720
Biochimica et Biophysica Acta, 428 ( 1 9 7 6 ) 7 2 0 - - 7 2 5 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in The N e t h e r l a n d s
BBA 27876
THE INTERACTION BETWEEN VITAMIN B-12 AND MICELLES IN AQUEOUS SOLUTION
L Y N N S. B E C K M A N N and D E N N I S G. BROWN *
Department of Chemistry, University of Idaho, Moscow, Idaho 83843 (U.S.A.) (Received N o v e m b e r 10th, 1975)
Summary The apparent pK for benzimidazole displacement of a number of cobalamins is markedly affected by the presence of sodium lauryl sulfate micelles. However, micelles of c e t y l t r i m e t h y l a m m o n i u m bromide or Triton X have little or no effect on the pK. By measuring the apparent pK as a function of sodium lauryl sulfate concentration, the association constants between the micelles and both base on and base off methylcobalamin were calculated. This calculation indicates that the base off form is strongly associated with the micelle while the base on form is not.
Introduction The binding of vitamin B-12 and its derivatives to macromolecules is an important process, not only with respect to the binding of B-12 coenzymes to active sites of B-12-dependent enzymes, but also to the binding between B-12 carrier or transport molecules [1--8]. Furthermore, binding of B-12 to proteins such as serum albumin has been studied [9,10]. In an early review Bonnett [11] discussed the various modes by which B-12 might bind to a macromolecule. Possible ways in which this binding could occur include cobalichrome formation, displacement of the benzimidazole nucleotide so that specific coordination to a functional group on the protein could occur, and hydrogen bonding involving the amide groups on the corrin ring. Hill et al. [12] have studied the binding of biologically important molecules to B-12 and its derivatives and concluded that formation of hydrogen bonds should be more important in forming complexes with proteins than specific coordination to the metal. There has been considerable work reported aimed at deducing specific sites at which B-12 binds to a protein. Even for serum albumin, a protein to which * Author t o w h o m c o r r e s p o n d e n c e s h o u l d b e a d d r e s s e d .
721
B-12 binds but for which there may be no B-12-dependent biological function, some work has been reported indicating a certain specificity for B-12 binding [9,10]. One of the reasons that most studies of B-12 binding seem to imply a specific site for the binding is u n d o u b t e d l y t h a t there are few ways to assess other non-specific forces which could contribute to formation of a complex between B-12 and a macromolecule. For example, the following type of question has n o t been addressed. Does B-12 exhibit a preference for a negatively charged surface or positively charged one? Does the charge configuration on the surface of a macromolecule even affect the B-12 binding tendency? We have been interested in this non-specific type of interaction and have been investigating possible B-12 binding to micelle surfaces as a model for the binding of B-12 to a macromolecule. In this article the interaction of B-12 with three types of aqueous micelles is discussed. The effects of surfactants on certain ligand exchange processes in aquocobalamin have recently been reported by Fendler et al. [13]. As these workers have pointed out previously, vitamin B-12 derivatives which are highly soluble in water and virtually insoluble in hydrocarbon solvents, would certainly not be solubilized in the interior of an aqueous micelle. Nevertheless, a substantial interaction with the micelle surface may still be possible. The cobalamin derivatives of vitamin B-12 all contain a 5,6-dimethylbenzimidazole ligand in the sixth axial coordination position. At normal pH values this ligand is coordinated to the central cobalt but it is displaced by a water molecule at low pH. The base on-base off equilibrium depicted in Eqn. 1 has been studied in detail in aqueous solution. The pK for this process depends L
L
N••
+ H+ " " ~ ~
(1)
upon the nature of the specific cobalamin, but is is particularly sensitive to the trans axial ligand. For methylcobalamin the pK has been reported to be 2.7 in water [14,15]. The effect of aqueous micelles on this equilibrium was determined by measuring the apparent pK in the presence of the anionic surfactant sodium lauryl sulfate, the cationia surfactant c e t y l t r i m e t h y l a m m o n i u m bromide, and the neutral detergent Triton X. The results indicate that vitamin B-12 derivatives have a marked affinity for the surface of the anionic micelle, sodium lauryl sulfate. However, there is very little interaction with the neutral or cationic micelles. Analysis of the variation in apparent pK with sodium lauryl sulfate concentration indicates that the protonated base off form of the cobalamin is largely responsible for binding to the sodium lauryl sulfate surface. Materials and Methods
Vitamin B-12 was purchased from Sigma Chemical Co. Methylcobalamin was prepared by a published procedure [16] and purified by column chromatog-
722 raphy on cellulose phosphate [17]. Aquocobalamin was prepared by exhaustive aerobic photolysis of pure methylcobalamin. Sodium lauryl sulfate and c e t y l t r i m e t h y l a m m o n i u m bromide were obtained from Eastman Chemical Co., Triton X from Calbiochem. All spectral measurements were performed on a Beckman DB-G spectrometer. pH measurements were made on a Coming Model 12 pH meter. The pK values for benzimidazole displacement were measured spectrophotometrically by monitoring the change in absorbance at 560 nm as a function of pH. The temperature of the samples was maintained at 25 ° C. In all determinations the concentration of the cobalamin derivative was 2.5 • 10 -4 M. Results Fig. 1 demonstrates the striking change in the apparent pK for benzimidazole displacement in methylcobalamin between pure water and a solution containing godium lauryl sulfate micelles. The change in apparent pK depends on the presence of micelles, n o t monomers of the surfactant. At sodium lauryl sulfate concentrations significantly below the critical micelle concentration, there is no change in the apparent pK from t h a t f o u n d in pure water. Similar determinations in apparent pK values have been made for methylcobalamin, cyanocobalamin, and aquocobalamin in the presence of micelles of sodium lauryl sulfate, c e t y l t r i m e t h y l a m m o n i u m bromide, and Triton X. The results of these determinations are summarized in Table I. In order to more thoroughly investigate the methylcobalamin-sodium lauryl sulfate system, the apparent pK values for benzimidazole in methylcobalamin were determined as a function of sodium lauryl sulfate concentration. That is, the serie of pH and absorbance measurements shown in Fig. 1 were carried out for several sodium lauryl sulfate concentrations ranging from below to well above the critical micelle concentration for sodium lauryl sulfate. Fig. 2 shows the change in the apparent pK as a function of surfactant concentration. The data shown in Fig. 2 can be analyzed to determine association constants for vitamin B-12 binding to the micelle.
oo
2.63 -
2
5.73 3
4
plf
~
6
7
-
Fig. 1. Plots o f p e r c e n t t r a n s m i s s i o n ( 5 6 0 n m ) vs. p H f o r m e t h y l c o b a l a m i n in w a t e r ( c u r v e a) a n d f o r m e t h y l c o b a l a m i n in t h e p r e s e n c e o f s o d i u m l a u r y l s u l f a t e m i c e l l e s ( c u r v e b). T h e a p p a r e n t p K v a l u e s f o r b e n z i m i d a z o l e d i s p l a c e m e n t are d e t e r m i n e d f r o m t h e i n f l e c t i o n p o i n t s o f t h e c u r v e s a n d a r e f o u n d t o be 2.63 and 5.73. respectively.
723
TABLE I APPARENT pK VALUES FOR BENZIMIDAZOLE PRESENCE OF A Q U E O U S MICELLES
DISPLACEMENT FOR
C O B A L A M I N S IN T H E
Cobalamin
H20
SLS a
CTAB b
Triton-X c
Methylcobalamin Cyanocobalamin Aquocobalamin
2.63 d --~0
Biochimica et Biophysica Acta, 428 ( 1 9 7 6 ) 7 2 0 - - 7 2 5 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in The N e t h e r l a n d s
BBA 27876
THE INTERACTION BETWEEN VITAMIN B-12 AND MICELLES IN AQUEOUS SOLUTION
L Y N N S. B E C K M A N N and D E N N I S G. BROWN *
Department of Chemistry, University of Idaho, Moscow, Idaho 83843 (U.S.A.) (Received N o v e m b e r 10th, 1975)
Summary The apparent pK for benzimidazole displacement of a number of cobalamins is markedly affected by the presence of sodium lauryl sulfate micelles. However, micelles of c e t y l t r i m e t h y l a m m o n i u m bromide or Triton X have little or no effect on the pK. By measuring the apparent pK as a function of sodium lauryl sulfate concentration, the association constants between the micelles and both base on and base off methylcobalamin were calculated. This calculation indicates that the base off form is strongly associated with the micelle while the base on form is not.
Introduction The binding of vitamin B-12 and its derivatives to macromolecules is an important process, not only with respect to the binding of B-12 coenzymes to active sites of B-12-dependent enzymes, but also to the binding between B-12 carrier or transport molecules [1--8]. Furthermore, binding of B-12 to proteins such as serum albumin has been studied [9,10]. In an early review Bonnett [11] discussed the various modes by which B-12 might bind to a macromolecule. Possible ways in which this binding could occur include cobalichrome formation, displacement of the benzimidazole nucleotide so that specific coordination to a functional group on the protein could occur, and hydrogen bonding involving the amide groups on the corrin ring. Hill et al. [12] have studied the binding of biologically important molecules to B-12 and its derivatives and concluded that formation of hydrogen bonds should be more important in forming complexes with proteins than specific coordination to the metal. There has been considerable work reported aimed at deducing specific sites at which B-12 binds to a protein. Even for serum albumin, a protein to which * Author t o w h o m c o r r e s p o n d e n c e s h o u l d b e a d d r e s s e d .
721
B-12 binds but for which there may be no B-12-dependent biological function, some work has been reported indicating a certain specificity for B-12 binding [9,10]. One of the reasons that most studies of B-12 binding seem to imply a specific site for the binding is u n d o u b t e d l y t h a t there are few ways to assess other non-specific forces which could contribute to formation of a complex between B-12 and a macromolecule. For example, the following type of question has n o t been addressed. Does B-12 exhibit a preference for a negatively charged surface or positively charged one? Does the charge configuration on the surface of a macromolecule even affect the B-12 binding tendency? We have been interested in this non-specific type of interaction and have been investigating possible B-12 binding to micelle surfaces as a model for the binding of B-12 to a macromolecule. In this article the interaction of B-12 with three types of aqueous micelles is discussed. The effects of surfactants on certain ligand exchange processes in aquocobalamin have recently been reported by Fendler et al. [13]. As these workers have pointed out previously, vitamin B-12 derivatives which are highly soluble in water and virtually insoluble in hydrocarbon solvents, would certainly not be solubilized in the interior of an aqueous micelle. Nevertheless, a substantial interaction with the micelle surface may still be possible. The cobalamin derivatives of vitamin B-12 all contain a 5,6-dimethylbenzimidazole ligand in the sixth axial coordination position. At normal pH values this ligand is coordinated to the central cobalt but it is displaced by a water molecule at low pH. The base on-base off equilibrium depicted in Eqn. 1 has been studied in detail in aqueous solution. The pK for this process depends L
L
N••
+ H+ " " ~ ~
(1)
upon the nature of the specific cobalamin, but is is particularly sensitive to the trans axial ligand. For methylcobalamin the pK has been reported to be 2.7 in water [14,15]. The effect of aqueous micelles on this equilibrium was determined by measuring the apparent pK in the presence of the anionic surfactant sodium lauryl sulfate, the cationia surfactant c e t y l t r i m e t h y l a m m o n i u m bromide, and the neutral detergent Triton X. The results indicate that vitamin B-12 derivatives have a marked affinity for the surface of the anionic micelle, sodium lauryl sulfate. However, there is very little interaction with the neutral or cationic micelles. Analysis of the variation in apparent pK with sodium lauryl sulfate concentration indicates that the protonated base off form of the cobalamin is largely responsible for binding to the sodium lauryl sulfate surface. Materials and Methods
Vitamin B-12 was purchased from Sigma Chemical Co. Methylcobalamin was prepared by a published procedure [16] and purified by column chromatog-
722 raphy on cellulose phosphate [17]. Aquocobalamin was prepared by exhaustive aerobic photolysis of pure methylcobalamin. Sodium lauryl sulfate and c e t y l t r i m e t h y l a m m o n i u m bromide were obtained from Eastman Chemical Co., Triton X from Calbiochem. All spectral measurements were performed on a Beckman DB-G spectrometer. pH measurements were made on a Coming Model 12 pH meter. The pK values for benzimidazole displacement were measured spectrophotometrically by monitoring the change in absorbance at 560 nm as a function of pH. The temperature of the samples was maintained at 25 ° C. In all determinations the concentration of the cobalamin derivative was 2.5 • 10 -4 M. Results Fig. 1 demonstrates the striking change in the apparent pK for benzimidazole displacement in methylcobalamin between pure water and a solution containing godium lauryl sulfate micelles. The change in apparent pK depends on the presence of micelles, n o t monomers of the surfactant. At sodium lauryl sulfate concentrations significantly below the critical micelle concentration, there is no change in the apparent pK from t h a t f o u n d in pure water. Similar determinations in apparent pK values have been made for methylcobalamin, cyanocobalamin, and aquocobalamin in the presence of micelles of sodium lauryl sulfate, c e t y l t r i m e t h y l a m m o n i u m bromide, and Triton X. The results of these determinations are summarized in Table I. In order to more thoroughly investigate the methylcobalamin-sodium lauryl sulfate system, the apparent pK values for benzimidazole in methylcobalamin were determined as a function of sodium lauryl sulfate concentration. That is, the serie of pH and absorbance measurements shown in Fig. 1 were carried out for several sodium lauryl sulfate concentrations ranging from below to well above the critical micelle concentration for sodium lauryl sulfate. Fig. 2 shows the change in the apparent pK as a function of surfactant concentration. The data shown in Fig. 2 can be analyzed to determine association constants for vitamin B-12 binding to the micelle.
oo
2.63 -
2
5.73 3
4
plf
~
6
7
-
Fig. 1. Plots o f p e r c e n t t r a n s m i s s i o n ( 5 6 0 n m ) vs. p H f o r m e t h y l c o b a l a m i n in w a t e r ( c u r v e a) a n d f o r m e t h y l c o b a l a m i n in t h e p r e s e n c e o f s o d i u m l a u r y l s u l f a t e m i c e l l e s ( c u r v e b). T h e a p p a r e n t p K v a l u e s f o r b e n z i m i d a z o l e d i s p l a c e m e n t are d e t e r m i n e d f r o m t h e i n f l e c t i o n p o i n t s o f t h e c u r v e s a n d a r e f o u n d t o be 2.63 and 5.73. respectively.
723
TABLE I APPARENT pK VALUES FOR BENZIMIDAZOLE PRESENCE OF A Q U E O U S MICELLES
DISPLACEMENT FOR
C O B A L A M I N S IN T H E
Cobalamin
H20
SLS a
CTAB b
Triton-X c
Methylcobalamin Cyanocobalamin Aquocobalamin
2.63 d --~0
