3.9 P h o s p h o l ipase T r e a t m e n t of I n t a c t Red Cells and Gh osts
3.9.1 E f f e c t of C a ^ ^ and EDTA C o n c e n t r a t i o n s on P h o s p h o l i p a s e A c t i v i t y
B e c a u s e lOmM C a ^ ^ was r e p o r t e d t o be the m a x i m u m c o n c e n t r a t i o n for a c t i v a t i o n of ph o s p h o l i p a s e f r o m bee v e n o m ( S h i p ollni, C a l l e w a e r t , C o t t r e l l , D o o n a n and V e r n on , 1971). This c o n c e n t r a t i o n was used in i n c u b a t i o n s
2
+i n s t e ad of 0 . 2 5m M of Ca r e c o m m e n d e d by Zwa a l and
R o e l o f s e n (1975). F i n a l c o n c e n t r a t i o n of 1 . 6 6 6 6m M EDTA Na^ was a d d ed to stop th e e n z y m e a c ti v i t y as r e c o m m e n d e d by Z w a a l and R o e l o f s e n (1975 ). H ow e v e r , the d e g r e e of h a e m o l y s i s (9.4%) was f o u n d to be much h i g h e r t h a n the a c c e p t a b l e 3%, and some PS d e g r a d a t i o n was f o u n d in i n tact cells due to this h a e m o l y s i s as PS b e c a m e a c c e s s i b l e to the added e n zy m e . S h i p o l l n i e ^ al^. (1971) i n d i c a t e d that
a d d i t i o n of both E D T A and c a l c i u m ions had a large a c t i v ating e f f ec t pro v i d i n g t hat c a l c i u m ions were p r esent in large e x c e s s . The a c t i v e e n t i t y of the e n z y m e was a
c a I c i u m - p r o t e i n c o m p l e x whi c h d i s s o c i a t e d g i v i n g i n a c t i v e p r o t e i n as the c a l c i u m ions f o r m e d a c o m p l e x with EDTA. H o w e v e r , a d d i t i o n of E D T A and e x c e s s c a l c i u m ions led to a very large r a t e e n h a n c e m e n t . It was p r o p o s e d that E D T A
or some E D T A - c a l c i u m c o m p l e x i n t e r a c t e d with the e n z y m e in such a way that the c a t a l y t i c a c t i v i t y of the c a l c i u m - e n z y m e c o m p l e x was I n c r e a s e d (i.e. acting as an a l l o s t e r i c a c t i v a t o r ) ( S h ip ol l n i et aly, 1971). C o t t r e l l (1981)
of E DTA and a g r e a t e x c e s s of Ca^* were pre s e n t t h e re was c h e l a t i o n of i n h i b i t i n g m e t a l ions that bind both t o E D T A and t o t he e n z y m e m o r e t i g h t l y t h an Ca^^ (as well as some of the Ca^^), r e s u l t i n g in e n h a n c e d a c t i v i t y of the
e n zy m e . Either of t h e s e h y p o t h e s e s may e x p la in the h i g h d e g r e e of h a e m o l y s i s % o b t a i n e d in this wor k , if, under
2
+the I n i t i al c o n d i t i o n s of Ca and EDTA, a d d i t i o n of E D T A