Characterization of the interaction between methylene blue and glycosaminoglycans

Characterization of the interaction between methylene blue and glycosaminoglycans

Spectrochimica Acta Part A 55 (1999) 1667 – 1673 Characterization of the interaction between methylene blue and glycosaminoglycans Qingcai Jiao, Qian...

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Spectrochimica Acta Part A 55 (1999) 1667 – 1673

Characterization of the interaction between methylene blue and glycosaminoglycans Qingcai Jiao, Qian Liu * Department of Biological Science and Technology, Nanjing Uni6ersity, Nanjing 210093, People’s Republic of China Received 3 September 1998; received in revised form 6 November 1998; accepted 6 November 1998

Abstract The methylene blue (MB) method for glycosaminoglycans (GAGs) assay is commonly used because of its sensitivity and convenience. The basis of the assay method is the binding of dye to GAGs, with production of a dye – GAGs complex which absorbs decrease at 664 nm, but the mechanism of the binding process is not well understood. The research attempts to gain better understanding of the assay system through spectrophotometric binding studies carried out on selected GAGs under normal GAGs assay. In this paper, two mathematical models for the binding process are proposed. These models are tested for their ability to estimate the number of dye-binding sites (n, N) and condition binding constant (K) from GAGs assay data. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Interaction; Methylene blue; Glycosaminoglycans

1. Introduction The assay of glycosaminoglycans (GAGs) by chemical means can be accomplished in several ways [1–7]. Among the most widely used techniques are assays based on metachromatic interactions with dyes [2 – 5]. However, no systematic investigation on the interaction between dyes and GAGs has been found in the literature [2,8,9]. A study of the GAGs – dyes combination should be interesting to improve the existing methods or to understand better the interaction between GAGs and ions or molecules. * Corresponding author. fax: + 86-25-6529492. E-mail address: [email protected] (Q. Liu)

GAGs are linear, strongly negatively charged sugar chains, consisting of repetitive sulphated and/or carboxylated disaccharide units [6,10,11]. To visualise GAGs, cationic dyes such as alcian blue, toluidine blue, methylene blue (MB), and azure A have been used [2–5]. In this study, we describe the binding properties of GAGs with MB, by a spectrophotometric method which is more rapid than the usual methods based on physiological activity [1], and takes advantage of the fact that the absorption spectra of the free and bound dyes are quite different. It will be shown that the MB–GAGs binding reaction can be successfully treated by using the methods proposed in this paper. The effect of increasing concentration of sodium chloride is considered, as well as the

1386-1425/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 1 4 2 5 ( 9 8 ) 0 0 3 2 3 - 0

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influence of the GAGs on the equilibria of the dye.

2. Experimental A Kontron Uvikon 860 spectrophotometer (Kontron, Switzerland) and a Hitachi U-3000 spectrophotometer (Tokyo, Japan) were used for recording absorption spectra, or measuring the absorbance at a given wavelength, using a 1-cm path length. A pH-HJ90B model portable acidity meter (Beijing hangtian computer, China) was used for the pH measurements. Heparin (Hep), sodium salt]160 IU mg − 1, was obtained from Shanghai, China and used without further purification. All calculations reported by Hep are in terms of a molecular weight of 12 000 Da [12]. The aqueous Hep solution (5.21× 10 − 5 mol l − 1) was prepared by dissolving 0.05 g Hep reagent in 80 ml deionised water. This stock solution of Hep was pipetted 2 ml into a 100 ml volumetric flask, and then diluted to the mark with water. This stock solution is stable for several weeks when kept in the dark at 4°C. Polysaccharide (PSP) isolated from a blue-green alga, Spirulina maxima, was kindly provided by the Department of Biology, with an average molecular weight of 15 000 Da [13,14]. The PSP stock solution (3.33×10 − 5 mol l − 1) was prepared by dissolving 0.05 g PSP in 100 ml deionised water. The operating solution of PSP was prepared by diluting 2 ml stock solution with water into 100 ml. The MB (Fig. 1) was purchased from Shanghai, China. The MB stock solution (1.34×10 − 3 mol l − 1) was prepared by dissolving 0.5 g dye in 1000 ml deionised water. The operating solution of MB was prepared by diluting 5 ml stock solution with water into 30 ml. Dye operating solution should be used soon after preparation. Due to light sensitivity of the dye, the MB stock solution must be stored in the dark. All other reagents were of analytical or guaranteed reagent grade. MB operating solution was transferred into a series of 12× 100 mm test tubes, then Hep or PSP solution, aliquots of NaCl solution were added to each test tube in different

amounts. The mixtures were diluted to a certain volume with water and mixed either by inversion or vortexing. After 4 min and before 1 h, spectra or absorbances of these solutions were measured with reference to water. All runs were thermostated at room temperature, and performed in triplicate.

3. Results and discussion Fig. 2 shows the absorption spectra of MB dye and MB–Hep complexes from 400 to 800 nm. They are obtained by keeping the pH and MB concentration constant, and changing the Hep concentration. The absorption peaks at 664 and 614 nm decrease, with the increase in Hep concentration, while a new absorption peak at 566 nm appears. These results are attributed to the Hep– dye complex, while decreasing in absorbance at 664 nm is in proportion to Hep concentration (These results also suggest that the assay at 664 nm is about twice as sensitive as that at 566 nm). Two isobestic points are formed at 590 and 703 nm. Fig. 2 indicates that there are interactions between MB and Hep [8,9]. In view of the molecular structure of MB, and Hep [10,11,16], it is not possible to reach a conclusion that MB combines preferentially with a particular group on Hep to form a complex. A reasonable explanation of these molecular events is that MB interacts with Hep by non-specific, electrostatic forces. Owing to the presence of the sulphate, carboxyl groups, the whole Hep molecules are negatively charged under the conditions of Fig. 2. However, the MB species have positive charges. Therefore, Hep and MB species should be bound together by electrostatic forces [2] (In the text ion charges will be omitted for simplicity): k1

DCl+ HepNal HepD+ NaCl

Fig. 1. Structure of MB dye.

(1)

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Fig. 2. Absorption spectra of MB-Hep mixtures. MB operating solution constants at 1.86 × 10 − 5 mol l − 1. pH 8.05. In order of decreasing peak absorbances at 664 nm, Hep concentrations are, 0.0, 8.68, 17.36, 26.03, 34.72, and 43.4 ×10 − 8 mol l − 1 in total assay volume.

k1 =[HepD][NaCl]/[DCl][HepNa] 2

= [HepD] /[DCl][HepNa]

(2)

where DCl represents the unbound MB species. where HepNa represents the concentration of unoccupied binding sites on Hep, and HepD refers to the concentration of MB bound dye DB. k1 is the equilibrium constant of Eq. (1). While: k2

DCll D + + Cl − +



(3) + 2

k2 =[D ][Cl ]/[DCl] = [D ] /[DCl]

(4)

D + refers to free MB dye DF, and k2 is the equilibrium constant of Eq. (3).Let: k = k1/k2 =[HepD]2/[D + ]2[HepNa] = D2B/D2F[HepNa]

(5)

According to Eqs. (1) and (3), the simplest mechanism able to describe the reversible interaction between Hep and dye is: K

DF + HepNal DB

(6)

K represents the condition binding constant when DT remains constant, and DT is the total analytical concentration of MB: DT = DF + DB

(7)

Dye-binding studies are facilitated by development of an equation describing the GAGs–dye binding process. A simple approach based on Beer’s law: DA = DoDB

(8)

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Define the average binding number of dye molecules per Hep molecule as: n = DB/CP

(9)

where CP represents the analytical concentration of Hep. Substituting Eq. (9) into Eq. (8), then Eq. (8) may be simplified as: DA =DonCP

(10)

Eq. (10) indicates that the sensitivity of the assay (the slope of the assay curve) depends on the molar absorptivities of free and bound dye and the average number (n) of dye molecules bound per Hep molecule. If oB and oF are determined from the absorbance of solutions with an excess of Hep and without Hep, then Do is a known quantity. Eq. (10) suggests a cause for the observed nonlinearity of the Hep in terms of the parameter n. This parameter must decrease or at best remain constant as Hep concentration CP increases because under constant dye conditions the number of dye molecules available per Hep molecule decreases. If n decreases, the assay must necessarily be non-linear. If the Hep molecule is saturated with dye, n becomes equal to N, the total number of binding sites [17]. At higher Hep concentration, the absorbance at 664 nm is increased when more than 17.36×10 − 6 mol l − 1 of Hep are added, while the absorption peak at 566 nm decreases and shifts to 575 nm (Fig. 3). Linearity is enhanced by maximising the dye concentration in the assay mixture to maintain the highest possible dye/Hep ratio over the entire Hep concentration range. However, the dye concentration cannot be too high because of the restriction of spectral measurement. In this case, if the experiment is conducted by keeping the dye concentration DT and other conditions constant and changing the Hep concentration CP, a group of CP  DA data will be measured and cause nonlinear plots. In the following section a simple linear regression equation is proposed for studying the MB Hep assay. According to Eqs. (6) and (5) gives: K=DB/DF[HepNa]= kDF/DB

(11)

K is the condition binding constant, where [HepNa] represents the concentration of unoccupied binding site on Hep: [HepNa]= NCP − DB = (N− n)CP

(12)

where CP is the analytical concentration of Hep, N the total number of binding sites per Hep molecule, and n the average binding number of dye molecules per Hep molecules. Substituting Eqs. (12), (7) and (9) into Eq. (11) gives: K= nCP/(DT − nCP)(N − n)CP

(13)

Substituting Eq. (13) into Eq. (10) yields: DA = Do(1+ KDT)/K −DoN(DTDo/DA − 1)CP (14) where N is the maximum number of binding sites per Hep molecule. Do is a known quantity, DoN is a constant, K is the condition binding constant and Do(1+ KDT)/K has a fixed value at given MB concentration, DT. There is a linear relationship between DA and (DTDo/DA −1)CP according to Eq. (14). Calculating Eq. (7)–Eq. (14) by using

Fig. 3. Spectra of Hep – MB mixtures at high Hep concentration. MB operating solution is 1.86× 10 − 5 mol l − 1, pH 8.05. From the top to the bottom at 664 nm Hep usage are, (a), 0.0; (b)17.36 ×10 − 6; and (c) 17.36× 10 − 7 mol l − 1 in total assay volume.

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Fig. 4. Absorbance of solutions containing increasing concentration of Hep. MB concentrations are, (d) 22.29 × 10 − 6; (c) 18.58 × 10 − 6; (b) 14.86 ×10 − 6; and (a) 11.15 × 10 − 6 mol l − 1 in total assay volume.

the data from Fig. 2, a DA  (DoDT/DA − 1) CP regression equation is obtained: DA =1.07− 2.88 × 106(DoDT/DA −1)CP R= − 0.998 From the slopes and intercepts of this equation, the values K= 1.15 ×106, and N =52.35. are obtained. A set of experiments is performed, keeping the dye concentration constant and increasing the Hep concentration. As an example, Fig. 4 reports the absorbance at 664 nm, of solutions with different concentrations of dye, with increasing Hep concentration at a pH of 8.05. The absorbance decrease with the Hep concentration increasing. At dye concentration lower than about 11.15× 10 − 6 mol l − 1 a turbidity is observed. It will be shown that the MB – GAGs binding reaction can been successfully treated by using a new method proposed in this paper (see Fig. 5). That is to say, the dye/GAGs concentration ratios could not effect this DA  (DoDT/DA − 1)CP linear regression equation. The experiment could be carried out under any dye/GAGs ratios conditions to determine the Hep concentration. The influence of MB concentration on the binding reaction is shown in Table 1. From this test we know that an increase in MB concentration causes an increase in K, while N, Do and R remain

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Fig. 5. Regression lines of DA(DoDT/DA− 1)CP at different DT. pH 8.05, at 664 nm, from the top to the bottom MB concentration are, (a) 22.29 ×10 − 6; (b) 18.58× 10 − 6; (c) 14.86 ×10 − 6; (d) 11.15 ×10 − 6 mol l − 1 in total assay volume.

practically constant. If the Hep molecule is nearly saturated with dye, DF will increase with excess DT, leading to DF \ DB, from which Eq. (11) K increase with DT. The absorbances of the solution at 664 nm are recorded. Some results are reported in Fig. 6. Increasing salt concentration causes an ascent of the absorbance at 664 nm [2,18]. This is due to a partial release of dye from Hep, as demonstrated by a few dialysis experiments (Table 2). It is shown in Table 2 that an increase in salt concentration causes a significant decrease in K and Do values, thus decreasing the sensitivity of the MB–Hep assay [2,8]. This effect may be explained as a competition between anion and Hep for the same binding sites on dye species. In this case the concentrations of the anions are 100-fold higher than that of the MB species [18,19], so the cationic dye species are actually surrounded by Table 1 Effect of MB concentration on binding of MB on Hep, pH 8.05, at 664 nm DT(mol l−1)

Do

N

K

R

1.12×10−6 1.49×10−6 1.86×10−6 2.23×10−6

5.48×104 5.46×104 5.50×104 5.50×104

57.71 51.54 52.35 52.41

7.02×105 8.46×105 11.14×105 31.36×105

−0.989 −0.996 −0.998 −1

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Table 3 Data from PSP–MB assay used for linear regressions. pH 6.90, DT =1.86×10−5 mol l−1, Do=4.32×104 l mol−1, at 664 nm

Fig. 6. Effect of salt concentration on the sensitivity of MB – Hep complex. MB operating solution constants at 1.86 ×10 − 5 mol l − 1. pH 8.05. Salt concentrations are, (a) 0; (b) 7.13 × 10 − 3; and (c) 14.26× 10 − 3 mol l − 1 in total assay volume.

anions which prevent the MB species from binding to Hep, thus decreasing assay response to Hep. According to Eq. (6), DB refers to bound dye. Here MB binds not only to Hep, but also to NaCl when colour reaction in salt solution. This causes DB to increase with NaCl concentration, accompanied by n elevated according to Eq. (9). [HepNa] could not be a negative number, according to Eq. (12), here N is increased with n, so the N is elevated with the salt concentration increase, and most likely due to NaCl bound to dye [15]. The combination of MB with PSP is investigated by using the above method. Table 3 shows the absorption data of MB – PSP mixtures. Using the absorbance measured at 664 nm, a DA  (DoDT/DA −1)CP linear regression equation is obtained: DA = 0.86−1.53 ×106(DoDT/DA −1)CP Table 2 Effect of NaCl concentration on binding of MB on Hep. pH 8.05, DT =1.86×10−5 mol l−1, at 664 nm CNaCl(mol l−1) Do 0 3.57×10−3 7.13×10−3 10.70×10−3 14.26×10−3

5.50×104 4.30×104 3.47×104 3.03×104 2.72×104

N

K

R

52.35 56.11 60.80 73.89 91.66

1.15×106 8.67×105 6.67×105 4.54×105 3.57×105

−0.998 −0.998 −0.998 −0.996 −0.995

CP(mol l−1)

DA

(DoDT/DA−1)CP

5.56×108 11.11×108 16.67×108 22.22×108 27.78×108 33.33×108 38.89×108 44.44×108 50.00×108 55.56×108 61.11×108

0.080 0.150 0.228 0.303 0.379 0.449 0.512 0.559 0.598 0.615 0.627

5.02×107 4.83×107 4.20×107 3.66×107 3.10×107 2.62×107 2.21×107 1.94×107 1.71×107 1.69×107 1.71×107

From the slope and intercept of this equation, the values K= 7.52× 105, and N= 35.31 are obtained.

4. Conclusions This method can improve the existing dye– GAGs binding assay, the experiment could be carried out under any conditions. That is to say, the dye/GAGs concentration ratios could not effect this DA  (DoDT/DA − 1)CP linear regression equation. The method is useful and convenient for the investigation of small substance–GAGs interaction, because the parameters defined in this paper can be determined easily. Experimental conditions such as ion strength, and concentration of dye have different effects on the maximum binding number, the condition binding constant, the molar absorptivities of free and bound dye, thereby influencing the sensitivity of a GAGs assay.

References [1] [2] [3] [4] [5]

P. Band, A. Lukton, Anal. Biochem. 120 (1982) 19. Q.C. Jiao, Q. Liu, Anal. Lett. 31 (1998) 1311. E.D. Dold, Anal. Biochem. 99 (1979) 183. S. Radoff, I. Danishefsky, Anal. Biochem. 120 (1882) 373. Y.Z. Wang, X.R. Huang, H.N. Hou, Z.F. Yin, Acta Acad. Med. Hebei 13 (1992) 65. [6] N. Volpi, Anal. Biochem. 240 (1996) 114.

Q. Jiao, Q. Liu / Spectrochimica Acta Part A 55 (1999) 1667–1673 [7] T. Katayama, K-I. Takai, R. Kariyama, Y. Kanemasa, Anal. Biochem. 88 (1978) 382. [8] Y.J. Wei, K.A. Li, S.Y. Tong, Talanta 43 (1996) 1. [9] R.W. Congdom, G.W. Muth, A.G. Splittgerber, Anal. Biochem. 213 (1993) 407. [10] U. Lindahl, K. Lindholt, D. Spillmann, L. Kjellent, Thromb. Res. 75 (1994) 1. [11] C.H.A. van de Lest, E.M.M. Versteeg, J.H. Veerkamp, T.H. van Kuppevelt, Anal. Biochem. 221 (1994) 356. [12] R. Malsch, M. Guerrini, G. Torri, G. Lohr, B. Casu, J. Harenberg, Anal. Biochem. 217 (1994) 255.

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[13] Q. Liu, Q.C. Jiao, Z.L. Liu, Chin. J. Mar. Drugs 17 (1998) 48. [14] K.M. Shekharam, L.V. Venkataraman, P.V. Salimath, Phytochemistry 26 (1987) 2267. [15] Q.C. Jiao, Q. Liu, Spectrosc. Lett., 31 (1998) 1353. [16] P. Tivant, A. Perera, P. Turq, Biopolymers 28 (1989) 1179. [17] H.J. Chial, A.G. Splittgerber, Anal. Biochem. 213 (1993) 362. [18] Q.C. Jiao, Q. Liu, C. Sun, H. Hua, Talanta, 1999, in press. [19] L.B. Jaques, S.M. Wice, L.M. Hiebert, J. Lab. Clin. Med. 115 (1990) 422.