Bioelectrochemistry: Ions, Surfaces, Membranes - American Chemical

17 Increased Ionization of Catecholamines in the Presence of Imidazole and Related Compounds

Downloaded by UNIV OF SYDNEY on May 3, 2015 | http://pubs.acs.org Publication Date: June 1, 1980 | doi: 10.1021/ba-1980-0188.ch017

M . B. A B R A M S O N and M . C H O I The Saul R. Korey Department of Neurology, Albert Einstein College of Medicine, Bronx, NY 10461

Spectrophotometric measurements of dopamine and norepinephrine show that at physiological pH the presence of imidazole or related proton acceptors causes an increase in the ionization of the phenolic OH. The absorbance at 295 nm permits an estimation of the concentration of the ionized species. While the pK ' of norepinephrine in 0.1M sodium phosphate is 8.9, in 0.1M imidazole, histidine, or tris buffer at pH 7.4, it is 8.6, 8.7, or 8.4, respectively. Increasing the concentration of imidazole or tris causes greater lowering of the pK '. The monophenolic compounds, normetanephrine, or tyramine did not show this increased ionization except at pH levels > 9. Using codispersions of imidazole in egg lecithin in phosphate buffer with a concentration of 3mM imidazole, the pK ' of norepinephrine was 8.6. This increased ionization may be important in some biological systems. a

a

a

T t has been assumed that the active molecular form of the catecholamines which is involved i n various processes i n the nervous system is the cation. This view is based upon the fact that at physiological p H about 97% is present as the cation, since the p K of the first phenolic hydroxyl is roughly 8.8 and remains essentially unionized. I n a recent study i n this laboratory (1), the uptake of norepinephrine by synaptosomes from rat brain increased with p H up to approximately p H 8.2, and presumably the active species was the zwitterion form of the molecule w i t h negative phenolate and positive amine. This chapter describes experiments which show that at a constant p H , the ionization of the phenolic group of the

A

0-8412-0473-X/80/33-188-273$05.00/l © 1980 American Chemical Society

In Bioelectrochemistry: Ions, Surfaces, Membranes; Blank, M.; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.


274

BIOELECTROCHEMISTRY:

IONS, SURFACES, M E M B R A N E S

catecholamine can be increased as a result of the interaction with certain N bases i n the medium. This presents the view that i n some actions of the catecholamines i n the nervous system, a reaction may occur with a specific agent such as histidine i n the medium or in the membrane surface at the uptake or receptor sites which could then alter the charged state of the catecholamine. The different behavior observed for the catecholamines and such related compounds as tyramine, octopamine, or normetanephrine could result from the inability of these latter compounds to form a zwitterion by ionization of a hydroxyl group at neutral p H . I n another area of interest, the active site of some hydrolytic enzymes is observed to include a histidine residue ( 2 ) . It is possible that the imidazole functions by catalyzing a proton transfer in the enzyme-substrate complex. In order to relate the reactions we describe to biological phenomena, we include some experiments i n which imidazole or some related compound is incorporated with egg lecithin and then dispersed i n water forming multilamellar structures. This provides a system possessing some of the characteristics of the biological membrane. Experimental Materials. Dopamine, L-norepinephrine, and normetanephrine were from Regis C o . ; for spectroscopic measurements solutions of these compounds were prepared daily at concentrations of either 1 X 10~ M or 2 X 10" M i n 0.01M H C l . Tris(hydroxymethyl)aminomethane base was from Fisher Scientific; imidazole was from Eastman Chemical; histamine was from Sigma, and L-histidine was from Calbiochem. E g g lecithin from Sylvana C o . was used for lipid preparations. I n preparing die buffer media, solutions of tris base or N a H P 0 were brought to the desired p H with H C l . Solutions of O.lOAf imidazole, histamine, or histidine were prepared i n a similar manner. A l l p H measurements were made with either a Corning M o d e l 12 or Orion 601A p H meter using a Thomas or Fisher combined glass and reference electrode. The water used was deionized, distilled, and redistilled from Pyrex. Lipsomes. L i p i d codispersions were made by dissolving a weighed amount of imidazole or related compound i n absolute ethanol, adding lecithin i n alcohol and mixing. The solvent was removed by a stream of nitrogen followed b y several hours i n vacuum. The required volume of water was added and the tube was shaken until the l i p i d was freed from the glass surface. A clear dispersion then was formed by exposure to ultrasonics using either an M S E or Branson sonifier. I n most instances, a 2-10 min exposure at 1-min intervals sufficed to reduce the turbidity to low levels. Aliquots of this system were added to O.lOAf sodium phosphate to give phosphate concentrations from 0.025-0.05M and the desired p H was established by the addition of H C l or N a O H . Spectroscopic Measurements. Most of the spectra reported here were obtained using a P e r k i n - E l m e r N o . 576 spectrophotometer equipped with a memory unit for the storing and correcting of a nonhorizontal 3

3

2

4



17.

A B R A M S O N A N D CHOI

275

Catecholamines

baseline. The cell compartment was temperature controlled and a l l spectra were obtained at 25°C. Additional spectra were obtained using a Cary Model 308 spectrophotometer. In obtaining the spectra, 3 m L of the buffer or test solution was added to each of two matched 10-mm quartz cuvets and the baseline was scanned from 320-260 n m and stored in the memory unit. Whenever the baseline departed significantly from the horizontal, the samples were discarded. 0.01M H C l (30 / i L ) was added to the solution in the reference cell and 30 pL of either l - 2 m M dopamine or norepinephrine in 0.01M H C l was added to the sample cuvet and rapidly stirred. The spectra were obtained within 2 min after mixing, and repeated to detect whether there were any changes resulting from oxidation. Spectroscopic studies of the dispersed lipids were run with cuvets in the compartment for turbid samples. E q u a l 3-mL portions of the codispersion in phosphate buffer were added to two matched cuvets and the baseline spectrum was obtained and stored i n the memory unit of the instrument. Whenever the baseline showed differences between the contents of the two cuvets, small changes in the concentrations were made until a satisfactory baseline was obtained. W h e n this could not be done, new samples were taken. The spectra then were obtained as before. Results Spectral Changes. The absorption spectra of aqueous solutions of dopamine and norepinephrine show prominent bands in the regions of 280 and 240 nm. In this study, our attention was directed only to hte changes i n the longer wavelength band. A t p H levels above 7, the maximum of this band shifts to the red and a shoulder increases i n intensity i n the 295-nm region (see Figure 1). A t p H 10, the maximum is at 295 n m (3). These changes are associated with the deprotonation of one phenolic O H with increasing p H . Above p H 10, the second phenolic O H is deprotonated and additional changes are observed i n the spectra. W h e n the p H is above 8, the spectra are complicated further by the formation of oxidation products w i t h absorbances i n the 300-nm region. Using 0.10M sodium phosphate buffer systems with 2 X 10" M norepinephrine or dopamine at p H 7.4, the maximum is at 279.6. However, this differs i n O.IOM tris buffer at p H 7.4, as these compounds then give spectra with the maximum at 281 nm and increased absorbance at approximately 295 n m (see Figure 2, Curves 1 and 2). These spectral shifts to the red are similar to those Observed with phosphate buffers at a somewhat higher p H although both systems are at the same p H . W i t h the increasing p H of the tris buffer, there is a further increase i n this red shift. This change in spectra was dependent upon the concentration of tris as shown by the following experiment. Several solutions of tris were prepared with concentrations ranging from 0.02M-0.10M, all at p H 7.4. The ionic strength was maintained at 0.10 by the addition of N a C l . The shift i n the wavelength of the maxi5



276

240 250



260 270 280 290 300

310 320 nm

Figure 1. Effect of pH on spectra of 2 X 10~ M norepinephrine. Spectra 1, 2, and 3 are in 0.10M sodium phosphate at pH: 1, 6.60; 2, 7.45; 3, 8.27. Spectra 2A and 3A are in 0.10M imidazole at pH: 2A, 7.41; 3A, 8.23. Note the shift in maximum to longer wavelength and the increased absorbance in the region of 295 nm with increased pH in the imidazole solutions. Spectra are displaced vertically for clarity. 5

240 250 260 270

280 290 300

310 320 nm

Figure 2. Spectra of 2 X 10 M norepinephrine at pH 7.4 in 0.10M solutions of I , sodium phosphate; 2, tris; 3, imidazole; 4, histamine; and 5, histidine. The change in wavelength of maximum and the increased absorbance at 295 nm in Solutions 2-5 compared with 1 can be seen. 5



17.

A B R A M S O N A N D CHOI

277

Catecholamines

mum and the absorbance near 295 nm increased with the tris concentrations. Similar changes were observed with mixed buffers prepared with tris and sodium phosphate at p H 7.4 with total buffer concentration equal to O.IOM. To determine whether these spectral changes resulted from oxidation, a number of spectra were obtained in the absence of oxygen. Using a Thurnberg cell with the buffer medium in the main portion of the cell and the acidified solution of catecholamine in the side arm, the solutions were deaerated by a stream of argon and the catecholamine solution was added and mixed under the argon atmosphere. Spectra obtained in this way showed the same characteristics as those obtained immediately after mixing in the normal atmosphere. Other experiments were performed to determine whether oxidation was involved i n the observed changes. In numerous instances, the buffer solutions with either tris or phosphate were prepared with added O . l m M N a E D T A and I m M sodium metabisulfite to minimize the oxidation of the catecholamine. N o difference was observed in the spectra taken with or without these added antioxidants. To further monitor the importance of oxidation during the course of these studies, the spectra were scanned immediately after adding the acidified catecholamine to the buffer and were completed within 2 min after mixing. The spectrum then was repeated and the absence of a change in the 280-295-nm region indicated negligible oxidation. 2

These studies were extended to other biologically relevant bases by using solutions of imidazole and some of its derivatives as the medium in place of the tris buffer. Spectra in imidazole resembled those obtained in tris at the same concentration and p H . Here, however, the displacement of the maximum towards the red and the broadening of the band on the long wavelength side were somewhat less than was seen with tris. Histamine and histidine also showed similar but decreased effects (see Figure 2 ) . Although the results reported here deal chiefly with norepinephrine, numerous experiments with dopamine showed similar effects. To obtain a better understanding of the interaction of tris or imidazole with the catecholamines, we studied the spectra of tyramine or normetanephrine in these systems. Tyramine and normetanephrine possess a single phenolic hydroxyl with higher p K values than that of the first phenolic O H i n the catecholamines. A t p H 7.4, the spectra of normetanephrine in 0.1M solutions of sodium phosphate, tris, imidazole, or histamine are alike with maxima at 278 nm and a weak shoulder i n the 285-nm region (see Figure 3, Curves 1 and 1 A ) . However, i n 0.1M tris at p H 8.4 and higher, normetanephrine shows increased intensity of the shoulder at 284 and 295 nm (see Curves 2 and 2 A ) . Larger differences are evident at p H 9.4 (see Curves 3 and 3 A ) . To study the effect of N bases when present on a membrane surface, we incorporated some of these compounds with egg lecithin into a multilamellar dispersed system. Figure 4 shows spectra of 2 X 10" norea

5



278


240

250

260

270

280

290


300

310

320 nm

Figure 3. Spectra of 1 X 10' M normetanephrine in either: I, 2, and 3; 0.10M tris; or I A, 2A, and 3A; 0.1 M sodium phosphate, at I, I A, pH 7.40; 2, 2A, pH 8.4; 3, 3A, pH 9.4. Significant changes between the spectra in the two buffer systems are evident only at high pH. 5

ii

I

240 250

I

I

I

I

260 270 280 290

I

I

300

I

L_

310 320 nm

Figure 4. Effect of either imidazole or histamine in codispersions with egg lecithin on the spectra of 2 X 10' M norepinephrine: (1) dispersion of 1.2 mg/mL lecithin and imidazole to give a concentration of 3.4mM imidazole in 0.025M sodium phosphate at pH 7.74; (2) dispersion of 1.4 mg/mL lecithin with histamine to give a concentration of 1.9mM histamine in 0.025M sodium phosphate at pH 7.80; and (2A) 0.025M sodium phosphate alone at pH 7.80. 5



17.

ABRAMSON A N D CHOI

Catecholamines

279

pinephrine in 0.025M sodium phosphate at p H 7.75. In Curve 1, a codispersion of egg lecithin and imidazole is added. The solution then contained 1.2 m g / m L lecithin and 3.4mM imidazole. Curve 2 depicts the spectrum in a codispersion of 1.4 m g / m L lecithin and 1.9mM histamine. Curve 2A is for 0.025M sodium phosphate without lipid dispersion. The shift in the wavelength of the maximum and the increased absorbance at 295 nm in Curves 1 and 2 compared with 2A can be noted. In another experiment in which a lecithin dispersion not containing any of the N bases was used, there was no change i n the spectrum of norepinephrine. In still another experiment, an attempt was made to incorporate tris into lecithin for codispersion, without success. Nevertheless, a dispersion of lecithin with 3 m M tris was prepared. This d i d not alter the spectrum of norepinephrine in 0.025M sodium phosphate at p H 7.4. Changes in pK . W e interpret these changes in the spectra of the catecholamines i n the presence of tris and other N bases studied as the result of an effective decrease in their p K values i n these media. These apparent p K ' values were estimated by the following procedure. A t p H 6.6, the catecholamines can be considered to be fully protonated, and the absorbance at 280 nm gives a measure of the concentration of the cationic species present. W e calculated the absorbance at 295 n m of the same concentration of catecholamine solution in the zwitterion form by using the observed absorbance at 280 nm at p H 6.6 multiplied by the ratio of the extinction coefficients for the zwitterion at 295 nm to the cation at 280 nm as given by Kappe and Armstrong (3). Using the same stock solution of acidified catecholamine and the same final concentration of catecholamine in all experiments in a series, we measured the absorbance at 295 nm under different conditions. This absorbance includes contributions from both forms of the catecholamines and therefore needed correction for the small absorbance of the unionized species at 295 nm. T h e absorbance at 295 nm (A os) of a sample gave the fraction a present i n the zwitterion form according to the following a

a

0

2

A

2 9 5

— (1 - a) B + «C

where B is the absorbance at 295 nm of the fully protonated form and C is for the zwitterion alone. The p K ' was obtained in the customary manner

P

K ' = pH +

log^^ a

The p K / values obtained in this way in solutions of tris, imidazole, histamine, and histidine are shown at several p H levels in Table I. Tris produces the greatest decrease of the pK

Bioelectrochemistry: Ions, Surfaces, Membranes - American Chemical

Recommend Documents