Effect of Environmental Factors on Starch Gel Electrophoretic Patterns

16 Effect of Environmental Factors on Starch Gel Electrophoretic Patterns of Human Erythrocyte Acid Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on March 17, 2018 | https://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/bk-1975-0013.ch016

Phosphatase CORNELIUS

G.

McWRIGHT,

JAMES

J.

KEARNEY,

and

JAMES

L.

MUDD

B i o l o g i c a l Science Research U n i t , FBI L a b o r a t o r y , W a s h i n g t o n , D.C. 20535

Human erythrocyte acid p h o s p h a t a s e (ΕΑΡ) p o l y m o r p h i s m was first described by H o p k i n s o n , S p e n c e r a n d Harris (1). ΕΑΡ c a n be classified by electrophoresis into six different phenotypes, A, AB, Β, CB, AC and C. From numerous distribution and family studies it has been determined that the six phenotypes are directed by three common alleles p , p , and p (2,3,4). Using crude hemolysates (5) and 1,000 fold pure homogeneous type AA and BB enzymes (6) some properties of ΕΑΡ, such as thermostability, pH and substrate specificity and molecular size, have been examined. From the time ΕΑΡ polymorphism was first described, the use of the enzyme as a means of typing human blood has been of interest to the forensic scientist. Investigators have described successes in typing dried bloodstains stored at 20 - 25ºC for 5-8 weeks and stored whole blood kept at 5 C was typed for as long as 15 months (7). Difficulties in typing ΕΑΡ types AB (1,3), Β and C (8) have also been described. Putrefactive bacteria, such as Clostridium welchii, which frequently invade human blood during the agonal period or immediately after death, produce the enzyme neuraminidase (9). Neuraminidase has been shown to effect the heterogeneity of electrophoretic banding patterns of the human prostate acid phosphatase (10). The effect of this enzyme on ΕΑΡ is not known. The purpose of this study was to compare two electrophoretic methods used to type ΕΑΡ and to examine the stability of ΕΑΡ phenotypes in red cell hemolysates, clotted blood and dried bloodstains stored at room temperature (25°C). A distribution study of the frequency of five ΕΑΡ phenotypes among ABO, MN, Rh blood groups and among 137 metropolitan Washington D. C. area residents was made. The effect of neuraminidase on ΕΑΡ was also studied. a

b

c

151

Davies; Forensic Science ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

152

FORENSIC SCIENCE

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on March 17, 2018 | https://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/bk-1975-0013.ch016

Methods a n d M a t e r i a l s P r e p a r a t i o n o f samples. F r e s h and outdated b l o o d samples used i n t h i s study were o b t a i n e d f r o m b l o o d banks o f s e l e c t e d m e d i c a l i n s t i t u t i o n s i n t h e m e t r o p o l i t a n W a s h i n g t o n , D. C. a r e a . Red c e l l h e m o l y s a t e s w e r e p r e p a r e d b y w a s h i n g c e n t r i f u g e d r e d c e l l s t w i c e w i t h a 0.87% s a l i n e s o l u t i o n a n d t h e n m i x i n g o n e v o l u m e o f d i s t i l l e d w a t e r t o one volume o f p a c k e d r e d c e l l s . T h i s m i x t u r e was t h e n f r o z e n a n d s u b s e q u e n t l y t h a w e d j u s t p r i o r to use. D r i e d b l o o d s t a i n s were prepared by p i p e t t i n g a mixed suspension o f r e d c e l l s onto a clean piece o f white cotton s h e e t i n g w h i c h was t h e n c o m p l e t e l y a i r d r i e d . A c c u r a t e l y c u t 10mm χ 2mm c u t t i n g s w e r e u s e d f o r e l e c t r o p h o r e s i s . Samples t o b e t a k e n f r o m l i q u i d h e m o l y s a t e s a n d p u l v e r i z e d c l o t t e d b l o o d w e r e p r e p a r e d b y s a t u r a t i n g 10mm χ 2mm c u t t i n g s w i t h hemolysates o r l i q u i d f r o m t h e p u l v e r i z e d c l o t and a l l o w i n g the cuttings t o dry before a p p l i c a t i o n . S t a r c h - g e l e l e c t r o p h o r e s i s . E l e c t r o p h o r e s i s was c a r r i e d o u t f o r t i m e i n t e r v a l s o f 4 h o u r s a n d 15 h o u r s u s i n g t h e method f i r s t d e s c r i b e d b y H o p k i n s o n a n d H a r r i s ( 1 1 ) w i t h one s l i g h t m o d i f i c a t i o n . The 0.245M N a H P 0 / 0 . 1 5 M N a o C H r O b r i d g e b u f f e r was p r e p a r e d w i t h 0.02M M g C l a n d 0.01M EDTA a t pH 5.5. Two m i l l i m e t e r t h i c k ( 1 2 ) 1 0 % s t a r c h - g e l s , pH 6.1, w e r e p r e p a r e d b y u s i n g a 1/100 d i l u t i o n o f t h e b r i d g e b u f f e r . Samples w e r e i n s e r t e d i n t o t h e g e l a p p r o x i m a t e l y HO m i n u t e s a f t e r t h e g e l was p o u r e d . Samples w e r e o v e r l a y e d w i t h 0.1M D i t h i o e r y t h r i t o l (Sigma) p r e p a r e d i n g e l b u f f e r and were a l l o w e d t o i n c u b a t e i n t h e p r e s e n c e o f t h e t h i o l r e a g e n t f o r 10 m i n u t e s b e f o r e e l e c t r o p h o r e s i s was s t a r t e d . F o u r h o u r e l e c t r o p h o r e s i s r u n s w e r e c a r r i e d o u t o n c o o l i n g p l a t e s a t 0 C a t 20 m a / p l a t e . F i f t e e n h o u r r u n s w e r e c a r r i e d o u t i n t h e r e f r i g e r a t o r a t 5°C a t 8 ma/plate. F o r b e s t r e s u l t s b r i d g e b u f f e r s were o n l y used once. 2

4

R

?

2

_Lf

Measurement o f ΕΑΡ A c t i v i t y . A 2 χ 1 0 M 4 - m e t h y l u m b e l l i f e r y l phosphate (Koch-Light L a b o r a t o r i e s ) s o l u t i o n p r e p a r e d i n 0.05M c i t r i c a c i d / N a O H b u f f e r , pH 5.0 (13) was u s e d t o s a t u r a t e a n a p p r o p r i a t e s i z e d p i e c e o f Whatman 1MM f i l t e r p a p e r . The s a t u r a t e d p a p e r was p l a c e d o n t h e g e l s u r f a c e c o v e r i n g t h e a r e a between t h e o r i g i n and a n o d i c b r i d g e . ΕΑΡ a c t i v i t y was d e v e l o p e d b y p l a c i n g t h e g e l i n a 37 C i n c u b a t o r f o r 30 m i n u t e s a n d t h e n o b s e r v i n g i t u n d e r l o n g w a v e l e n g t h UV light. Less a c t i v e samples r e q u i r e d l o n g e r i n c u b a t i o n t i m e s . Photography and Densitometer T r a c i n g s . ΕΑΡ a c t i v i t y was p h o t o g r a p h e d u n d e r UV l i g h t t h r o u g h a T i f f e n P h o t a r , #61, s e r i e s 7 g r e e n g l a s s f i l t e r u s i n g P o l a r o i d t y p e 55 f i l m a n d a c o n s t a n t f o c u s G r a p h i c camera box b u i l t by t h e S p e c i a l Photo U n i t o f t h e FBI Laboratory. D e n s i t o m e t e r t r a c i n g s w e r e made d i r e c t l y f r o m


16.

M C WRIGHT

E T

AL.

Human

Erythrocyte

Acid

153

Phosphatase

t h e c l e a r e d n e g a t i v e s u s i n g a J o y c e L o e b l m o d e l 3CS d e n s i t o meter. N e u r a m i n i d a s e , N e u r a m i n i d a s e ( S i g m a ) c o n t a i n e d 500 u n i t s of a c t i v i t y p e r ml.


Results C o m p a r i s o n o f 4 h o u r a n d 15 h o u r s t a r c h - g e l e l e c t r o p h o r e s i s . The c o m p a r i s o n o f t h e b a n d i n g p a t t e r n s o f f i v e ΕΑΡ p h e n o t y p e s o b t a i n e d d u r i n g e l e c t r o p h o r e s i s w i t h t h e same b u f f e r b u t f o r d i f f e r e n t t i m e i n t e r v a l s i s shown i n f i g u r e 1. B a n d i n g p a t t e r n s o b t a i n e d d u r i n g 4 h o u r e l e c t r o p h o r e s i s a t 0 ~C w e r e c o n s i s t e n t l y s h a r p e r a n d b e t t e r d e f i n e d t h a n t h e more d i f f u s e p a t t e r n s o b t a i n e d d u r i n g 15 h o u r e l e c t r o p h o r e s i s a t 5 C . T h e a i s o z y m e b a n d i s c l e a r l y s e p a r a t e d f r o m t h e f a s t e r m o v i n g components t y p e s AB a n d AC. I n t h e 15 h o u r e l e c t r o p h o r e s i s r u n t h e a isozyme band appears a s a s h o u l d e r t o t h e f a s t e r moving b i s o z y m e b a n d i n t y p e s A B a n d AC, m a k i n g t h e s e t y p e s more d i f f i c u l t t o interpret. P a t t e r n s o b t a i n e d from b o t h systems a r e c o n s i s t e n t w i t h those described by other i n v e s t i g a t o r s (1,3,4). Densitometer t r a c i n g s o f t h e e l e c t r o p h o r e s i s banding p a t t e r n s o f f i v e ΕΑΡ p h e n o t y p e s f r o m a 4 h o u r e l e c t r o p h o r e s i s a r e shown i n f i g u r e 2. A l l f o u r i s o z y m e b a n d s m a k i n g u p t h e f i v e ΕΑΡ p h e n o t y p e s a r e c l e a r l y d e f i n e d . The s t r o n g s t o r a g e band, s , i s always a s s o c i a t e d w i t h those phenotypes having s t r o n g b i s o z y m e b a n d s s u c h a s t y p e s B, CB a n d AB. A w e a k e r s s t o r a g e b a n d a p p e a r s i n p h e n o t y p e A. The s s t o r a g e band i s a s s o c i a t e d w i t h t h e s t r o n g a i s o z y m e b a n d o f t y p e A. T h e w e a k e r s b a n d a l s o a p p e a r s i n t y p e s AC a n d AB b u t i s s o weak i t i s d i f f i c u l t t o observe. S t r o n g s bands c a n be seen i n f i g u r e s 1 and 3, b u t t h e s i s t o o l i g h t t o b e s e e n o r t o b e r e a d w i t h t h e d e n s i t o m e t e r e x c e p t i n t y p e A. The a p p e a r a n c e a n d i n t e n s i t y o f t h e s a n d s b a n d s c a n b e c o n t r o l l e d somewhat b y i n c u b a t i o n o f samples i n t h i o l reagents b e f o r e e l e c t r o p h o r e s i s . I f t h e s b a n d becomes a s s t r o n g a s i t a p p e a r s i n f i g u r e 3, i n t h e a b s e n c e o f a c o n t r o l showing t h e placement o f t h e a isozyme band, a type Β may b e f a l s e l y r e a d a s a t y p e AB b y a n u n t r a i n e d e x a m i n e r . C

1

f

T

f

f

T

f

C

S t a b i l i t y o f ΕΑΡ d u r i n g s t o r a g e a t 2 5 C . T h e l o s s o f ΕΑΡ a c t i v i t y m d r i e d b l o o d s t a i n s , f r e s h c e l l hemolysates, and c l o t t e d b l o o d d u r i n g s t o r a g e a t 25°C i s shown i n f i g u r e 4. H e m o l y s a t e s a n d c l o t t e d b l o o d l o s t n e a r l y a l l ΕΑΡ a c t i v i t y a f t e r s t o r a g e f o r 5 d a y s a t 25°C w h i l e ΕΑΡ a c t i v i t y i n d r i e d s t a i n s p e r s i s t e d f o r much l o n g e r p e r i o d s o f t i m e . The l o s s o f a c t i v i t y o f a l l f i v e d i f f e r e n t ΕΑΡ p h e n o t y p e s was t h e same i n h e m o l y s a t e s a n d c l o t t e d b l o o d s t o r e d a t 25*C. I n d r i e d b l o o d s t a i n s , h o w e v e r , ΕΑΡ p h e n o t y p e s CB a n d AC l o s e a c t i v i t y a t a s l o w e r r a t e t h a n d o t y p e s A B , A a n d B. T y p e A i s t h e l e a s t s t a b l e o f t h e p h e n o t y p e s . Type A B was i n t e r m e d i a t e b e t w e e n t y p e s Β a n d A.



FORENSIC SCIENCE

Figure 1. The comparison of the ΕΑΡ phenotype electrophoretic patterns ob tained during electrophoresis for 4 hr at 0°Candl5hr at5°C

2

1

03

2

1

0

·#— MIGRATION OF ΕΑΡ cm

Figure 2. Densitometer tracings illustrating the isozyme migration patterns of five ΕΑΡ phenotypes during electrophoresis for 4 hr at 0°C. The s and s' storage bands can also be seen.


M C WRIGHT

Human

E T AL.

Erythrocyte


16.

Acid

Phosphatase

155

Figure 3. Densitometer tracings and ac companying photographs of ΕΑΡ phenotype Β illustrating the loss of activity of the h isozyme and increase in activity of the c isozyme during storage at 25°C. Samples were taken from clotted blood. Electrophoresis was carried out for 15 hr at 5 ° C . The successive densitometer tracings shown were made from the ΕΑΡ phenotype Β pattern located on the left in the photograph.

*

20 μ 10 η

I

0

12

I

I

3

I

I

U, 5 6

I

I I

?

8

L_J

I

L

10

11

12

9

Figure 4. Comparison of the loss of ΕΑΡ activity present in red cell hemoly sates, clotted blood, and dried blood stains stored at 25°C. ΕΑΡ phenotypes shown are types A, AB, B, CB, and AC.

DAYS



156

FORENSIC SCIENCE

A s shown i n t a b l e 1, d r i e d b l o o d s t a i n s r e t a i n ΕΑΡ a c t i v i t y f o r p e r i o d s a s l o n g a s 15 m o n t h s . D r i e d b l o o d s t a i n s w e r e t y p e d b l i n d up t o 4 months w i t h o u t d i f f i c u l t y b u t b e y o n d t h a t p e r i o d t y p i n g became v e r y d i f f i c u l t . D r i e d b l o o d s t a i n s t h a t c o u l d be t y p e d a f t e r 4 months w e r e s a t u r a t e d a n d r e q u i r e d l o n g i n c u b a t i o n periods i n the presence o f f r e s h l y prepared t h i o l reagents. D u r i n g t h e c o u r s e o f s t o r a g e s t u d i e s , changes i n t h e i n t e n s i t i e s o f v a r i o u s components o f t h e d i f f e r e n t ΕΑΡ p h e n o t y p e s were observed. The most s t r i k i n g c h a n g e s a r e i l l u s t r a t e d i n f i g u r e s 3 a n d 5. The f a s t e r m o v i n g components o f t y p e s Β and CB became w e a k e r w h i l e t h e s l o w e r components became s l i g h t l y s t r o n g e r . A s shown i n f i g u r e 3, f r o m t h e a c c o m p a n y i n g d e n s i t o m e t e r t r a c i n g s , i t i s apparent t h a t a f t e r 4 days a t y p e Β c o u l d be m i s i n t e r p r e t e d a s a weak t y p e C. Changes i n t h e i n t e n s i t i e s o f t h e d i f f e r e n t components o f t h e ΕΑΡ p h e n o t y p e s did not occur o f t e n during the course o f t h i s study but d i d o c c u r o f t e n enough t o w a r r a n t d i s c u s s i o n . Whole b l o o d c o n t a i n i n g c i t r a t e p h o s p h a t e d e x t r o s e a n t i c o a g u l a n t and s t o r e d a t 5°C was t y p e d up t o 10 m o n t h s . The e f f e c t o f n e u r a m i n i d a s e o n ΕΑΡ b a n d i n g p a t t e r n s d u r i n g , electrophoresis" The e f f e c t o f n e u r a m i n i d a s e o n f i v e ΕΑΡ p h e n o t y p e p a t t e r n s i s shown i n f i g u r e 6. Selected fresh red c e l l h e m o l y s a t e s w e r e t r e a t e d w i t h n e u r a m i n i d a s e a n d s t o r e d a t 25°C. Samples 1-4 c o n t a i n e d no n e u r a m i n i d a s e w h i l e s a m p l e s 5-9 c o n t a i n e d 10 u n i t s o f n e u r a i n i n i d a s e / m l . A f t e r 114 h o u r s , a l t h o u g h a l l 9 s a m p l e s h a d l o s t s i g n i f i c a n t amounis o f ΕΑΡ a c t i v i t y , s a m p l e s 1-4 r e m a i n e d t y p e a b l e . Samples 5-9, c o n t a i n i n g n e u r a m i n i d a s e h a d l o s t a l l o b s e r v a b l e ΕΑΡ a c t i v i t y . The e f f e c t o f n e u r a m i n i d a s e o n t h e f i v e ΕΑΡ p h e n o t y p e p a t t e r n s n e e d s t o be e x a m i n e d more c l o s e l y b u t may i n v o l v e t h e r e m o v a l o f s i a l i c a c i d r e s i d u e s f r o m t h e ΕΑΡ enzyme. A l t h o u g h n e u r a m i n i d a s e a f f e c t s t h e h e t e r o g e n e i t y o f ΕΑΡ p h e n o t y p e p a t t e r n s , i t d o e s n o t a l t e r t h e m i n a manner w h i c h w o u l d l e a d t o m i s i n t e r p r e t a t i o n o f pattern types. D i s t r i b u t i o n o f f i v e ΕΑΡ p h e n o t y p e s among a r a n d o m l y s e l e c t e d number o f m e t r o p o l i t a n W a s h i n g t o n , D. C. a r e a r e s i d e n t s and among t h e ABO, MN and Rh b l o o d g r o u p s . The i n c i d e n c e o f f i v e ΕΑΡ p h e n o t y p e s i n 137 r a n d o m l y s e l e c t e d n e g r o a n d C a u c a s i a n m e t r o p o l i t a n W a s h i n g t o n , D. C. a r e a r e s i d e n t s i s shown i n t a b l e 2. The computed f r e q u e n c i e s o f 10.2% t y p e A, 44.5% t y p e B, 38.0% t y p e AB, 4.4% t y p e CB and 2.9% t y p e AC d i f f e r s f r o m t h o s e f i g u r e s o b t a i n e d by G i b l e t t a n d S c o t t (_3). They o b s e r v e d among 193 S e a t t l e C a u c a s i a n s f r e q u e n c i e s o f 1 7 . 1 % t y p e A, 31.6% t y p e B, 39.4% t y p e AB, 6.73% t y p e CB and 5.18% t y p e AC. Among 164 n e g r o s f r e q u e n c i e s o f 7.32% t y p e A, 60.4% t y p e B, 29.3% t y p e A B , 1.83% CB and 1.21% t y p e AC w e r e o b s e r v e d . No ΕΑΡ t y p e Cte w e r e o b s e r v e d i n e i t h e r s t u d y . I f the data obtained b y G i b l e t t a n d S c o t t i n t h e s e p a r a t e s t u d i e s o n n e g r o e s and


16.

M C WRIGHT

E T

Human

AL.

Erythrocyte

Acid

Phosphatase

157


TABLE 1· ΕΑΡ TYPING OF AGED-DRIED BLOODSTAINS STORED AT 25 C No. attempted 6 6 8 6 5 6 8 8

Age of stain months

1

2 k

5 7 10 12 15

Νο· having ΕΑΡ a c t i v i t y

No. typeable

6 6 8 6 5 6 7 3


6 6 8 4 k

3 2 0


FORENSIC

SCIENCE

Figure 6. The effect of neuraminidase on the ΕΑΡ phenotype patterns obtained during electrophoresis for 4 hr at 0°C. Samples 1-4 contained no neuramini dase. Samples 5-9 contained 10 units of neuraminidase activity per ml. All sam ples were incubated at 25°C. The photo graph taken at 114 hr was exposed for 6 min to enhance ΕΑΡ activity observed in samples 1—4. A 2-min exposure was used for photographs taken at the other times. The only ΕΑΡ activity observed after incubation with neuraminidase for 114 hr was the c isozyme band of sample 5 (ΕΑΡ phenotype AC). The c isozyme is the most stable of the four ΕΑΡ isozymes (5).


16.

M C

WRIGHT

E T

AL.

Human

Erythrocyte

Acid

Phosphatase

159


Caucasians

are c o m b i n e d , i t w o u l d then represent an a p p r o x i m a t e 60/40 m i x t u r e o f Caucasians to negroes. I f new phenotype f r e q u e n c i e s a r e t h e n c a l c u l a t e d , p e r c e n t a g e s o f 12.2% t y p e A , 44.8% t y p e B, 34.4% t y p e AB, 4.20% t y p e CB a n d 3.36% t y p e AC a r e o b t a i n e d . The f i g u r e s o b t a i n e d b y t h i s c o m b i n a t i o n a r e s t r i k i n g l y s i m i l a r t o the frequencies obtained i n t h i s study. The d i s t r i b u t i o n o f f i v e ΕΑΡ p h e n o t y p e s among b l o o d g r o u p s ABO, MN a n d Rh i s shown i n t a b l e 3 . A l t h o u g h t h e s a m p l e s t a k e n i n some g r o u p s a r e s m a l l i t i s a p p a r e n t t h a t t h e i n c i d e n c e o f ΕΑΡ i s n o t a s s o c i a t e d g e n e t i c a l l y w i t h b l o o d g r o u p s ABO, MN a n d Rh. Discussion ΕΑΡ i s a s t a b l e p o l y m o r p h i c enzyme w h i c h can, b y e l e c t r o p h o r e s i s , b e t y p e d i n t o s i x p h e n o t y p i c p a t t e r n s — A , A B , B, CB, A C , a n d C ( t h e r a r e t y p e C was n o t o b s e r v e d i n t h i s s t u d y ) . E l e c t r o p h o r e s i s f o r 4 h o u r s a t 0 'C c o n s i s t e n t l y g a v e s h a r p e r ΕΑΡ b a n d i n g p a t t e r n s t h a n e l e c t r o p h o r e s i s f o r 15 h o u r s a t 5 C The a i s o z y m e o f t y p e s AB a n d AC was w e l l d e f i n e d d u r i n g 4 h o u r e l e c t r o p h o r e s i s a n d made t h e t y p i n g o f ΕΑΡ p h e n o t y p e AB r e l i a b l e and simple. D u r i n g e l e c t r o p h o r e s i s r u n s , i t i s recommended t h a t a f r e s h t y p e A B o r t y p e s A a n d Β b e u s e d a s c o n t r o l s t o mark t h e m i g r a t i o n a n d i n t e n s i t y o f t h e v a r i o u s components o f t h e s i x d i f f e r e n t ΕΑΡ p h e n o t y p e s . These c o n t r o l s w i l l a l s o a i d i n d i f f e r e n t i a t i n g t h e t r u e i s o z y m e s o f t h e ΕΑΡ t y p e s f r o m s t o r a g e bands s a n d s w h i c h a r e a s s o c i a t e d r e s p e c t i v e l y w i t h t h e strong b and a isozymes. The r a t e o f l o s s i n a c t i v i t y o f t h e f i v e d i f f e r e n t ΕΑΡ p h e n o t y p e s d u r i n g s t o r a g e a t 25'C i n h e m o l y s a t e s a n d c l o t t e d b l o o d was d e t e r m i n e d t o b e t h e same. A l l f i v e t y p e s l o s t e s s e n t i a l l y a l l t y p e a b l e a c t i v i t y i n 5-6 days under t h e s e c o n d i t i o n s . I ndried bloodstains, differences i n t h e r a t e s o f l o s s i n a c t i v i t y o f t h e f i v e ΕΑΡ t y p e s was observed. T y p e s CB, AC a n d Β w e r e o b s e r v e d t o b e t h e most s t a b l e , w h i l e t y p e A was t h e l e a s t s t a b l e . Type AB i s i n t e r m e d i a t e b e t w e e n t y p e s A a n d B. D r i e d b l o o d s t a i n s w e r e t y p e d b l i n d up t o 20 weeks b u t b e y o n d t h a t p o i n t c a u t i o n s h o u l d be e x e r c i s e d . O c c a s i o n a l changes i n a c t i v i t y o f t h e b isozyme o f t y p e s Β a n d CB d u r i n g s t o r a g e a t 2 5 C i n c l o t t e d a n d d r i e d b l o o d s t a i n s were o b s e r v e d . T h i s phenomerrn i s c o n s i s t e n t w i t h o b s e r v a t i o n s made b y L u f f m a n a n d H a r r i s (3). During thermostability studies w i t h f i v e ΕΑΡ p h e n o t y p e s t h e y f o u n d t h a t t h e a c t i v i t y o f t h e f a s t e r m o v i n g component ( b i s o z y m e ) o f t y p e Β became w e a k e r a s i t was h e a t e d f o r 5 m i n u t e s a t 52 C w h i l e t h e a c t i v i t y o f t h e s l o w e r component ( c i s o z y m e ) became s t r o n g e r . B o t h i s o z y m e s l o s t e s s e n t i a l l y a l l a c t i v i t y a f t e r 10 m i n u t e s . S m i t h a n d W h i t b y ( 1 0 ) f o u n d t h a t i n c u b a t i o n o f t h e human p r o s t a t i c a c i d phosphatase i n the presence o f neuraminidase T

f

r


FORENSIC


160

SCIENCE

TABLE 2. INCIDENCE OF ΕΑΡ PHENOTYPES IN 137 RANDOMLY SELECTED METROPOLITAN WASHINGTON D.C. RESIDENTS Incidence

No. of individuals 14 52 61 6 4 0 137

ΕΑΡ phenotypes A AB Β CB AC C Total

*

10.2 38.0 44.5 4.4 2.9

TABLE 3. DISTRIBUTION OF FIVE ΕΑΡ PHENOTYPES AMONG THE ABO, MN AND Rh BLOOD GROUPS Phenotypes A AB Β CB AC Totals

A* Β 3 6 15 21 7 4 0 0 3 46 15

1

ABO AB

~τ 5 1 0 1 7

0 1 5 4 0 0 10

MN ~Z 11 9 2 1 25

MN M Τ 9 10 0 1 21

Rh Ν

15 6 2 1 15

+ -

Τ 16 11 21 10 3 1 3 0 47 23

"Contains 2 Α > each m e having a d i f f e r e n t ΕΑΡ phenotype. 2


16.

M C WRIGHT

E T

AL.

Human

Erythrocyte

Acid

Phosphatase

161


caused a r e d i s t r i b u t i o n o f t h e f a s t e r moving e l e c t r o p h o r e t i c components of t h i s enzyme among i t s s l o w e r m o v i n g c o m p o n e n t s . A l t h o u g h n e u r a m i n i d a s e a l t e r s t h e h e t e r o ^ n e i t y o f ΕΑΡ e l e c t r o p h o r e t i c p a t t e r n s i t d o e s n o t a l t e r t h e m i n a manner w h i c h w o u l d l e a d t o m i s i n t e r p r e t a t i o n of t y p e s . The f r e q u e n c y o f o c c u r e n c e o f f i v e ΕΑΡ p h e n o t y p e s was d e t e r m i n e d t o be 10.2% A , 4 4 . 5 % B, 38.0% A B , 4.4% CB a n d 2.9%

CA among 137 randomly selected n e g r o and Caucasian metropolitan W a s h i n g t o n , D. C. r e s i d e n t s . These f i g u r e s a r e i n agreement w i t h t h e r e s u l t s o b t a i n e d b y G i b l e t t a n d S c o t t when t h e d a t a o b t a i n e d f r o m s e p a r a t e s t u d i e s among S e a t t l e n e g r o a n d Caucasians w e r e c o m b i n e d a n d new p h e n o t y p e frequencies calculated. ΕΑΡ i s n o t g e n e t i c a l l y l i n k e d t o t h e ABO, MN o r Rh b l o o d g r o u p s y s t e m s .

Literature Cited 1. Hopkinson, D. Α., Spencer, Ν., and Harris, Η., Nature, (1963), 199, 969. 2. Hopkinson, D. A., Spencer, Ν., and Harris, Η., Human Genetics, (1964), 16, 141. 3. Giblett, E. R., and Scott, Ν. M., Amer. J. Hum. Genet., (1965), 1 7 , 425. 4. S o r e n s e n , S. A., Human Heredity, ( 1 9 7 3 ) , 2 3 , 4 7 0 . 5. L u f f m a n , J . E. a n d Harris, Η., A n n . Hum. G e n e t . , ( 1 9 6 7 ) , 30, 387. 6. Scott, Ε. M., J. Biol. Chem., (1966), 241, 3049. 7. Brinkmann, Β., Gunnemann, M. and Koops, E., J. For. Med., (1972), 70, 68. 8. Fiedler, Η., Arztl. Lab. (1967), 13, 507. 9. MacLennan, J. D., Bact. Rev., (1962), 26_, 177. 10. Smith, J. K. and Whitby, L. G., Biochim. Biophys. Acta., (1968), 151, 607. 11. Hopkinson, D. A. and Harris, Η., Biochemical Methods in Red Cell Genetics, 337, Editor J. J. Yunis, Academic Press, NY, 1969. 12. Wraxall, B. G. D. and Culliford, B. J., J. For. Sci. Soc., (1968), 8, 81. 13. Wraxall, B. G. D. and Adams, E. G., For. Sci., (1974), 3, 57.


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