4 Refractive Indices of Amino Acids, Proteins, and Related Substances THOMAS L. McMEEKIN, MERTON L. GROVES, Amino Acids and Serum Proteins Downloaded from pubs.acs.org by MIDWESTERN UNIV on 01/23/19. For personal use only.
AND NORBERT J. HIPP Eastern Regional Research Laboratory, Philadelphia 18, Pa. The molar refractions of the amino acids were determined by measurements on their aqueous solutions and the expanded LorenzLorentz equation. The refractive indices of a number of proteins were calculated from their amino acid compositions and the values for the refraction of the amino acid residues. These calculated results are in good agree ment with those experimentally determined, demonstrating that refractive index is a unique characteristic of a protein. A com parison of the refractive index of heat dena tured β-lactoglobulin with the native protein demonstrated that changes in structure pro duced a small change in refractive index, not associated with a change in volume.
A l t h o u g h the r e f r a c t i v e i n d e x of a s o l u t i o n c a n be s i m p l y and p r e c i s e l y m e a s u r e d , i t has been l i t t l e u s e d i n c h a r a c t e r i z i n g p r o t e i n s . T h e r e f r a c t i o n of p r o t e i n s i s , however, frequently i n v o l v e d i n m e a s u r e m e n t s on p r o t e i n s o l u t i o n s by s u c h methods as l i g h t s c a t t e r i n g , sedimentation, and e l e c t r o p h o r e s i s . P r e v i o u s i n v e s t i g a t i o n s , s u m m a r i z e d by Doty and G e i d u s c h e k (11), i n d i c a t e the i m p o r t a n c e of c o m p o s i t i o n , d e n s i t y , c h a r g e , and e n v i r o n m e n t a l f a c t o r s i n d e t e r m i n i n g the r e f r a c t i v e i n d i c e s of p r o t e i n s . T h e y note that the v a l u e s r e p o r t e d f o r the r e f r a c t i v e i n d i c e s of p r o t e i n s a r e c l o s e to 1.60 and a r e n e a r l y constant. A d a i r and R o b i n s o n (1) i n d i c a t e that the r e f r a c t i v e i n d e x of a p r o t e i n o r a n a m i n o a c i d i s a p p r o x i m a t e l y d e t e r m i n e d by i t s e l e m e n t a r y c o m p o s i t i o n ; h o w e v e r , the s t r u c t u r e of a m o l e c u l e i s a l s o of i m p o r tance. T h e v a l u e s r e p o r t e d f o r a m i n o a c i d s a r e s c a t t e r e d and f r a g m e n t a r y (1, 10), and p r i o r to o u r p r e l i m i n a r y c o m m u n i c a t i o n (25) no s y s t e m a t i c i n v e s t i g a t i o n had accounted q u a n t i t a t i v e l y f o r the r e l a t i o n s h i p 54
4.
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between the a m i n o a c i d c o m p o s i t i o n of a p r o t e i n and i t s r e f r a c t i v e i n dex. In t h i s p a p e r , the r e s u l t s of a s y s t e m a t i c study of the r e f r a c t i v e i n d i c e s of the a m i n o a c i d s , and s o m e peptides and p r o t e i n s , a r e d e s c r i b e d . The v a l u e f o r the r e f r a c t i v e index of a p r o t e i n c a l c u l a t e d f r o m the r e f r a c t i v e i n c r e m e n t s of i t s a m i n o a c i d r e s i d u e s and s o l u t i o n v o l u m e a g r e e s w i t h the e x p e r i m e n t a l v a l u e and i s a c h a r a c t e r i s t i c of the p r o t e i n . The change i n the r e f r a c t i v e i n d e x of a p r o t e i n as a r e s u l t of denaturation has a l s o been i n v e s t i g a t e d . Materials
and
Methods
A m i n o A c i d s and P e p t i d e s . T h e a m i n o a c i d s and peptides u s e d were high grade c o m m e r c i a l products, further purified by r e c r y s t a l l i z a t i o n f r o m a l c o h o l - w a t e r m i x t u r e s , except when they w e r e c h r o m a t o graphically pure. Glycolamide was prepared by passing d r y ammonia into c o l d , f r e s h l y d i s t i l l e d e t h y l g l y c o l l a t e . P u r e l a c t a m i d e w a s o b tained f r o m Ε . H . H a r r i s of t h i s l a b o r a t o r y . P r o t e i n s . C r y s t a l l i n e l y s o z y m e , bovine s e r u m a l b u m i n , r i b o n u c l e a s e , a n d p e p s i n w e r e obtained f r o m the A r m o u r L a b o r a t o r i e s and c r y s talline β -lactoglobulin was prepared f r o m s k i m m e d m i l k , α - L a c t a l b u m i n w a s obtained f r o m W . G . G o r d o n of t h i s l a b o r a t o r y . C r y s t a l l i n e o v a l b u m i n and human s e r u m a l b u m i n w e r e obtained f r o m N u t r i t i o n a l B i o c h e m i c a l C o r p . and p u r i f i e d p i g s k i n g e l a t i n f r o m the E a s t m a n Kodak C o . R e f r a c t i v e Index D e t e r m i n a t i o n . T h e r e f r a c t i v e i n d i c e s of a m i n o a c i d s w e r e d e t e r m i n e d by means of a d i p p i n g r e f r a c t o m e t e r , u s i n g a s o d i u m l i g h t , and a l s o w i t h the B r i c e - H a l w e r (4) d i f f e r e n t i a l r e f r a c t o m e t e r . C o n c e n t r a t i o n s of s o l u t i o n s w e r e based on the d r y weight of an aliquot at 1 1 0 ° C . and a l s o on m o i s t u r e d e t e r m i n a t i o n s . T h e v a l u e s obtained w e r e i n e s s e n t i a l a g r e e m e n t . N o d i f f e r e n c e w a s found between the D L - a m i n o a c i d s and the c o r r e s p o n d i n g o p t i c a l l y a c t i v e a m i n o a c i d s ; consequently, m o s t of the m e a s u r e m e n t s w e r e made on the D L - a m i n o a c i d s . R e f r a c t i v e index m e a s u r e m e n t s w e r e made on s o l u t i o n s v a r y i n g i n c o n c e n t r a t i o n f r o m 1 to 10%, depending on the s o l u b i l i t y of the a m i n o a c i d . N o difference i n r e f r a c t i v e i n c r e m e n t w a s found due to v a r i a t i o n s i n c o n c e n t r a t i o n , except i n the c a s e of g l y c i n e s o l u t i o n s , w h e r e the d i f ference c o u l d be c o r r e l a t e d w i t h v a r i a t i o n s i n s p e c i f i c v o l u m e and w e r e e s s e n t i a l l y e l i m i n a t e d b y a p p l y i n g the a p p r o p r i a t e v o l u m e f o r a g i v e n c o n c e n t r a t i o n of g l y c i n e . C y s t i n e , t y r o s i n e , and a s p a r t i c a c i d a r e not sufficiently s o l u b l e i n w a t e r f o r a c c u r a t e m e a s u r e m e n t of r e f r a c t i v e i n d i c e s . The m o l a r r e f r a c t i o n of c y s t i n e w a s c a l c u l a t e d b y u s i n g the m o l a r a t o m i c r e f r a c t i o n s (in c c . ) g i v e n by F a j a n s ( 1 2 ) : C 2.418, Η 1.10, 0 - 1 . 5 2 5 , Ο 2.211, and Ν 2.322, and S 8.11 g i v e n by Cohen (8). The m o l a r r e f r a c t i o n s of t y r o s i n e and a s p a r t i c a c i d w e r e e s t i m a t e d f r o m the m o l a r r e f r a c t i o n s of g l y c y l t y r o s i n e and g l y c y l a s p a r t a t e , r e s p e c t i v e l y , by s u b t r a c t i n g the r e f r a c t i o n due to the g l y c y l r e s i d u e . P r o t e i n s o l u t i o n s of 1 and 2% c o n c e n t r a t i o n s w e r e u s e d f o r m a k i n g r e f r a c t i v e i n d e x m e a s u r e m e n t s . T h e v a l u e s f o r w a t e r a s a function of wavelength of l i g h t and t e m p e r a t u r e w e r e taken f r o m the I n t e r n a t i o n a l - 2
ADVANCES IN CHEMISTRY SERIES
56
C r i t i c a l T a b l e s and f r o m T i l t o n and T a y l o r (32). T h e wavelength of light i s g i v e n i n m i l l i m i c r o n s ( π ι μ ) . C a l c u l a t i o n s . R e f r a c t i v e I n d i c e s and M o l a r R e f r a c t i o n s of S o l u t i o n s of A m i n o A c i d s and P r o t e i n s . T h e m e a n r e f r a c t i v e i n d i c e s of the a m i n o a c i d s and p r o t e i n s w e r e c a l c u l a t e d by means of the f o l l o w i n g e x panded L o r e n z - L o r e n t z equation as g i v e n by Doty and G e i d u s c h e k (11):
n
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. fa» - 1) # T 2 j
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w h e r e n , fy, and η i n d i c a t e the r e f r a c t i v e i n d i c e s of the w a t e r - f r e e p r o t e i n o r a m i n o a c i d , the solvent and the s o l u t i o n r e s p e c t i v e l y , ν i s the s p e c i f i c v o l u m e of the p r o t e i n o r a m i n o a c i d and c o n c e n t r a t i o n , c, i s expressed i n grams per cubic centimeter. M o l a r refraction, R , i s c a l c u l a t e d b y E q u a t i o n 2. p
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w h e r e n i s the mean r e f r a c t i v e index of the a m i n o a c i d o r p r o t e i n , M , i s the m o l e c u l a r weight of the a m i n o a c i d (for p r o t e i n s , 100 g r a m s has been u s e d i n s t e a d of the m o l e c u l a r weight of the p r o t e i n ) , and ρ i s the d e n s i t y of the a m i n o a c i d o r p r o t e i n , 1/v. R e f r a c t i v e Index of P r o t e i n f r o m A m i n o A c i d C o m p o s i t i o n . T h e method u s e d f o r c a l c u l a t i n g the r e f r a c t i v e i n d e x of a p r o t e i n f r o m i t s a m i n o a c i d c o m p o s i t i o n i s e s s e n t i a l l y the s a m e as that d e s c r i b e d by Cohn and E d s a l l (9, Chap. 16) f o r c a l c u l a t i n g the s p e c i f i c v o l u m e of a protein from its amino acid composition. T h e weight p e r cent of e a c h a m i n o a c i d found by a n a l y s i s i s c o n v e r t e d into the weight p e r cent of i t s r e s i d u e by m u l t i p l y i n g by the r a t i o of the m o l e c u l a r weight of the r e s i d u e (amino a c i d m i n u s 1 m o l e of water) d i v i d e d by the m o l e c u l a r weight of the a m i n o a c i d . T h e n the weight p e r cent of e a c h a m i n o a c i d r e s i d u e i s m u l t i p l i e d b y the v a l u e of the r e f r a c t i o n of 1 g r a m of r e s i d u e , a s g i v e n i n T a b l e I, to g i v e the total* r e f r a c t i v e v o l u m e i n 100 g r a m s of the p r o t e i n due to a g i v e n a m i n o a c i d r e s i d u e . T h e t o t a l r e f r a c t i v e v o l u m e of the a m i n o a c i d r e s i d u e s i n 100 g r a m s of p r o t e i n i s obtained by adding the r e f r a c t i v e v o l u m e s of the i n d i v i d u a l a m i n o a c i d r e s i d u e s . S i n c e an a m i n o a c i d a n a l y s i s i s s e l d o m p e r f e c t , a c o r r e c t i o n i s made by m u l t i p l y i n g the r e f r a c t i o n p e r 100 g r a m s of p r o t e i n by percentage r e c o v e r y , w h i c h i s obtained by a d d i n g the weight p e r c e n t a g e s of a m i n o a c i d r e s i d u e s and d i v i d i n g by 100. T h e m e a n r e f r a c t i v e index of the p r o t e i n , n ^ , i s then c a l c u l a t e d by s o l v i n g f o r n i n E q u a t i o n 2, w h e r e [R] i s the v a l u e obtained f o r the r e f r a c t i v e v o l u m e of 100 g r a m s of p r o t e i n and M i s equal to 100. A m i d e n i t r o g e n i s a r b i t r a r i l y c a l c u l a t e d as b e i n g c o m b i n e d w i t h the g l u t a m i c a c i d r e s i d u e s ; any e x c e s s i s a s s i g n e d to a s p a r t i c a c i d r e s i dues. p
p
McMEEKIN ET AL
Refractive Indices of Amino Acids
Table I. Refractive Indices, Molar Refractions of Amino Acids 25°C
Specific Volume , Cc.
Refractive Index
Glycine
0.58
1.685
Alanine a -Aminobutyric acid Valine a-Aminovaleric acid' Leucine
0.68 0.74 0.79 0.79 0.83
1.606 1.587 1.571 1.577 1.565
Isoleucine a-Aminocaproic acid Serine
0.83 0.83 0.58
1.568 1.565 1.676
Threonine
0.66
1.618
Hydroxyproline
0.64
1.618
Proline
0.70
1.596
Methionine
0.71
1.646
Cystine
0.59
-
Phenylalanine
0.74
1.682
Tyrosine
0.68
-
Tryptophan
0.71
1.754
Histidine
0.64
1.700
Arginine
0.67
1.664 1.615
a
Amino Acid
5
Lysine
0.74
Aspartic acid
0.56
-
Glutamic acid
0.63
1.655
Asparagine
0.59
1.691
Glutamine
0.64
1.670
a
b
c
(9). Calculated from data of Craig and Schmidt (10) 2 moles of water subtracted (7.46 c c ) .
ADVANCES IN CHEMISTRY SERIES and Calculated Refractions of Amino Acid Residues - 589
Πΐμ)
Amino Acid Observed
Residue (Ref. of Amino Acid - 3.73)
Refraction per G. Residue (Mol. Ref. Res.) (Mol. Wt. Res.), Cc.
16.54 ± 0.1
12.81
0.225
20.88 ± 0.15 25.67 ± 0.15 30.46 ± 0.13 30.72 ± 0.15 35.32 ± 0.15
17.15 21.94 26.73 26.99 31.59
0.242 0.258 0.270 0.272 0.279
35.60 ± 0.20 35.17 ± 0.15 22.89 ± 0.10
31.87 31.44 19.16
0.282 0.278 0.220
27.55 ± 0.10
23.82
0.236
29.57 ± 0.10
25.84
0.229
27.47 ± 0.10
23.74
0.245
38.18 ± 0.05
34.45
0.263
56.04
48.58
45.94 ± 0.15
42.21
0.287
48.07
44.34
0.272
58.97 ± 0.30
55.24
0.297
38.35 ± 0.15
34.62
0.253
43.20 ± 0.10
39.47
0.253
c
0.238
37.83 ± 0.2
34.10
0.266
28.54
24.81
0.216
33.80 ± 0.15
30.07
0.233
29.82 ± 0.20
26.09
0.229
34.10 ± 0.20
30.37
0.237
4.
McMEEKIN £7 AL
Refractive Indices of Amino Acids
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Results A m i n o A c i d s . T h e v a l u e s f o r the r e f r a c t i v e i n d i c e s and m o l a r r e f r a c t i o n s of the a m i n o a c i d s c a l c u l a t e d f r o m the r e f r a c t i v e i n d i c e s by L o r e n z - L o r e n t z Equations 1 and 2 a r e r e c o r d e d i n T a b l e I . V a l u e s f o r m o l a r r e f r a c t i o n s of the a l i p h a t i c a m i n o a c i d s a r e i n good a g r e e m e n t w i t h v a l u e s c a l c u l a t e d f r o m a t o m i c r e f r a c t i o n f a c t o r s . H o w e v e r , the m o l a r r e f r a c t i o n s of tryptophan, t y r o s i n e , p h e n y l a l a n i n e , and h i s t i d i n e a r e l a r g e r than those c a l c u l a t e d f r o m a t o m i c r e f r a c t i o n f a c t o r s and l a r g e r than might be expected f r o m t h e i r c o m p a r a t i v e s p e c i f i c v o l u m e s . T h e r e f r a c t i v i t y p e r g r a m of a m i n o a c i d r e s i d u e (Table I) i s o b tained f r o m the m o l a r r e f r a c t i o n of the a m i n o a c i d by s u b t r a c t i n g the value of 3.73 due to the l o s s of a m o l e c u l e of w a t e r i n f o r m i n g the r e s idue f r o m the a m i n o a c i d . T h i s v a l u e i s the s u m of i t s a t o m i c r e f r a c t i o n f a c t o r s , 2 H = 2.2 and Ο = 1.53. It i s i n e s s e n t i a l a g r e e m e n t w i t h the v a l u e deduced f r o m the m o l a r r e f r a c t i o n s of g l y c i n e and i t s p e p t i d e s . T h e m o l a r r e f r a c t i o n of g l y c i n e i s 16.54, of d i g l y c i n e 29.89, of t r i g l y c i n e 41.33 and of g l y c y l l e u c i n e 48.04 (Table Ι Π ) . T h u s , by d i f f e r ence, the l o s s of a m o l e of w a t e r i n m a k i n g d i g l y c i n e d e c r e a s e s m o l a r r e f r a c t i o n by 3.19 and the l o s s of 2 m o l e s of w a t e r i n m a k i n g t r i g l y c i n e d e c r e a s e s r e f r a c t i o n by 4.15 p e r m o l e of w a t e r . A v a l u e of 3.8 c c . i s obtained f o r w a t e r i n the f o r m a t i o n of g l y c y l l e u c i n e , g i v i n g an a v e r a g e value of 3.72 f o r w a t e r f r o m the t h r e e peptides. S p e c i f i c V o l u m e s and R e f r a c t i v e I n d i c e s of P r o t e i n s . T h e s p e c i f i c v o l u m e s and r e f r a c t i v e i n d i c e s , a s w e l l as the r e f r a c t i v e i n d i c e s c a l c u lated f r o m the a m i n o a c i d c o m p o s i t i o n s of a n u m b e r of p r o t e i n s , a r e r e c o r d e d i n T a b l e Π . T h e r e f r a c t i v e i n d i c e s c a l c u l a t e d b y u s i n g the a m i n o a c i d c o m p o s i t i o n of the p r o t e i n a r e i n good a g r e e m e n t w i t h the v a l u e s as d e t e r m i n e d i n the c a s e of m o s t p r o t e i n s , except i n the c a s e s of α - c a s e i n , g e l a t i n , and α - l a c t a l b u m i n , w h e r e the m e a s u r e m e n t s w e r e made away f r o m the i s o e l e c t r i c point. T h i s difference c o u l d be due to a c h a r g e effect, a s w a s found by P e r l m a n n and L o n g s w o r t h (26), o r to i n a c c u r a t e s p e c i f i c v o l u m e s , s i n c e the b l a n k c o r r e c t i o n u s e d i n s p e c i f i c v o l u m e c a l c u l a t i o n s f r o m d e n s i t y d e t e r m i n a t i o n s i s of q u e s t i o n able a p p l i c a b i l i t y on p r o t e i n s o l u t i o n s c o n t a i n i n g a l k a l i . T h e c l o s e a g r e e m e n t between the d e t e r m i n e d v a l u e f o r the r e f r a c t i v e index of o v a l b u m i n w a s unexpected, s i n c e o v a l b u m i n contains c a r b o h y d r a t e and no e s t i m a t e i s made of the r e f r a c t i o n due to c a r b o h y d r a t e . T h e c a l c u lated r e f r a c t i v e i n d e x i s , h o w e v e r , based on the a s s u m p t i o n that o v a l b u m i n i s c o m p o s e d e n t i r e l y of a m i n o a c i d r e s i d u e s , w h i c h i n d i c a t e s that the r e f r a c t i o n of the c a r b o h y d r a t e does not g r e a t l y d i f f e r f r o m that of the a v e r a g e a m i n o a c i d . T h e i m p o r t a n c e of s p e c i f i c v o l u m e o r d e n s i t y i n d e t e r m i n i n g r e f r a c t i v e i n d e x i s apparent i n E q u a t i o n s 1 and 2, w h i c h a r e u s e d i n c a l c u l a t i n g r e f r a c t i v e index and m o l a r r e f r a c t i o n . T h i s i n v e r s e r e l a t i o n s h i p between s p e c i f i c v o l u m e and r e f r a c t i v e index i s i l l u s t r a t e d i n T a b l e Π (cf. c o l u m n s 2 and 4). T h e n e c e s s i t y of obtaining an a c c u r a t e v a l u e f o r the s p e c i f i c v o l u m e of a p r o t e i n i n o r d e r to obtain a g r e e m e n t between i t s r e f r a c t i v e i n d e x c a l c u l a t e d f r o m the a m i n o a c i d c o m p o s i t i o n and the d e t e r m i n e d v a l u e c a n be i l l u s t r a t e d i n the c a s e of r i b o n u -
60
ADVANCES IN CHEMISTRY SERIES Table II. Specific Volumes and Refractive Indices of Proteins 25° (λ = 589 πΐμ)
Refractive Index
Protein
Solvent, pH 7.0 5.0 7.0 5.2 6.0 5.0 5.0 4.8 5.0 5.0 5.0
NaOH, a -Casein Water, Gelatin a -Lactalbumin NaOH, β - Lactoglobul in 0.1M NaCl, Lysozyme Water, Ovalbumin Water, Pepsin Water, Ribonuclease Water, Bovine serum albumin Water, Horse serum albumin Water, Human serum albumin Water, a
(14); b( 3); c ( i ) d 3
5
;
( 1 3 ) ;
e ) ( 3
;
Refrac Specific tion/100 Volume, G. Cc. 25.50 24.27 25.64 25.44 25.34 25.35 24.91 24.66 25.32 25.12 25.28
0.728 0.682 0.735 0.751 0.718 0.745 0.725 0.693 0.734 0.734 0.736
Calcd. from amino acid Detd. comp. 1.618 1.630 1.615 1.594 1.624 1.596 1.603 1.630 1.606 1.600 1.603
a
1.607 1.618b 1.601C 1.590d 1.620° 1.593° 1.605β 1.630 f 1.599g 1.602°
f 0); S(30). (2
c l e a s e , w h e r e a n u m b e r of different v a l u e s f o r i t s s p e c i f i c v o l u m e have been r e p o r t e d . R o t h e n ' s (28) value of 0.709 on the r i b o n u c l e a s e p r e p a r e d by K u n i t z i s i n good a g r e e m e n t w i t h that c a l c u l a t e d f r o m i t s a m i n o a c i d c o m p o s i t i o n . U s i n g r i b o n u c l e a s e obtained f r o m the A r m o u r L a b o r a t o r y , B u z z e l l and T a n f o r d (6) found 0.728 f o r i t s s p e c i f i c v o l u m e , w h i l e H a r r i n g t o n and S c h e l l m a n (17) r e p o r t e d 0.692 t o 0.696. S i n c e the r e p o r t e d v a r i a t i o n s i n the s p e c i f i c v o l u m e of r i b o n u c l e a s e a r e l a r g e , i t w a s d e s i r a b l e to d e t e r m i n e the s p e c i f i c v o l u m e on the s a m p l e u s e d . T h e v a l u e of 0.695 found i s i n e x c e l l e n t a g r e e m e n t w i t h the v a l u e of H a r r i n g t o n and S c h e l l m a n (17). I t s n i t r o g e n content, found t o be 16.6%, i s a l s o i n a g r e e m e n t w i t h 16.8% found b y H a r r i n g t o n and S c h e l l m a n . T h e v a l u e f o r the s p e c i f i c v o l u m e of r i b o n u c l e a s e of 0.693 w a s u s e d i n c a l c u l a t i n g i t s r e f r a c t i v e i n d e x f r o m the a m i n o a c i d c o m p o s i t i o n . I n the c a s e of p e p s i n , no v a l u e f o r i t s s p e c i f i c v o l u m e c o u l d be l o c a t e d . T h e s p e c i f i c v o l u m e of a 2% s o l u t i o n w a s found to be 0.725 c c , a l s o i n a g r e e m e n t w i t h the 0.725 c a l c u l a t e d f r o m i t s a m i n o a c i d c o m p o s i t i o n . T h e r e m a i n d e r of the v a l u e s f o r s p e c i f i c v o l u m e s g i v e n i n T a b l e Π w e r e taken f r o m o u r p r e v i o u s c o m p i l a t i o n (24). R e f r a c t i v e I n d i c e s of P e p t i d e s . T o d e t e r m i n e the effect of peptide f o r m a t i o n on r e f r a c t i v e i n d e x , the r e f r a c t i v e i n d i c e s of s e v e r a l p e p t i d e s w e r e d e t e r m i n e d (Table Π Ι ) . T h e a v e r a g e m o l a r r e f r a c t i o n of w a t e r p r o d u c e d i n peptide f o r m a t i o n c a n be e s t i m a t e d , e m p i r i c a l l y , b y s u b t r a c t i n g the o b s e r v e d m o l a r r e f r a c t i o n of the peptide f r o m the s u m of m o l a r r e f r a c t i o n s of i t s constituent a m i n o a c i d s . T y r o s i n e and a s p a r t i c a c i d a r e not s u f f i c i e n t l y s o l u b l e f o r a c c u r a t e m e a s u r e m e n t s of the r e f r a c t i v e i n d i c e s of t h e i r s o l u t i o n s . C o n s e quently, t h e i r m o l a r r e f r a c t i o n s have been e s t i m a t e d f r o m t h e i r m o r e s o l u b l e g l y c y l peptides. F r o m the r e s u l t s g i v e n i n T a b l e Ι Π , the m o l a r r e f r a c t i o n s of t y r o s i n e (48.07) and a s p a r t i c a c i d (28.54) a r e obtained b y
4.
McMEEKIN ET AL
Refractive Indices of Amino Acids
61
Table III. Molar Refractions of Some Peptides 25° (λ = 589 ιημ)
Substance
Specific Volume, Cc.
Glycine Diglycine Triglycine Glycylleucine Glycyltyrosine Glycylaspartate
0.581 0.584 a 0.600 0.741 0.664 0.557
a
a
a
Molar Refraction Refractive Index
Obsd.
Calcd. with Atomic factors
1.685 1.702 1.649 1.606 1.694 1.706
16.54 29.89 41.33 48.04 60.73 41.2
16.38 29.09 41.38 47.52 54.10 39.82
(9).
s u b t r a c t i n g the c a l c u l a t e d m o l a r r e f r a c t i o n of the g l y c y l r e s i d u e [12.66 ( m o l a r r e f r a c t i o n of g l y c i n e m i n u s the r e f r a c t i o n of 1 m o l e of water)] f r o m the r e f r a c t i o n of the peptide. T h e m o l a r r e f r a c t i o n s c a l c u l a t e d for the peptides i n T a b l e Ι Π a r e i n good a g r e e m e n t w i t h the o b s e r v e d except i n the c a s e of the t y r o s i n e peptide and to a l e s s e r extent the a s p a r t i c a c i d peptide; consequently, i t i s felt that the v a l u e s f o r the m o l a r r e f r a c t i o n of t y r o s i n e and a s p a r t i c a c i d deduced f r o m e x p e r i m e n t a l m e a s u r e m e n t s on t h e i r peptides a r e m o r e a c c u r a t e than the v a l u e s c a l c u l a t e d f r o m a t o m i c f a c t o r s . S i n c e o u r p r e l i m i n a r y p u b l i c a t i o n (25), the m o l a r r e f r a c t i v i t y of g l y c y l a s p a r t a t e h a s been r e d e t e r m i n e d on a h i g h l y p u r i f i e d s a m p l e . T h e v a l u e of 41.2 c c . f o r i t s m o l a r r e f r a c t i o n i s about 2% l o w e r than that p r e v i o u s l y r e p o r t e d . T h i s has r e d u c e d the value f o r the m o l a r r e f r a c t i v i t y of the a s p a r t i c a c i d r e s i d u e f r o m 26.06 to 24.81. S i n c e a s p a r t i c a c i d i s p r e s e n t i n c o n s i d e r a b l e amounts i n p r o t e i n s , the l o w e r i n g of the v a l u e f o r i t s r e f r a c t i v i t y h a s s l i g h t l y r e duced the c a l c u l a t e d v a l u e s f o r the r e f r a c t i v e i n d i c e s of p r o t e i n s g i v e n i n T a b l e Π , as c o m p a r e d to the p r e v i o u s l y p u b l i s h e d v a l u e s (25). C o m p a r i s o n of M o l a r R e f r a c t i o n s of A m i n o A c i d s w i t h T h e i r U n c h a r g e d I s o m e r s . N u m e r o u s c o m p a r i s o n s have been made between the p r o p e r t i e s of a m i n o a c i d s and the p r o p e r t i e s of t h e i r n o n z w i t t e r i o n i s o m e r s (9). B y c o m p a r i n g the s o l u t i o n d e n s i t i e s of g l y c i n e and i t s i s o m e r , g l y c o l a m i d e , and alanine w i t h i t s i s o m e r , l a c t a m i d e , i t has been found that the a m i n o a c i d o c c u p i e s about 13 c c . p e r m o l e l e s s v o l u m e than i t s uncharged i s o m e r . Consequently, i t i s a l s o of i n t e r e s t to c o m p a r e the s o l u t i o n r e f r a c t i v e i n d i c e s of these a m i n o a c i d s w i t h t h e i r i s o m e r s . T a b l e I V shows that the r e f r a c t i v e i n d i c e s of the a m i n o a c i d s a r e c o n s i d e r a b l y g r e a t e r than those of t h e i r u n c h a r g e d i s o m e r s ; however, the m o l a r r e f r a c t i o n s a r e the s a m e w i t h i n the e x p e r i m e n t a l v a r i a t i o n . T h i s i s because the l a r g e r s p e c i f i c v o l u m e of the u n c h a r g e d i s o m e r compensate f o r i t s l o w e r r e f r a c t i v e i n d e x i n c a l c u l a t i n g the m o l a r r e f r a c t i o n by E q u a t i o n 2 . It c a n be c o n c l u d e d that é l e c t r o s t r i c t i o n of w a t e r b y a n a m i n o a c i d i n s o l u t i o n i n c r e a s e s i t s r e f r a c t i v e i n dex but has no effect on i t s m o l a r r e f r a c t i o n and that the m o l a r r e f r a c tions do not change s i g n i f i c a n t l y w i t h t e m p e r a t u r e .
ADVANCES IN CHEMISTRY SERIES
62
Table IV. Comparison of Molar Refractions of Amino Acids and Their Uncharged Isomer ( λ = 589 Πΐμ)
5
25
40
Specific Volume , Cc.
Refractive Index
Molar Refraction, Cc.
Glycine Glycolamide
1.691 1.516
16.85 17.00
Alanine Lactamide
1.615 1.496
21.13 21.47
a
Temp., •C.
Substance
Glycine Glycolamide
0.58 0.75
1.685 1.506
16.58 16.73
Alanine Lactamide
0.68 0.83
1.606 1.490
20.88 20.99
Glycine Glycolamide
1.676 1.506
16.38 16.73
Alanine Lactamide
1.612 1.489
21.06 21.21
Specific volumes obtained at 25° also used to calculate refractive index values at 5° and 40°. Effect of I o n i z a t i o n on the R e f r a c t i v e Index and M o l a r R e f r a c t i o n of A m i n o A c i d s and P r o t e i n s . Since the é l e c t r o s t r i c t i o n p r o d u c e d b y an a m i n o a c i d does not affect i t s m o l a r r e f r a c t i o n , the i o n i z a t i o n of an a m i n o a c i d m i g h t be expected to p r o d u c e no s i g n i f i c a n t change i n m o l a r r e f r a c t i o n . T a b l e V i n d i c a t e s that this i s the c a s e , p r o v i d e d the l a r g e change i n the v o l u m e of the a m i n o a c i d a s a r e s u l t of i o n i z a t i o n , found by K a u z m a n n , B o d a n s z k y , and R a s p e r (23), i s u s e d i n c a l c u l a t i n g m o l a r r e f r a c t i o n . T h e r e f r a c t i v e index of an equivalent c o n c e n t r a t i o n of h y Table V. Effect of Ionization on the Refractive Index and Molar Refraction of Alanine and Ovalbumin 25° (λ = 589 πΐμ)
Substance Alanine Alanine* CI" Alanine" Na Ovalbumin ΡΗ4.6 ΡΗ 3.8 PH 3.2 PH 2.5 PH 2.0 +
Refractive Index
Specific Volume, Cc.
Molar Refraction, Cc.
1.615 1.539 1.473
0.682 0.767 0.924
21.1 21.4 22.0
1.600 1.598 1.593 1.592 1.588
0.745 0.747 0.749 0.753 0.757
11,470 11,475 11,425 11,466 11,466
4.
McMEEKIN ET AL.
Refractive Indices of Amino Acids
63
d r o c h l o r i c a c i d o r s o d i u m h y d r o x i d e was u s e d i n s t e a d of w a t e r i n c a l c u l a t i n g m o l a r r e f r a c t i o n of the s a l t s of a l a n i n e . S i m i l a r l y , the data on the r e f r a c t i v e index and m o l a r r e f r a c t i o n of o v a l b u m i n i n T a b l e V i n d i cate that a c i d i f i c a t i o n r e d u c e s the r e f r a c t i v e i n d e x of o v a l b u m i n ; but i f i t s v o l u m e changes, r e p o r t e d by K a u z m a n n , a r e taken into c o n s i d e r a t i o n , the m o l a r r e f r a c t i o n of o v a l b u m i n does not change on a c i d i f i c a t i o n . T h i s l a c k of change of m o l a r r e f r a c t i o n w i t h c h a r g e m a y a p p e a r to be c o n t r a r y to the findings of P e r l m a n n and L o n g s w o r t h (26) on the effect of c h a r g e on the r e f r a c t i v e i n c r e m e n t of p r o t e i n s . H o w e v e r , t h i s i s not n e c e s s a r i l y t r u e , s i n c e they only c a l c u l a t e d r e f r a c t i v e i n c r e m e n t s . T h e i r r e s u l t s do not take into c o n s i d e r a t i o n v o l u m e changes w h i c h take p l a c e w i t h change i n c h a r g e , r e p o r t e d by K a u z m a n n (22). S p e c i f i c R e f r a c t i v e I n c r e m e n t s of P r o t e i n s . The s p e c i f i c r e f r a c t i v e i n c r e m e n t s , ( n - n ) c , of a n u m b e r of p r o t e i n s have been d e t e r m i n e d by A r m s t r o n g et a l . (2), P e r l m a n n and L o n g s w o r t h (26), H a l w e r , N u t t i n g , and B r i c e (16), and C h a r l w o o d (7). T h e s e a r e of p r a c t i c a l value i n d e t e r m i n i n g the c o n c e n t r a t i o n of p r o t e i n s o l u t i o n s b y m e a n s of the r e f r a c t i v e i n d e x of the s o l u t i o n and have an a c c u r a c y of about ± 0.5%, as stated by H a l w e r , N u t t i n g , and B r i c e (16). Consequently, v a l u e s i n T a b l e V I a r e g i v e n to o n l y t h r e e s i g n i f i c a n t f i g u r e s . I n g e n e r a l , the 0
Table VI. Specific Refractive Increments of Certain Proteins at 25° ( n - n ) c , g/ml 0
Wavelength Protein
589 ητμ
546 ητμ
436 ητμ
Gelatin of-Lactalbumin β -Lactoglobulin Lysozyme Ovalbumin Pepsin Ribonuclease Bovine serum albumin Horse serum albumin Human serum albumin
0.184 0.188 0.180 0.184 0.178 0.177 0.185 0.183 0.177 0.180
0.186 0.189 0.181
0.191 0.195 0.187
-
0.181 0.182 0.186 0.188 0.185 0.186
-
0.185 0.188 0.192 0.193 0.191 0.188
v a l u e s l i s t e d i n T a b l e V I a g r e e w i t h those r e p o r t e d by H a l w e r , N u t t i n g , and B r i c e (16) and C h a r l w o o d (7) f o r the s a m e p r o t e i n s . R e f r a c t i v e Index of P r o t e i n s at D i f f e r e n t W a v e l e n g t h s . The r e f r a c t i v e i n d i c e s of a m a n d i n and h e a m o c y a n i n w e r e found b y P u t z e y s and B r o s t e a u x (27) to v a r y w i t h the i n v e r s e s q u a r e of the wavelength. A s i m i l a r r e l a t i o n f o r the s p e c i f i c r e f r a c t i v e i n c r e m e n t s of a n u m b e r of p r o t e i n s w a s r e p o r t e d by P e r l m a n n and L o n g s w o r t h (26). T h e r e f r a c t i v e i n d i c e s of s e v e r a l p r o t e i n s a r e plotted a s a function of the i n v e r s e s q u a r e of wavelength f o r t h r e e wavelengths ( π ι μ ) i n F i g u r e 1. The s l o p e s a r e e s s e n t i a l l y the s a m e f o r the p r o t e i n s l i s t e d and differ
64
ADVANCES IN CHEMISTRY SERIES 1.66
1.64
κ
a 1.62
UJ >
