Transport of amino acids through liquid membranes III. The alkaline ion role

journal of MEMBRANE SCIENCE ELSEVIER

Journal of Membrane Science 133 (1997) 127-131

Transport of amino acids through liquid membranes III. The alkaline ion role C.V. Uglea*, Mariana Croitoru Institute of Biological Research, Blvd. Copou 20A, lassy, Romania Received 11 July 1996; received in revised form 11 February 1997; accepted 25 March 1997

Abstract This paper discusses the alkaline ion (Na÷) role in the uphill transport of amino acids through a bulk liquid membrane. The aqueous phases (source phase - S and receiving phase - R) are made up of equimolar concentrations of amino acid (4.38 mM p-aminobenzoic acid (PABA)) and alkaline ion (75 mM Na+). A chloroform solution containing 5 mM dibenzo-18-crown-6 (DB18C6) represents the bulk liquid membrane (M). The data obtained show that at the S-M interphase, the amino acid is coupled with the carrier via the H3N+ group rather than being transported to the R-M interphase, where Na + substitutes the amino acid. If Na + is absent, the amino acid is transported to the opposite direction. These results support the hypothesis that the presence of Na÷ ion in the aqueous phases assures the 'biological' direction of aminobenzoic acids transport through membranes.

Keywords: Coupled facilitated transport; Facilitated transport; Liquid membranes; Biomembrane analogues

1. Introduction Participation of amino acids in a great variety of metabolic processes makes them some of the most studied compounds, especially regarding their transport through biological membranes. It is well known that the permeation of these compounds through biological membranes requires the involvement of a carrier capable of assuring their liposolubility [1]. For a better understanding of amino acids' transport through biological membranes, it is necessary to build

*Corresponding author. Fax: +40 32 147202 0376-7388/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0376-7388(97)00079-3

a model system capable of demonstrating some aspects that refer to the mechanism of transport. Bulk liquid membrane and synthetic carriers [2] provide an adequate experimental model employed in the study of amino acids' transmembrane transport. The aim of this paper is to evaluate the role of the alkaline ion (Na +) in the uphill transport of p-aminobenzoic acids (PABA) through organic liquid membrane using DB18C6 as a carrier. Selection of the p-aminobenzoic acid has been determined both by its biological significance and by the data we obtained on the intensification of the antitumoral activity of some anionic polymers (copolymers of maleic anhydride, carboxymethylcellulose) modified with amino acids (PABA) [3,4].

128

C.V Uglea, M. Croitoru/Journal of Membrane Science 133 (1997) 127-131

2. Experimental

\'

,/

/.

2.1. Materials Chloroform was used as an organic liquid membrane. The following reagent grade chemicals: PABA; ethyl-p-aminobenzoate (anesthesine or benzocaine); HC1; NaCI; NaOH; dibenzo-18crown-6 (DB18C6); brom; pyridine; potassium thiocyanate and acetic acid were obtained from Sigma-Aldrich.

"~--_---"-2----"-"-_

•i'i ~i~!~)':i~.

- ---'---- 2 - - - - " - - - "

- " --- ---

1. Type "V" glass vetsel (r~2 era) 2. Temperature control balh (25' C) J. Magnetic stirrer (1 $0 rpm) $ - Source aqueous phase (~0 mL) R - Receiving aqueous phase (20 mL) M - Organic liquid phase O 0 mL)

2.2. Procedure

Fig. 1. Liquid membrane device.

A schematic representation of the device employed for experiments is shown in Fig. 1. The composition of source and receiving phases, for the three different experiments that were performed are given in Table 1. The organic phase contains 5 mM of DB 18C6 in chloroform for all the three experiments. Variation of the aqueous phases' compositions during the transport experiments was determined both by spectrometry (PABA and benzocaine) and by flame photometry (Na+). 100 ~tl PABA (benzocaine)-containing sample was diluted with 2 ml distilled water, then 1 ml solution of pyridine (10%) and 2 ml reactive saturated solution of brom, treated up to decolouration with 10% potassium thiocyanate, were added.

After 5 min, 2 ml of glacial acetic acid have been added, then the samples were left still for 75 min, and the absorbtion at 410 nm was read using a Spekol 10 (Carl Zeiss Jena); a sample of distilled water, treated in the same manner as the samples, was employed as a reference. Evaluation of the Na + content involved taking over of 1 ml samples from the aqueous phases. The ionic Na + was determined by the classical method using a Carl Zeiss Flammenphotometer (Model III). Thus, the values of PABA, benzocaine and Na + concentration in aqueous phases were determined; the pH values for the aqueous phases were measured with a Piccolo pH-meter (by Hanna, HI 1280). The results obtained are presented in Tables 2-4.

Table 1 Composition of the aqueous source and receiving phases Substances

Aqueous source phase (S) (mM)

Aqueous receiving phase (R) (mM)

a

PABA NaC1 HC1 NaOH

4.37 75 100 --

4.37 --75

b

Benzocaine NaC1 HCI NaOH

4.37 75 100

4.37 --75

PABA HC1

4.37 100

c

--

4.37 --

129

C.V Uglea, M. Croitoru/Journal of Membrane Science 133 (1997) 127-131 Table 2 Characteristics of aqueous phases during the uphill transport of PABA with Na +, Experiment 1 Composition of aqueous phases Time (h) 0 24 48

Receiving (R)

Source (S) pH

PABA (mM)

Na+ (mM)

pH

PABA (mM)

Na + (mM)

2.15 2.23 2.14

4.38 3.58 2.25

74 -84

11.86 11.20 10.02

4.38 5.40 6.34

74 -64

Table 3 Characteristics of aqueous phases during the substitution of benzocaine for PABA, Experiment 2 Composition of aqueous phases Time (h)

0 24 48

Source (S)

Receiving (R)

pH

Benzocaine (mM)

pH

Benzocaine (mM)

1.85 1.79 1.78

4.36 0.27 0.00

11.80 8.92 6.73

4.36 4.51 4.36

Table 4 Characteristics of aqueous phases during transport of PABA in the reverse direction, without Na+, Experiment 3 Composition of aqueous phases Time (h)

0 24 48

Source (S)

Receiving (R)

pH

PABA (mM)

pH

PABA (mM)

2.38 2.47 2.60

4.38 5.76 5.98

3.78 3.76 3.55

4.38 1.87 1.50

3. Results and discussion The results for the first experiment showing the transport of PABA are listed in Table 2. These results permit the evaluation of the PABA transport mechanism through the liquid membrane according to the scheme plotted in Fig. 2. At the S - M interface one may observe the formation of Complex I (Fig. 2), the structure of which is based on the amino acid's penetration inside the crown ether, as induced by the interactions between the carrier's negatively charged cavity and the H O O C C6H4-H3 N+ positive ion. Mention should be made here of the fact that in the region of contact between

the two phases (S and M), the existing conditions favour the formation of hydrogen bonds between the oxygen atoms from the crown ether and the hydrogen atoms of the H3N+--C6H4-COOH species. Thus, even if in phase M the crown ether is a neutral molecule, at interface, it may play the role of an opposite ion, versus the H3N+-C6H4-COOH species. Complex I represents the molecular species that assures the amino acid's transport between the S and the R phase. At the M - R interphase, the amino acid is substituted by the Na + ion, thus forming Complex II which transports the Na ÷ ion from the R towards the S phase.

130

C.V. Uglea, M. Croitoru/Journal of Membrane Science 133 (1997) 127-131

.~otts

source ~

Orl~uc mm'n~le

Aquemmrece~m8pl~se

~

the experiments where the aqueous phases had benzocaine are presented in Table 3. It is generally known that the esters of amino acids are stronger bases than their amino acid counterparts [6-8]. Table 3 shows a decrease of benzocaine concentration in S phase, while the R phase's concentration remains essentially constant. The pH of R phase decreases. The experiment may be represented by the following reactions:

I

ltooc-

C~

l~-e~l¢-c'oorl + N*OH

-~lt~

I I I I I ~ I

I I I I I --,,wry-

I

,

I

C- + HaN +-C6H4-COOC2H5 ~ C - q- H +

Fig. 2. Schematicrepresentationof amino acid transportmechanism.

+H2N-C6H4-COOC2H5 (at interface S - M )

(7)

(C- H3N+) - C6H4-COOC2H5 The above-described mechanism may be represented by the following reactions: C - ÷ H3N+-C6H4-COOH --~ (C-H3N+)-C6H4-COOH

(at interface S - M ) (1)

( C - H a N + ) - C 6 H a - C O O H ~ (C-Na+)+ HaN+-C6H4-COOH

(at interface M - R )

(2)

C1- ÷ N + H 3 - C 6 H 4 - C O O H q- (C-Na +) (C-H3N +) - C 6 H 4 - C O O H + NaC1 (at interface M - S )

(3)

where C=DB18C6 The fact that amino acids' transport is not quantitatively equivalent to the Na + transport (expressed in moles) may be explained by the existence of a coupled transport mechanism between H + and Na + [5]. Consequently, the following series of reactions may be assumed: C- + H + ~ (C-H +)

(at interface S - M )

(4)

NaOH + (C-H +) ~ (C-Na +) ÷ H20 (at interface M - R )

(5)

C1-H + + (C-Na +) --* (C-H +) + NaC1 (at interface M - S )

(6)

Formation of Complex I at the S-M interphase (Fig. 2) is supported indirectly by the data obtained from the second experiment in which we keep the same aqueous phases contents only PABA being substituted with benzocaine (Table lb). The results for

(C-H3N +)-C6H4-COOC2H5 and H + + OH- ~ H20

(at interface R - M )

(8) (9)

Due to its solubility in water, benzocaine remains in the organic phase. Consequently, benzocaine may be extracted from the acidic condition via the H3N+ group. The role of Na + in the amino acids' transport is still to be discussed. Is the presence ofNa + compulsory for this transport? The answer to such a question may be found from the data obtained in a third experiment in which the Na + ion is absent in the composition of the aqueous phases (Table lc). The above mentioned data are listed in Table 4. They show that in the absence of Na + ion, the PABA transport occurs in an opposite direction to that observed in Experiment 1. The decrease of pH in phase R and increase of PABA concentration in phase S show that the H + is transported towards the R phase, while the amino acid is transported back towards the S phase. One may observe that the transfer of amino acid through a membrane is not conditioned by the presence of Na +. However, the fact that in the absence of Na +, PABA is carried in an opposite direction when compared with the experiment developed in the presence of Na +, permits some conclusion to be drawn on the amino acids' transport in the living cell. It is known that the exterior portion of the cell has lower pH values (acidic), comparatively with the intracell pH ones (basic). Consequently, the presence of Na + ion inside the cell allows the amino acids' transport through the cell membrane from outside the cell to its internal part.

C.V Uglea, M. Croitoru/Journal of Membrane Science 133 (1997) 127-131

It is worth mentioning here that, in a previous paper in which a series of fatty acids had been employed as carriers - the PABA transport through the same type of liquid membrane occurred in an opposite direction to that of the Experiment 1. Such a behaviour may be explained only by a careful analysis of all the system's compounds at the SP and RP interfaces. When employing fatty acids as carriers, at the SPOP and RP-OP interfaces, they are dissociated forming the R C O O - and H + species. Such a process induces the formation at the OP-SP interface of the H3N+-R-COO - and H3N+-R-COOH species, that assure the formation of the RCOO-H3N+-C6H4 C O O - N a + and RCOO-H3N+-C6Ha-COOH complexes, according to the following reactions:

131

4. C o n c l u s i o n s

-

R-COOH ~ R-COO- + H÷

(10)

R - C O O - + Na + ~ R C O O - N a +

(11)

H3N + -C6H4-COO-

-~-H+ .-_,

H3 N+ - C 6 H 4 - C O O H

(12)

H 2 N - C 6 H 4 - C O O - + H+ H3N + - C 6 H 4 - C O O -

(13)

R C O O - + H3 N+ -C6H4COOH ---+ R C O O - H3N + - C6H4- COOH

(14)

R C O O - + H 3 N + - C 6 H 4 - C O O - --* RCOO-H3N + - C 6 H 4 - C O O -

(15)

The basic medium of the S phase assures the formation of the complex with the R C O O - H 3 N + - C 6 H a - C O O - N a + structure, which is diffused towards phase R. When using the crown ether as a carrier, the S - M acid interface permits the formation of hydrogen bonds between the oxygen atoms from the crown ether and hydrogen from the H3N+--C6H4--COOH species. The complex thus formed diffuses towards the basic phase.

The liquid membrane provides an adequate model for study of PABA transport through biological membranes. The obtained data showed that the liquid organic membrane acts as a carrier - PABA complex (I, Fig. 2). Also, the presence of Na + in the composition of the aqueous phases assures the 'biological' direction of the PABA transport from the aqueous phase with an acidic pH towards the phase with basic pH.

References

[1] J.M. Lehn, Chemistry of transport processes - design of synthetic carder molecules, the 36th Meeting of the Societe de Chimie Physique, Paris, 1982. [2] E. Weber, Progress in crown-ether chemistry (post NE): New applications of crown compounds in chemical analysis, Kontakte, 26 (1984). [3] C.V. Uglea, A. Vatajanu, H. Offenberg, A. Grecianu, R.M. Ottembrite and I.I. Negulescu, Benzocaine modified maleic anhydride-cyclohexyl-l,3-dioxepin copolymer: preparation and potential medical applications, Polymer (London), 34 (1993) 3298-3301. [4] C.V. Uglea, I.N. Albu, A. Vatajanu, M. Croitoru, D. Iurea, M. Isac and R.M. Ottembrite, Polyanionic polymers. I. Synthesis, characterization and potential medical application of benzocaine modified carboxymethylcellulose, J. Bioact. Compatible Polym., 9 (1994) 448-461. [5] E.M. Choy, D.E Evans and E.L. Cussler, A selective membrane for transporting sodium ion against its concentration gradient, J. Am. Chem. Soc., 96(22 Oct.) (1974). [6] C.D. Nenitescu, Treatis of Organic Chemistry, EDP Bucharest, Vol. 2, 1982, p. 360 (in Romanian). [7] C.V. Uglea and C.V. Zanoaga, Transport of amino acids through organic liquid membranes. II. Aspects of the mechanism, J. Membr. Sci., 65 (1992) 47-50. [8] C.V. Uglea, Oligomers as physical catalyst in biological processes, in: S. Dumitriu (Ed.), Polymeric Biomaterials, Marcel Dekker, New York, 1994, pp. 795-828.