Trace Elements Associated with Proteins - American Chemical Society

Chapter 21

Trace Elements Associated with Proteins Neutron Activation Analysis Combined with Biological Isolation Techniques 1

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Susan F. Stone, Rolf Zeisler, Glen E. Gordon, Raphael P. Viscidi, and Erich H. Cerny 4

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National Institute of Standards and Technology, Center for Analytical Chemistry, Gaithersburg, MD 20899 Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742 The Eudowood Division of Infectious Diseases, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205 Fondation pour la Recherche Medicale, University of Geneva, 64 Avenue de la Roseraie, 1211 Geneva 4, Switzerland

Downloaded by UNIV OF ARIZONA on August 1, 2012 | http://pubs.acs.org Publication Date: December 26, 1991 | doi: 10.1021/bk-1991-0445.ch021

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Combinations of b i o l o g i c a l techniques with neutron activation analysis (NAA) can be em ployed for protein quantification in biological samples. A protein of interest can be isolated physically by a high resolution separation tech nique such as polyacrylamide gel electro phoresis (PAGE) or by an immunochemical techni que, such as a specific antibody-antigen reac tion. NAA is then used for quantification of the isolated protein by determining either an element that is structurally i n t r i n s i c , or one that has been introduced as a label. An ex ample of the f i r s t method is the determination of phosphoproteins by PAGE-NAA. This method is discussed and results for two phosphoproteins, α-casein and phosvitin, are presented for several materials, including an α - c a s e i n con centration of 26 mg/mL in low-fat milk. The second method presented is an immunoassay with a colloidal gold tag and detection by NAA. Neutron a c t i v a t i o n analysis (NAA) has been used to d e t e r m i n e t r a c e e l e m e n t c o n c e n t r a t i o n s i n many t y p e s o f b i o l o g i c a l samples. We h a v e e m p l o y e d N A A a n d r e l a t e d nuclear methods f o r the analysis o f a wide v a r i e t y of b i o l o g i c a l samples, ranging from the determination of more than twenty trace elements i n human l i v e r s (JL) a n d 45 e l e m e n t s i n b i v a l v e samples (2.) , to the deter mination of u l t r a t r a c e concentrations of Pt i n human livers (ϋ) and Cr i n blood (4_) . It is also possible to quantify specific macromolecules i n b i o l o g i c a l samples by combining NAA w i t h protein " i s o l a t i o n " techniques. We d e s c r i b e h e r e the application o f NAA combined with two such t e c h n i q u e s : p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s 0097-6156/91/0445-0265$06.00/0 © 1991 American Chemical Society

In Biological Trace Element Research; Subramanian, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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(PAGE), w h i c h g i v e s s p a t i a l p r o t e i n s e p a r a t i o n , and an enhanced immunoassay with colloidal gold, which u t i l i z e s t h e s p e c i f i c i t y o f an a n t i b o d y - a n t i g e n react i o n to i s o l a t e the desired analyte.


PAGE-NAA For Détermination of I n t r i n s i c Elements I n c o n t r a s t t o p r e v i o u s w o r k , w h e r e t h e e m p h a s i s was on the determination of trace elements associated with i s o l a t e d p r o t e i n s o n a n i n d i v i d u a l b a s i s , we h a v e d e v e l o p e d a method t o s i m u l t a n e o u s l y a n a l y z e elements i n p r o t e i n s t h a t have been i s o l a t e d from complex p r o t e i n m i x t u r e s . The m e t h o d was used to determine concentrat i o n s o f e l e m e n t s a s s o c i a t e d w i t h s p e c i f i c p r o t e i n s and thus determine the concentrations of the p r o t e i n s . The s h a r p r e s o l u t i o n o f PAGE, c o m b i n e d w i t h t h e sensitive detection of NAA, was used to detect and quantify proteins i n c o m p l e x s a m p l e s . The feasibility of the P A G E - N A A m e t h o d was shown by o u r a n a l y s i s o f c o v a l e n t l y bound Ρ i n phosphoproteins (ϋ). The a d v a n t a g e s o f PAGE-NAA c a n be summarized as follows : (1) A p r o t e i n sample i s separated i n t o i t s com ponents by electrophoresis on a single polyacrylamide gel matrix. This takes advan tage o f t h e h i g h r e s o l u t i o n and simultaneous multi-component analysis that is possible w i t h t h i s system. (2) The whole gel is subjected to neutron activation; hence, each a c t i v a t a b l e element contained i n the gel matrix, in principle, c a n be d e t e c t e d , so t h e m e t h o d i s n o t limited to selectively introduced labels. Since the analytical separation is done prior to n e u t r o n bombardment, and t h e p o s i t i o n s of t h e proteins are f i x e d i n the g e l , the SzilardChalmers effects induced by recoil from prompt γ-ray emission do not affect this technique. The separated proteins are visualized v i a the a c t i v a t e d element (in t h i s case, P) a f t e r exposure to x-ray film and subsequent development, yielding an autoradiograph of the a c t i v a t e d g e l with i t s separated proteins. (3) An a d d i t i o n a l d i m e n s i o n f o r i d e n t i f i c a t i o n i s added t o the u s u a l molecular weight parameter o f PAGE, as t h e i n t r i n s i c e l e m e n t s a r e spe cific for certain proteins. This relation s h i p makes t h e t e c h n i q u e superior to staining techniques. (4) Q u a n t i t a t i v e assay of p r o t e i n s i s p o s s i b l e from determination of the associated trace elements i f the protein-element stoichiometry i s known. The a r e a l d e n s i t i e s , measured as absorbance readings of the v i s u a l i z e d areas, 3 2


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are p r o p o r t i o n a l t o t h e mass o f t h e e l e m e n t p r e s e n t i n t h e s e p a r a t e d band and/or stan d a r d . By c o m p a r i n g the areal densities of s t a n d a r d s , w h i c h c o n t a i n known amounts o f t h e element, with the areal densities of the s e p a r a t e d p r o t e i n s , t h e mass o f t h e e l e m e n t in t h e p r o t e i n band i s determined. I f the element-protein stoichiometry of the p a r t i c u lar p r o t e i n i s w e l l - c h a r a c t e r i z e d , t h e mass of t h e p r o t e i n can be a s s a y e d .


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Materials . A c r y l a m i d e , Ν,Ν ,Ν -methylenebisacrylamide (bis), ( b o t h a n a l y t i c a l g r a d e r e a g e n t s ) and TEMED were obtained f r o m SERVA Fine Biochemica (Westbury, NY) . Glycine, t r i s hydroxymethyl-aminomethane (Tris), am monium persulfate, phosvitin, α-casein and sodium c a s e i n a t e were p u r c h a s e d f r o m Sigma C h e m i c a l Company (St. L o u i s , MO), and sodium d o d e c y l s u l f a t e (SDS), f r o m P i e r c e Company (Rockford, I L ) . The two r e f e r e n ce m a t e r i a l s u s e d were t h e N a t i o n a l I n s t i t u t e o f S t a n dards and Technology S t a n d a r d R e f e r e n c e M a t e r i a l (SRM) 1845, C h o l e s t e r o l i n E g g Powder, a n d t h e I n t e r n a t i o n a l Atomic Energy Agency (IAEA) Certified Reference Material (CRM) A - l l , M i l k Powder. Ammonium d i h y d r o g e n phosphate ( p u r i t y 99.0%) was f r o m B a k e r . A l l water used t o prepare the gels and b u f f e r s was purified t h r o u g h a Nanopure t r i p l e exchange-column system. To m i n i m i z e c o n t a m i n a t i o n , a l l p r e p a r a t i o n and sample h a n d l i n g were done u n d e r a C l a s s 10 (< 10 p a r t i c l e s p e r c u b i c f o o t ) l a m i n a r f l o w c l e a n hood ( E n v i r o n m e n t a l A i r Co.). The H o e f e r s e r i e s 600 u n i t f o r s l a b g e l s was u s e d f o r t h e e l e c t r o p h o r e s i s . The f i l t e r p a p e r s u s e d as gel b a c k i n g s were e i t h e r Whatman 41 o r B i o - R a d filter paper. The X - r a y f i l m u s e d was Kodak X-OMAT AR ™ f i l m , 20- χ 25-cm (8 χ 10- i n . ) , and t h e d e v e l o p i n g and f i x i n g c h e m i c a l s were t h e Kodak GBX b r a n d . An LKB 2202 UltroScan laser densitometer ( L K B - P r o d u c k t e r , Bromma, Sweden) e q u i p p e d w i t h a h e l i u m - n e o n l a s e r s o u r c e (632.8 nm) was used t o scan the areas on t h e d e v e l o p e d autoradiographs. M e t h o d . Samples were s e p a r a t e d by g r a d i e n t polyacrylamide g e l e l e c t r o p h o r e s i s i n a discontinuous buffer system (jL) . P o l y a c r y l a m i d e s l a b g e l s , 15 χ 15 χ 0.15 cm, were p r e p a r e d i n T r i s / g l y c i n e b u f f e r s w i t h a s t a c k ing g e l o f 5% Τ a n d 2.6% C (where Τ = w t / v o l % o f monomers, and C = g b i s / 1 0 0 g ( a c r y l a m i d e + b i s ) ) and a r e s o l v i n g g e l o f 10 - 17% Τ a n d 2.6 % C. The gradient gels were p r e p a r e d u s i n g a gradient maker w i t h a p e r i s t a l t i c pump. The f i n a l molar con c e n t r a t i o n s o f T r i s were 0.375 M (pH 8.8) i n t h e s e p a r a t i n g g e l a n d 0.125 M (pH 6.8) i n t h e s t a c k i n g g e l . The c o n c e n t r a t i o n o f SDS i n b o t h g e l s was 0.1%. The g e l s were p o l y m e r i z e d c h e m i c a l l y b y t h e a d d i t i o n o f 0.025% TEMED (by volume) a n d 0.02% ( w t / v o l %) ammonium p e r s u l -


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fate. D u p l i c a t e g e l s , one f o r i r r a d i a t i o n a n d one f o r n o n - s p e c i f i c p r o t e i n s t a i n i n g were c a s t simultaneously. P r o t e i n samples, g e n e r a l l y o b t a i n e d a s l y o p h i l i z e d pow d e r s , needed t o be d i s s o l v e d i n sample b u f f e r p r i o r t o a p plication t o the gels. The T r i s / g l y c i n e sample b u f f e r s a l s o c o n t a i n e d t h e d e t e r g e n t SDS (10 %, w t / v o l ) and a reducing agent, such as 2-mercaptoethanol o r d i t h i o threitol, to a c h i e v e uniform, r o d shaped d e t e r g e n t p o l y p e p t i d e complexes. Ethylenediamine t e t r a a c e t i c a c i d (EDTA, sodium s a l t ) was a l s o i n c l u d e d i n t h e sample b u f fer t o chelate Ca , which promotes f o r m a t i o n o f c a s e i n aggregates o r m i c e l l e s . The p r e p a r e d sample s o l u t i o n s contained e i t h e r a p p r o x i m a t e l y 1 mg/mL o f t h e i s o l a t e d p r o t e i n s o r 2 mg/mL o f t h e l y o p h i l i z e d c o m p o s i t e s . The d a i r y p r o d u c t s were s a m p l e d d i r e c t l y a s l i q u i d s a n d d i l u t e d 1:1 i n a sample b u f f e r c o n t a i n i n g 50 mM T r i s , 0.3 mM EDTA, 3% SDS a n d 12.5% m e r c a p t o e t h a n o l ( 2 ) . Sample l o a d i n g s r a n g e d f r o m 2 μL t o 50 μL, t h e maximum p o s s i b l e l o a d i n g i n t h e e l e c t r o p h o r e s i s s y s t e m employed. E l e c t r o p h o r e s i s was r u n a t c o n s t a n t amperage, between 15 a n d 20 mA p e r g e l , f o r 6-8 h o u r s , u n t i l t h e l e a d i n g b u f f e r l i n e was c l o s e t o t h e b o t t o m o f t h e g e l s . The g e l s were c o o l e d t o 12-15 °C d u r i n g t h e e l e c t r o p h o r e s i s run by c i r c u l a t i n g c o l d water from a refrigerated circulator through t h e heat exchanger on t h e electrophoresis unit. Two i d e n t i c a l g e l s were r u n s i m u l t a n e o u s l y s o t h a t t h e PAGE-NAA r e s u l t s c o u l d be compared w i t h s t a n d a r d p r o t e i n d e t e c t i o n methods s u c h as Coomassie Brilliant Blue™ staining. Following electrophoresis, t h e separated proteins were f i x e d i n t h e g e l s b y s o a k i n g i n 50% e t h a n o l / 0.1% formaldehyde, u s u a l l y overnight. One g e l was s t o r e d i n 20% ethanol p r i o r t o drying f o r irradiation,and the o t h e r g e l was s t a i n e d w i t h C o o m a s s i e B r i l l i a n t Blue™. I f d e t e r g e n t (SDS) r e m o v a l was needed, t h e p r o c e d u r e o f O l d e n a n d Yamada, a s m o d i f i e d b y S c h i b e c i a n d M a r t o n o s i (£)/ was f o l l o w e d . P r i o r t o d r y i n g a n d i r r a d i a t i o n , a l l g e l s were c o n d i t i o n e d w i t h 10% a c e t i c a c i d / 1% g l y c e r o l . They were t h e n v a c u u m - d r i e d on a low b a c k g r o u n d f i l t e r p a p e r a t 80 °C f o r two h o u r s . E a c h g e l was i n d i v i d u a l l y p a c k a g e d in l i n e a r polyethylene (a h i g h p r e s s u r e e x t r u d e d f i l m o f p o l y e t h y l e n e made from v i r g i n m a t e r i a l s , n o t t r e a t e d w i t h c a t a l y s t s ) , a n d t h e n a l l g e l s were wrapped t o g e t h e r i n another p o l y e t h y l e n e bag f o r i r r a d i a t i o n . The phosphoproteins p h o s v i t i n (10.0 % P) a n d a casein (1.10% P) were s e l e c t e d b e c a u s e o f t h e i r h i g h Ρ content ( r e l a t i v e t o m o l e c u l a r weight) and because they were c o m m e r c i a l a v a i l a b l e i n p u r i f i e d f o r m (>90%). A c o m m e r c i a l l y a v a i l a b l e p h o s p h o p r o t e i n composite, sodium c a s e i n a t e , as w e l l a s two r e f e r e n c e m a t e r i a l s , S RM 1845, C h o l e s t e r o l i n E g g Powder, a n d IAEA CRM A - l l , M i l k Pow d e r , were a l s o s t u d i e d . Finally, to establish the method f o r a n a l y s i s o f n a t u r a l samples, s e v e r a l d a i r y p r o d u c t s were a n a l y z e d f o r t h e i r c a s e i n c o n t e n t : l o w - f a t 2 +


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(2%) m i l k , h o m o g e n i z e d m i l k , a n d " h a l f - a n d h a l f " (50% regular m i l k , 50% cream). I r r a d i a t i o n s were p e r f o r m e d i n a s p e c i a l l y d e s i g n e d i r r a d i a t i o n c a s s e t t e at the U n i v e r s i t y of V i r g i n i a r e a c tor i n C h a r l o t t e s v i l l e , VA (Jï) . The c o n t a i n e r c o n s i s t s o f an aluminum c a s s e t t e , w h i c h i s p l a c e d u n d e r w a t e r i n the p o o l , adjacent to the r e a c t o r core. A continuously c i r c u l a t e d d r y gas (N ) s u r r o u n d s t h e g e l p a c k a g e i n t h e s e a l e d c a s s e t t e and p r o v i d e s p o s i t i v e p r e s s u r e t o r e d u c e the p o s s i b i l i t y o f water leakage i n t o the c a s s e t t e . The c a s s e t t e i s 20 cm s q u a r e and h o l d s a sample s t a c k up t o 3 cm i n t h i c k n e s s . The r e a c t o r was operated at h a l f power (1 MW) a t a f l u e n c e r a t e o f 1.7 · 1 0 n «cm" «s" / s i n c e at f u l l power, γ-ray h e a t i n g damaged t h e g e l s so much that t h e y were u n u s a b l e . Irradiation times ranged between 4 and 6 h o u r s . F o l l o w i n g a 9-10 day d e c a y p e r i o d , a u t o r a d i o g r a p h s were made by e x p o s i n g t h e g e l s t o X - r a y f i l m . The a u t o r a diographs were u s u a l l y done by direct exposure in cardboard c a s s e t t e s . E x p o s u r e t i m e s v a r i e d from one day t o t h r e e weeks, d e p e n d i n g on t h e t y p e o f p r o t e i n and t h e amount o f p r o t e i n t h a t was a p p l i e d . A s t a n d a r d p r o c e s s f o r f i l m development was f o l l o w e d . The f i l m was d e v e l o p e d f o r 5 min, w i t h g e n t l e a g i t a t i o n e v e r y m i n u t e f o r a p p r o x i m a t e l y 15 seconds, r i n s e d i n d i s t i l l e d w a t e r f o r 30 s e c , and f i x e d f o r 3 min with constant a g i t a t i o n . The f i l m was r i n s e d i n r u n n i n g d i s t i l l e d w a t e r f o r 5 min, t h e n d r i e d i n a i r f o r a t l e a s t 1 hr. F i l m was s t o r e d on f l a t s u r f a c e s i n p r o t e c t i v e p l a s t i c covers. A l a s e r d e n s i t o m e t e r was u s e d t o s c a n t h e a r e a s on the developed autoradiographs. E a c h b a n d was scanned a c r o s s t h e m i d - s e c t i o n and t h r o u g h t h e l e n g t h of the b a n d a t a s c a n n i n g s p e e d o f 20-40 cm/min. Peak a r e a s were i n t e g r a t e d on an A p p l e l i e computer t h a t was inter f a c e d t o t h e d e n s i t o m e t e r u s i n g t h e LKB 2190 G e l S c a n software. The peak a r e a s a r e g i v e n i n a b s o r b a n c e units (au), where a f u l l s c a l e peak w i t h a f u l l w i d t h a t h a l f maximum o f 1.0 has an i n t e n s i t y o f 1000 u n i t s . These ab s o r b a n c e u n i t s have been r e l a t e d t o s t a n d a r d c a l i b r a t e d g l a s s f i l t e r s (Si) . The peaks were i n t e g r a t e d o v e r a s e l e c t e d b a c k g r o u n d w i t h a f i t t e d G a u s s i a n c u r v e and t h e a r e a under t h e c u r v e was t h e n c a l c u l a t e d . I f i t was n o t p o s s i b l e t o d e s c r i b e the peak w i t h a s i n g l e c u r v e , s e v e r a l G a u s s i a n c u r v e s were f i t t e d and t h e i n t e g r a t e d a r e a s were summed. The q u o t e d a c c u r a c y l e v e l o f t h e G a u s s i a n f i t method i s 5% from t h e G e l S c a n program d o c u m e n t a t i o n (X&). The a c c u r a c y of the software f i t is visually demonstrated by r e c o n s t r u c t i n g t h e c a l c u l a t e d c u r v e and c o m p a r i n g i t t o the a c t u a l s c a n n e d a b s o r b a n c e d a t a as an o v e r l a y . Peak f i t s can a l s o be done m a n u a l l y , w i t h r e s u l t s v i r t u a l l y i d e n t i c a l t o the software f i t . Ammonium d i h y d r o g e n p h o s p h a t e s o l u t i o n s were p r e p a r e d 2


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as Ρ s t a n d a r d s . D i l u t i o n s were p r e p a r e d f r o m a p r i m a r y s t o c k s o l u t i o n and r a n g e d f r o m 20 ng P/mL t o 1000 ng P/mL. Two μL o f e a c h s o l u t i o n was p i p e t t e d o n t o t h e d r i e d g e l s c o n t a i n i n g t h e s e p a r a t e d p r o t e i n s t o be i r r a d i a t e d . Results. P h o s p h o r u s s t a n d a r d s were a p p l i e d t o t h e g e l s b e f o r e i r r a d i a t i o n and a u t o r a d i o g r a p h s were developed from the i r r a d i a t e d g e l s f o l l o w i n g the d e s c r i b e d p r o c e d u r e s . S i n c e t h e p r o t e i n s and t h e Ρ s t a n d a r d s were on t h e same g e l , t h e r e was no concern about v a r i a t i o n s between s t a n d a r d s and samples i n t e r m s o f e x p o s u r e t i m e s to the x-ray f i l m or times r e q u i r e d f o r f i l m develop ment. A b s o r b a n c e m e a s u r e m e n t s were made o f b o t h the r e s u l t a n t s p o t s from t h e P of the a c t i v a t e d standards and the p r o t e i n bands, and calibration curves were p l o t t e d f o r each set of standards. In t h e a u t o r a d i ograph, the absorbance of the t o t a l area i s p r o p o r t i o n a l t o t h e mass o f P. The a b s o r b a n c e o f t h e t o t a l a r e a , w h i c h can be c a l l e d t h e a r e a l d e n s i t y , must be o b t a i n e d i n o r d e r t o compare t h e s t a n d a r d s and samples b e c a u s e o f t h e d i f f e r e n t s h a p e s o f t h e bands and s p o t s . The spots a r e c y l i n d r i c a l l y s y m m e t r i c w i t h an i n t e n s i t y d i s t r i b u t i o n t h a t i s g a u s s i a n about the c e n t e r o f the circle. The b a n d s a r e s y m m e t r i c a c r o s s t h e m a j o r a x i s w i t h a G a u s s i a n d i s t r i b u t i o n about t h a t a x i s . A r e p r e s e n t a t i v e standard c a l i b r a t i o n curve of t o t a l a r e a l d e n s i t i e s v e r s u s mass o f Ρ i n ng i s given i n F i g u r e 1. The mass o f Ρ i s o b t a i n e d by c o m p a r i n g t h e a r e a l d e n s i t y o f an unknown t o t h e s e a r e a l d e n s i t i e s o f standards with known Ρ masses, on the same a u t o r a d i o g r a p h . The mass o f p r o t e i n c o r r e s p o n d i n g t o t h e mass o f Ρ can t h e n be c a l c u l a t e d f r o m t h e s t o i c h i o m e t r y of the p a r t i c u l a r p h o s p h o p r o t e i n b e i n g a n a l y z e d . For i n s t a n c e , t h e r e i s 1.10% Ρ i n t h e p h o s p h o p r o t e i n α - c a s e i n , so one w o u l d d i v i d e t h e mass o f Ρ by 0.0110 t o o b t a i n t h e mass o f α - c a s e i n . The method was first t e s t e d with commercially a v a i l a b l e , i s o l a t e d phosphoproteins. The r e s u l t s o f t h e PAGE-NAA m e t h o d a p p l i e d t o p r e v i o u s l y i s o l a t e d phos phoproteins a l s o y i e l d e d absorbance values p r o p o r t i o n a l t o t h e mass o f sample a p p l i e d (5.) . R e p l i c a t e a l i q u o t s o f an α - c a s e i n s o l u t i o n were s e p a r a t e d on a number o f g e l s and t h e g e l s were t h e n a n a l y z e d t o a s s e s s t h e r e p r o d u c i b i l i t y o f t h e method. The t e s t was s e n s i t i v e t o e r r o r s due t o sample l o a d i n g , f l u e n c e c o r r e c t i o n s w h i c h were b a s e d on a c t i v i t i e s o f Fe f o i l s p l a c e d a t v a r i o u s l o c a t i o n s i n t h e c a s s e t t e , and e s t i m a t e s o f a r e a l d e n s i t y f o r a band (.2.) . A c o m p a r i s o n o f r e s u l t s from two g e l s i s shown i n T a b l e I . The mass o f α - c a s e i n in the original protein solution was used to calculate mass o f Ρ t h a t was applied. T h i s c a l c u l a t e d mass was compared w i t h t h e ex p e r i m e n t a l r e s u l t s f r o m t h e PAGE-NAA method. A 0.96 mg s o l i d / m L b u f f e r s o l u t i o n was p r e p a r e d , w i t h t h e α - c a s e i n


32


21.

STONE ET AL.


in


material c e r t i f i e d b y t h e m a n u f a c t u r e r t o have a t l e a s t a 90% p u r i t y , so t h e e x p e c t e d mass o f Ρ was 47.5 ng Ρ (assuming 90% p r o t e i n ) , a t a 5 mL a p p l i c a t i o n , a n d a Ρ c o n t e n t o f 1.10%. Due t o t h e p r e s e n c e o f some o f t h e reagents i n t h e b u f f e r s o l u t i o n used t o d i s s o l v e t h e p r o t e i n , such as T r i s , m e r c a p t o e t h a n o l , SDS, a n d EDTA, s e v e r a l d i f f e r e n t p r o t e i n d e t e r m i n a t i o n s were n o t s u c c e s s f u l because o f the i n t e r f e r e n c e caused by t h e s e reagents. From T a b l e I , t h e o v e r a l l mean f r o m t h e r e p l i c a t e a p p l i c a t i o n s , 48.7 ng, i s i n g o o d a g r e e m e n t w i t h t h e e x p e c t e d mass o f Ρ. A s i m i l a r c a l c u l a t i o n f o r t h e s e c o n d α - c a s e i n s o l u t i o n y i e l d e d a v a l u e o f 225 ng P, c l o s e t o t h e e x p e c t e d v a l u e o f 195 ng P.

Table

I.

R e p r o d u c i b i l i t y assessment o f PAGE-NAA method u s i n g r e p l i c a t e aliquots of α-casein Means a r e q u o t e d as ± t h e s t a n d a r d e r r o r , w h e r e

1σ

SE =• VÎT α-casein

Gel #

(replicates)

Measured

1 2 3 4 Mean = 1 2 3 Mean =

47.1 47.2 55.7 40.8 47.7 ± 3.0 53.7 42.2 54.2 50.0 ± 3.8

Overall

Mean

=

48.7

±

Ρ

(ησ)

2.3

A sample o f sodium c a s e i n a t e was s e p a r a t e d on a d i s c o n t i n u o u s g r a d i e n t g e l w i t h sample a l i q u o t s o f 10 - 50 μL from a p r o t e i n s o l u t i o n o f 2 mg/mL. An a u t o r a d i o g r a p h from a 9 day d i r e c t e x p o s u r e was o b t a i n e d f r o m t h e i r r a d i a t e d g e l and i s shown i n F i g u r e 2. The results from the determination of phos p h o p r o t e i n s i n s e v e r a l samples a r e g i v e n i n T a b l e I I . The u n c e r t a i n t i e s i n t h e t a b l e r e p r e s e n t an o b s e r v e d standard deviation relative t o t h e mean of four measurements, except f o r IAEA A - l l , which i s t h e un c e r t a i n t y f o r a s i n g l e measurement. A t t h e p r e s e n t d e t e c tion limits, o n l y t h e α - c a s e i n was q u a n t i f i e d i n t h e m i l k samples. The i d e n t i f i c a t i o n o f t h e p r o t e i n on t h e a u t o r a d i o g r a p h was made on t h e b a s i s o f m o l e c u l a r w e i g h t d e t e r m i n a t i o n s , and i t s known h i g h Ρ c o n t e n t . T h e s e measurements r e p r e s e n t a d i r e c t q u a n t i t a t i o n o f t h e c o n s t i t u e n t i n m i l k . In s t u d i e s r e p o r t e d i n t h e


272


8000-j

ε u a 33

70006000-

w

5000-

*35 S α> Q "w

3000-

Trace Elements Associated with Proteins - American Chemical Society

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