s p e c t r o p h o t o m e t r i c or a t o m i c a b s o r p t i o n a n a l y s i s could also b e used. T h e m e a n v a l u e o b t a i n e d for t h e n u m b e r of m e r c u r y a t o m s c o m b i n i n g w i t h t h e h a e m o g l o b i n molecule is in good a g r e e m e n t w i t h t h o s e o b t a i n e d b y o t h e r workers 1, 3,1~, 1~ a n d a p p e a r s t o c o n f i r m t h e o b s e r v a t i o n 2 t h a t r e a c t i o n of t h e n a t i v e , as o p p o s e d t o t h e d e n a t u r e d , p r o t e i n r e p r e s e n t s a n e q u i l i b r i u m i n v o l v i n g all t h e sulphy d r y l g r o u p s p r e s e n t . T h e l i n e a r i t y of t h e p l o t in t h e F i g u r e i n d i c a t e s t h a t t h e b i n d i n g sites are all e q u i v a l e n t a n d e q u a l l y a v a i l a b l e for m e r c u r y .

Zusammen/assung. Mittels 2~ als H g - C h l o r i d w u r d e n die freien S H - G r u p p e n des H~Lmoglobins in e i n e m s t r o m a f r e i e n H g m o l y s a t t i t r i e r t u n d festgestellt, dass die d u r c h s c h n i t t l i c h e Z a h l freier, r e a k t i v e r S H - G r u p p e n p r o Molekiil 2,5 b e t r u g . D. ]~ARLTROp17 a n d A. M. SMITH St. Mary's Hospital Medical School, Paediatric Unit, London W. 2 (England), 12 2Vlarch 1973.

O0 05

10 1'5 2'0 2'5 V

Plot and regression line for v/M against v, where v denotes moles of bound mercury per total moles of haemoglobin and M is the molar concentration of free metal (Mter SCATCHARD12).The 95% confidence limits are included.

10 A. J. HAINLINE, in Statistical Methods in Clinical Chemistry (Academic Press, New York 1958), vol. 2, p. 49. 11 W. H. DAUGHADAY, O. H. LOWRY, N. J. ROSEBROUGIt and W. S. FIELDS, J. Lab. clin. Med. 39, 663 (1952). 12 G. SCATCHARD,Ann. N. Y. Acad. Sei. 51,660 (1949). la j. E. COLEMANand B. L. VALEE, J. biol. Chem. 236, 2244 (1961). 1~ B. L. VALEE and D. D. ULMER, A. Rev. Bioehem. 47, 91 (1972). 15 W. L. HUaHES JR., Cold Spring Harbor Symp. quant. Biol. 14, 79 (1949). 16 R. BENESCH and R. E. BEN~SC~, Methods o/ Biochemical Analysi= (Ed. D. GLICK; Interscience, N.Y. 1962), vol. 10, p. 43. 17 Lead-203 was kindly supplied by the M. R. C. Cyclotron Unit, Hammersmith, London. D. ]3. is a Wellcome Senior Research FeUow in Clinical Science.

A Technique for Establishing Parabiosis between Houseflies (Musca domestica L.) E x c h a n g e of ' m i l i e u i n t e r n e , of l i v i n g a n i m a l s b y t h e t e c h n i q u e of p a r a b i o s i s affords u n i q u e o p p o r t u n i t i e s for v a r i e d e x p e r i m e n t a t i o n . WlGGLESWORTHFS 1 a n d WILLIAM'S ~ w o r k w i t h Rhodnius a n d Plalysamia respectively, u s i n g t h i s t e c h n i q u e , are classics i n t h e a n n a l s of e n t o m o logical research. BEAMENT a d e t e c t e d b l o o d - b o r n e p a r a l y z ing a g e n t s in s t r e s s - p r o s t r a t e d A m e r i c a n r o a c h e s b y u s i n g t h i s t e c h n i q u e . COLHOUN 4. h a s also used t h i s a p p r o a c h for s t u d y i n g i n s e c t i c i d a l action. I n t h e course of a n i n v e s t i g a t i o n 5 of t h e n a t u r e of r e s i s t a n c e m e c h a n i s m s a g a i n s t t h e insecticide dieldrin, a n a t t e m p t was m a d e to effect a p a r a b i o t i c l i n k b e t w e e n houseflies, Musca domestica L. E x p e r i m e n t s c o n d u c t e d for e s t a b l i s h i n g successful p a r a b i o s i s b e t w e e n flies are r e l a t e d below.

T h e a n i m a l used as t h e ' p r i m a r y ' was d e p r i v e d of its wings a n d its m e s o t h o r a c i c t e r g u m was clipped n e a t l y so as to expose t h e dorsal l o n g i t u d i n a l muscles l y i n g u n d e r n e a t h (Figure A). A s m a l l d r o p of p h y s i o l o g i c a l saline (ASPERN a n d VoN ESCH 6) was d e p o s i t e d in t h e incision t o keep it moist. T h e a n i m a l was t h e n i m m o b i l i z e d o n a b l o c k of w a x u s i n g staples (Figure ]3). T h e fly used as

1 V. B. WIGaLESWORTR,Q. J1. microsc. Sci. 77, 191 (1934). C. M. WILLIAMS,Biol. Bill. 93, 89 (1947). J. W. L. BEAMENT,J. Insect Physiol. 2, 199 (1958). 4 E. H. COLHOUN,J. agric. Food Chem. 8, 252 (1960). 5 R. N. SHARMA,Ph.D. Thesis, Delhi University (1959). 6 K.V. ASPERN and vo~ EscH, Archs nedrl..Zool. 11,342 (1956).