Interaction of cationic cyanine dye with algal alginates: evidence for a polymer bound dye dimer ()
1. INTRODUCTION
Algal alginates are structural polysaccharides found in high concentration in various types of brown seaweeds, which because of their gelling properties command considerable commercial interest. Chemically they are 1, 4-linked block polymers of β-D-mannuronate (M for short) and α-L-guluronate (G) or of alternating (MG) sequences with composition varying according to source and treatment. Due to the presence of carboxylate groups, which absorb at about 215 nm circular dichroism (CD)—the differential absorbance between left and right circularly polarized light—has played a major role in elucidating the structure of alginates and their cation mediated aggregation properties [1,2]. Interaction with dyes can shift the uvand the CD absorptions into the visible region and allow the study of biopolymers in a wavelength range, which is spectroscopically more easily accessible [3]. Seely and Hart have investigated the interaction between methylene blue and several types of sodium alginate by uv/vis and CD spectroscopy at high polymer to dye molar ratios [4], and Pal and Mandal have used several cationic dyes, among them pinacyanol chloride, to study the binding to potassium alginate [5]. Using this approach we have studied the interaction of pinacyanol with algal alginates from different sources in different concentrations. We have found what we believe is an alginate bound dye dimmer, which holds promise as an indicator for the various processes involving alginate chain conformations.
Peak analysis was used to analyse and separate the various new absorption bands that resulted by such interaction for the most optimal spectrum [6]. All measurements in this study are conducted mainly in 7.5% v/v ethanol/water solutions.
2. MATERIALS AND METHODS
2.1. Materials
Sodium alginate was purchased from Kelco, UK. According to manufacturer’s specifications, the alginate has a molecular mass of 90,000 g/mol and consists of approximately 450 monomers. The molar ratio of mannuronate to guluronate in the sample is 60 - 65 to 40 - 35, corresponding to an M/G ratio between 1.5 and 1.8 to 1. Mannuronate rich and guluronate rich alginates were prepared in our labs [7]; in the former, the M/G ratio was 5:1, in the latter it was 0.36:1. Pinacyanol chloride (1,1’- diethyl-2,2’-carbocyanine chloride) was obtained from Sigma and used as received. For the spectra, we used spectroscopy grade ethanol from Merck, and water was distilled three times.
2.2. Experimental Methods and Instruments Used
Standard pinacyanol and sodium alginate solutions were prepared in 25 ml volumetric flasks. Alginate-dye solutions (4.00 ml) were prepared in stoppered rolled rim glasses of 10 ml capacity. To prevent the dye from precipitating at the glass walls the alginate solutions were added first, followed by the required amount of the dye solution. The tendency of cyanine dyes to aggregate in aqueous solution is wellknown. In order to extend the concentration range we added a constant low concentration of 7.5% (v/v) ethanol in all our spectroscopic investigations.
UV/vis spectra were recorded with a Perkin-Elmer Lambda 5 spectrophotometer, and CD spectra were measured with an AVIV circular dichroism spectrometer, Model 62 DS. Both instruments were connected to a personal computer for data collection in ASCII-file format.
3. RESULTS AND DISCUSSION
3.1. Sodium Alginate; Characterization by CD
CD spectroscopy has been used to evaluate the alginate composition based on the carboxylate n®π* absorption at about 215 nm [2]. Figure 1 shows the CD spectrum of sodium alginate between 195 and 250 nm. The spectrum has been taken in aqueous solution, and the concentration of sodium alginate is 0.80 mg/ml, corresponding to 4.0 × 10–4 M (if monomeric sodium mannuronate or guluronate, C6H7O6Na, are taken as the molecular mass unit). The spectrum shows a positive peak at 200 nm, and a negative trough at 215. From the ratio of the peak to the trough amplitude the composition of sodium alginate, in terms of mannuronate and guluronate, can be calculated by using a specific equation [2]. The values obtained from such non-destructive method were found to be 63% and 37% for mannuronate and guluronate, respectively. Therefore, sodium alginate that was used in the following aggregation experiments contains an excess of mannuronate over guluronate in the ratio of 1.7:1.
3.2. UV/vis and CD Spectra at Different Alginate/Dye Ratios
Figure 2(a) shows how increasing the molar ratio of alginate to dye affects the UV/vis spectrum of an aqueous pinacyanol solution (7.5% v/v ethanol). At the pinacyanol concentration chosen (1.5 × 10–5 M) most of the dye molecules are present in the form of monomers (maximum wavelength = 600 nm) or dimers (546 nm). Addition of the alginate destroys the monomer/dimer band structure of the dye and shifts the absorbance to a new