Biochimie ( 1991 ) 73, 551-556 © Socirt6 fran~zaise de biochimie et biologie molrculaire / Elsevier, Paris
551
Chlorpheniramine binding to human serum albumin by fluorescence quenching measurements J Gonzalez-Jimenez, G Frutos, I Cayre, M Cortijo Departamento de Qufmica Fisica Farmacetitica, Facultad de Farmacia, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain
(Received 29 August 1990; accepted 10 January 1991 )
Summary - - The binding of chlorpheniramine to human serum albumin has been studied by fluorescence quenching, as a function of temperature; the experimental data could only be fitted to the Stern-Volmer modified equation. A statistical analysis of the results was performed in order to determine the significance of the constants calculated by this equation, as well as their thermodynamic parameters. The chlorpheniramine binding to human serum albumin accounts for almost half of the binding of this antihistaminic agent to human plasma proteins. chlorpheniramine / antihistaminic / albumin / fluorescence / quenching / binding
Introduction C h l o r p h e n i r a m i n e m a l e a t e ( C P M ) is a potent and h i g h l y e f f e c t i v e a n t i h i s t a m i n i c agent, w i d e l y used to alleviate s y m p t o m s o f the c o m m o n cold and allergic rmm~-t;r~ne I t hme h m ~ n in~hlri~rl l~i..sLJkqk~lklYt..lll:.J. I l k llLJl.t.~ i . , ¢ v ~ k ~ l l l l l ~ l l ~ g ~ J - ~ t J
~ n m~n~; 111 lliCtAkilJ
n hmrr~mr-nlr~eri_ ~llgdJ.lill~*.Jk~Orll,.,g~l
cal preparations during recent years, either as a single active p r i n c i p l e or m i x e d with other drugs. Several p h a r m a c o k i n e t i c studies h a v e b e e n carried out d u r i n g recen! years in a n i m a l s and h u m a n s with C P M ([1-3]; and r e f e r e n c e s quoted therein). It is clear from these studies that C P M r e m a i n s for several hours in p l a s m a , p o s s i b l y b o u n d to s o m e proteins. It is well k n o w n that p l a s m a protein b i n d i n g m i g h t regulate to a certain extent the p h a r m a c o l o g i c a l action o f a drug, a v o i d i n g its b i o t r a n s f o r m a t i o n and acting as a drug reservoir. This b i n d i n g could also a v o i d possible toxic effects due to h i g h drug intake. H u m a n s e r u m a l b u m i n ( H S A ) is a very a b u n d a n t p l a s m a protein. It is therefore a good candidate for C P M binding. This protein strongly binds m a n y hydrop h o b i c substances, but is also able to b i n d m a n y other c h e m i c a l s , a l t h o u g h with l o w e r affinity. Despite the i m p o r t a n c e o f this drug, data on C P M b i n d i n g to isolated p l a s ~ m proteins h a v e not b e e n p u b l i s h e d . In
Abbreviations: CPM, chlorpheniramine maleate; HSA, human serum albumin; ANOVA, analysis of variance
this paper, we d e m o n s t r a t e by fluorescence q u e n c h i n g m e t h o d s that H S A b i n d s C P M in vio'o at temperatures up to 41 °C.
Materials and Methods Chemicals
CMP was generous donated by ISFAS, and used without additional purification. Fatty acid-free HSA was purchased from Sigma. All other chemicals used were of analytical grade. Binding measurements
Stock solutions of HSA (1 mg/ml) and CPM (0.01 M) were prepared in 0.05 M phosphate buffer (pH 7.4), at ionic strength 0.6 by adding NaCI. The actual concentrations of these stock solutions were determined by dilution with the same buffer and by measuring their absorption at 278 nm (HSA) and 261.5 nm (CPM). The molar absorption coefficients used were 3.66 103 [41 and 4.54 103 1 mol -I cm -I (Gonzalez, unpublished observations) for HSA and CPM, respectively. It was checked that both solutions followed the Lambert-Beer law within the range used in this study. A typical binding experiment was performed at a constant protein concentration, as follows: 2 HSA solutions (at I laM concentration) were prepared by dilution of the corresponding stock solution with either: A), buffer; or B), a CPM solution with a final drug concentration (after mixing with HSA) of 0.05 M. These 2 solutions (A and B) were kept in the dark. About 2 ml of solution A were added to a fluorescence cuveue and after a constant solution temperature was achieved, the
552
J Gonzalez-Jimenez et al
fluorescence output of the instrument was adjusted until a 100 reading was observed. The zero value of ihe instrument was then set by using a solution containing buffer only. The 100 and 0 knobs were alternately adjusted until no more adjustments were required. The buffer solutions were kept to ascertain that there was no drift in instrument response and to readjust the fluorometer if necessary. In practice, the 100 and zero settings did not need any readjusement during a single experiment. Another fluorescence cuvette with = 2 ml of solution A served as the titration cuvette, to which aliquots (usually 10 or 20 l.tl) of the B solution were added. After a constant temperature was achieved, fluorescence was measured and a new aliquot was added. The ! 00 and 0 values were checked every 2 or 3 additions. In this manner, the experiment was performed at a constant HSA concentration in several rain. The absorption spectrum of the titration solution was recorded at the end of the experiment when necessary, the inner filter effect correction was performed as described by Swadesh et al [5]. The shutter allowing light to reach the solution was only opened when reading fluorescence. This precaution prevented significant decomposition from taking place. Titrations were highly reproducible by this experimental procedure. However, each titration was repeated from 4-6 times in an attempt to decrease the experimental uncertainties of the parameters fitting each of the titration curves.
Statistical analysis A weighted non-linear regression program BMDP-AR [6] was used in all computations, although the details are not given here for those cases in which there was a lack of fitting. This program estimates the parameters by a pseudo Gauss-Newton I71 algorithm. When the Cochran statistics (C = S~,/E. S? showed that there was no variance homogeneity a weiglited function was used. S is the estimated variance. This function, when used, was Wi = I/t~2 where t~,2 stands for the variance of the ith observation. The initial parameter values used in the nonlinear regression were estimated by application of linear regression methods to the linearized forms of the mathematical models.
O n e o f our f l u o r e s c e n c e titrations is s h o w n in figure 1, in w h i c h it can be seen that the p r o t e i n fluorescence is almost a b o l i s h e d by the a d d i t i o n o f the a n t i h i s t a m i n i c agent. T h e o r i g i n o f this q u e n c h i n g is not r e l e v a n t to our b i n d i n g e x p e r i m e n t s . H o w e v e r , it can be said that it is not c l e a r l y s e l f - q u e n c h i n g b e c a u s e we u s u a l l y used a b s o r p t i o n s < 0.1, either at e x c i t a t i o n or e m i s s i o n w a v e l e n g t h s . It is n e i t h e r trivial n o r r e s o n a n c e e n e r g y transfer due to the n o n s i g n i f i c a n t o v e r l a p p i n g bet w e e n the H S A e m i s s i o n f l u o r e s c e n c e and the C P M a b s o r p t i o n band. It c o u l d be due to c o n t a c t q u e n c h i n g . A p r o p o r t i o n a l i t y b e t w e e n the drug b o u n d to the protein (PL) a n d the o b s e r v e d c h a n g e in o n e particular e x p e r i m e n t a l p r o p e r t y (the q u e n c h i n g , Q = Fo-F, in this case) is u s u a l l y i m p l i c i t l y a s s u m e d in the b i n d i n g e x p e r i m e n t s . F o r an e q u i l i b r i u m b e t w e e n a protein, P, and a ligand, L: P+L
(1)
551
Chlorpheniramine binding to human serum albumin by fluorescence quenching measurements J Gonzalez-Jimenez, G Frutos, I Cayre, M Cortijo Departamento de Qufmica Fisica Farmacetitica, Facultad de Farmacia, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain
(Received 29 August 1990; accepted 10 January 1991 )
Summary - - The binding of chlorpheniramine to human serum albumin has been studied by fluorescence quenching, as a function of temperature; the experimental data could only be fitted to the Stern-Volmer modified equation. A statistical analysis of the results was performed in order to determine the significance of the constants calculated by this equation, as well as their thermodynamic parameters. The chlorpheniramine binding to human serum albumin accounts for almost half of the binding of this antihistaminic agent to human plasma proteins. chlorpheniramine / antihistaminic / albumin / fluorescence / quenching / binding
Introduction C h l o r p h e n i r a m i n e m a l e a t e ( C P M ) is a potent and h i g h l y e f f e c t i v e a n t i h i s t a m i n i c agent, w i d e l y used to alleviate s y m p t o m s o f the c o m m o n cold and allergic rmm~-t;r~ne I t hme h m ~ n in~hlri~rl l~i..sLJkqk~lklYt..lll:.J. I l k llLJl.t.~ i . , ¢ v ~ k ~ l l l l l ~ l l ~ g ~ J - ~ t J
~ n m~n~; 111 lliCtAkilJ
n hmrr~mr-nlr~eri_ ~llgdJ.lill~*.Jk~Orll,.,g~l
cal preparations during recent years, either as a single active p r i n c i p l e or m i x e d with other drugs. Several p h a r m a c o k i n e t i c studies h a v e b e e n carried out d u r i n g recen! years in a n i m a l s and h u m a n s with C P M ([1-3]; and r e f e r e n c e s quoted therein). It is clear from these studies that C P M r e m a i n s for several hours in p l a s m a , p o s s i b l y b o u n d to s o m e proteins. It is well k n o w n that p l a s m a protein b i n d i n g m i g h t regulate to a certain extent the p h a r m a c o l o g i c a l action o f a drug, a v o i d i n g its b i o t r a n s f o r m a t i o n and acting as a drug reservoir. This b i n d i n g could also a v o i d possible toxic effects due to h i g h drug intake. H u m a n s e r u m a l b u m i n ( H S A ) is a very a b u n d a n t p l a s m a protein. It is therefore a good candidate for C P M binding. This protein strongly binds m a n y hydrop h o b i c substances, but is also able to b i n d m a n y other c h e m i c a l s , a l t h o u g h with l o w e r affinity. Despite the i m p o r t a n c e o f this drug, data on C P M b i n d i n g to isolated p l a s ~ m proteins h a v e not b e e n p u b l i s h e d . In
Abbreviations: CPM, chlorpheniramine maleate; HSA, human serum albumin; ANOVA, analysis of variance
this paper, we d e m o n s t r a t e by fluorescence q u e n c h i n g m e t h o d s that H S A b i n d s C P M in vio'o at temperatures up to 41 °C.
Materials and Methods Chemicals
CMP was generous donated by ISFAS, and used without additional purification. Fatty acid-free HSA was purchased from Sigma. All other chemicals used were of analytical grade. Binding measurements
Stock solutions of HSA (1 mg/ml) and CPM (0.01 M) were prepared in 0.05 M phosphate buffer (pH 7.4), at ionic strength 0.6 by adding NaCI. The actual concentrations of these stock solutions were determined by dilution with the same buffer and by measuring their absorption at 278 nm (HSA) and 261.5 nm (CPM). The molar absorption coefficients used were 3.66 103 [41 and 4.54 103 1 mol -I cm -I (Gonzalez, unpublished observations) for HSA and CPM, respectively. It was checked that both solutions followed the Lambert-Beer law within the range used in this study. A typical binding experiment was performed at a constant protein concentration, as follows: 2 HSA solutions (at I laM concentration) were prepared by dilution of the corresponding stock solution with either: A), buffer; or B), a CPM solution with a final drug concentration (after mixing with HSA) of 0.05 M. These 2 solutions (A and B) were kept in the dark. About 2 ml of solution A were added to a fluorescence cuveue and after a constant solution temperature was achieved, the
552
J Gonzalez-Jimenez et al
fluorescence output of the instrument was adjusted until a 100 reading was observed. The zero value of ihe instrument was then set by using a solution containing buffer only. The 100 and 0 knobs were alternately adjusted until no more adjustments were required. The buffer solutions were kept to ascertain that there was no drift in instrument response and to readjust the fluorometer if necessary. In practice, the 100 and zero settings did not need any readjusement during a single experiment. Another fluorescence cuvette with = 2 ml of solution A served as the titration cuvette, to which aliquots (usually 10 or 20 l.tl) of the B solution were added. After a constant temperature was achieved, fluorescence was measured and a new aliquot was added. The ! 00 and 0 values were checked every 2 or 3 additions. In this manner, the experiment was performed at a constant HSA concentration in several rain. The absorption spectrum of the titration solution was recorded at the end of the experiment when necessary, the inner filter effect correction was performed as described by Swadesh et al [5]. The shutter allowing light to reach the solution was only opened when reading fluorescence. This precaution prevented significant decomposition from taking place. Titrations were highly reproducible by this experimental procedure. However, each titration was repeated from 4-6 times in an attempt to decrease the experimental uncertainties of the parameters fitting each of the titration curves.
Statistical analysis A weighted non-linear regression program BMDP-AR [6] was used in all computations, although the details are not given here for those cases in which there was a lack of fitting. This program estimates the parameters by a pseudo Gauss-Newton I71 algorithm. When the Cochran statistics (C = S~,/E. S? showed that there was no variance homogeneity a weiglited function was used. S is the estimated variance. This function, when used, was Wi = I/t~2 where t~,2 stands for the variance of the ith observation. The initial parameter values used in the nonlinear regression were estimated by application of linear regression methods to the linearized forms of the mathematical models.
O n e o f our f l u o r e s c e n c e titrations is s h o w n in figure 1, in w h i c h it can be seen that the p r o t e i n fluorescence is almost a b o l i s h e d by the a d d i t i o n o f the a n t i h i s t a m i n i c agent. T h e o r i g i n o f this q u e n c h i n g is not r e l e v a n t to our b i n d i n g e x p e r i m e n t s . H o w e v e r , it can be said that it is not c l e a r l y s e l f - q u e n c h i n g b e c a u s e we u s u a l l y used a b s o r p t i o n s < 0.1, either at e x c i t a t i o n or e m i s s i o n w a v e l e n g t h s . It is n e i t h e r trivial n o r r e s o n a n c e e n e r g y transfer due to the n o n s i g n i f i c a n t o v e r l a p p i n g bet w e e n the H S A e m i s s i o n f l u o r e s c e n c e and the C P M a b s o r p t i o n band. It c o u l d be due to c o n t a c t q u e n c h i n g . A p r o p o r t i o n a l i t y b e t w e e n the drug b o u n d to the protein (PL) a n d the o b s e r v e d c h a n g e in o n e particular e x p e r i m e n t a l p r o p e r t y (the q u e n c h i n g , Q = Fo-F, in this case) is u s u a l l y i m p l i c i t l y a s s u m e d in the b i n d i n g e x p e r i m e n t s . F o r an e q u i l i b r i u m b e t w e e n a protein, P, and a ligand, L: P+L
(1)
