The reversibility of the interaction between SNP and methionine synthase was
studied further using the method of dilutions (described in Materials and Methods). The
results are shown in table 4.2.
SNP % inhibition
concentration p.M predicted irreversible predicted reversible experimental
4Ô 89 ± 3 80 ± 8 74 ± 1 6
20 8 9 ± 3 6 2 ± 7 58 ± 12
10 89 ± 3 46 ± 10 50 ± 1 9
5 89 ± 3 20 ± 8 40 ± 2 0
Table 4.2 Study of the nature of the S W induced inactivation of methionine synthase. E n zym e sa m p le (sp e c ific a c tiv ity 1 3 0 0 n m o l/h o u r/m g p r o te in ) w a s tr e a te d f o r 3 0 m in u tes a t 3 7 °C, u n d er r e d u c in g co n d itio n s, w ith 6 0 p M SN P. A liq u o ts o f th is m ixtu re (1 0 0 p i, 5 0 p i a n d 2 5 p i) w e r e a s s a y e d f o r en zym e a c tiv ity . A fte r a p r e -in c u b a tio n p e r i o d o f 5 m in u tes r e a c tio n s w e re in itia te d b y
th e a d d itio n o f h o m o c y ste in e (fin a l co n c e n tra tio n 4 0 0 p M ). R e a c tio n s w e r e in c u b a te d f o r 3 0 m in u tes a t
3 7 in 5 0 m M p h o s p h a te buffer. C o n tr o l v a lu e s w e r e o b ta in e d b y r e p e a tin g th e e x p e rim e n t su b s titu tin g 5 0 m M p h o s p h a te bu ffer f o r SN P. The p r e d i c te d v a lu e s w e r e o b ta in e d fr o m f i g 4.4, e ith e r d ir e c tly o r f r o m th e e q u a tio n f o r the lin e o f b e s t f it, in th is c a se th e s ta n d a r d d e v ia tio n s w e r e e s tim a te d f r o m th e s ta n d a r d d e v ia tio n s o f th e tw o n e a r e s t p o in ts . The re s u lts w e r e e x p r e s s e d a s th e m ea n p e r c e n ta g e o f th e c o n tr o l
v a lu e ± SD . C o m p a rin g the r e s u lts w ith th o se p r e d i c te d u sin g a tw o s a m p le d t-test, no s ig n ific a n t d ifferen ce w a s f o u n d f o r e ith e r th e p r e d i c te d ir r e v e r s ib le o r re v e r s ib le v a lu e s (a lth o u g h v a lu e s w e r e c lo s e r to sig n ific a n c e w h en c o m p a r e d w ith ir r e v e r s ib le in h ibition ); n = 3 .
The results show that the interaction between SNP and methionine synthase did
not match the predicted reversible or irreversible values. At all concentrations the
experimental values were lower than expected for irreversible inhibition. At a
concentration o f 20 pM SNP, the inhibition was less than expected for a reversible
Chapter 4._______________________________________ Nitric Oxide.
interaction; at the other concentrations, inhibition was slightly greater than expected.
Thus, it seems that the actual effect o f SNP might have some reversible and irreversible
inhibitory effects. As reversible inhibition is expected for the interaction o f SNP with
methionine synthase (from section 4.5), but irreversible inhibition is expected for the
effect o f NO upon methionine synthase, that the experiment may be show a mixed effect
due to both o f these interactions occurring.
It may be that SNP may bind to the enzyme and protect it from the effects of
nitric oxide. If this reaction is reversible as shown by previous results it would
effectively reduce the concentration o f enzyme available for NO to bind to. If, whilst
bound to the enzyme, SNP could not release nitric oxide, the concentration o f nitric
oxide would be reduced, and the rate o f formation of nitric oxide would also be slower
than expected. These two hypotheses are consistent with the results obtained in sections
4.4. and 4.5. Thus, the results found in this experiment, may be due to the following
situation. Reversible binding o f SNP to the enzyme would prevent release of NO, and
protect the enzyme from released NO. The concentrations o f NO would then increase
slowly and be due to a pool o f unbound SNP. Any nitric oxide formed would only be
able to attack the enzyme when the concentration of SNP had decreased sufficiently to
produce free enzyme in the mixture. It may have been that a longer pre-incubation
period would have produced results closer to the predicted irreversible inhibition.
Other conditions in the treatment period, such as omission o f reducing agent and
thiol groups may not have been ideal for SNP degradation, and these factors may have
contributed to the unexpectedly low percentage inhibitions, found experimentally.
4.8. The effect of sodium nitroprusside upon methionine synthase activity over