Investigation on Molecular Non-covalent Interaction in the Sodium Dodecyl Benzene Sulfonate-polychrome Blue B-protein Replacement Reaction

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1 hinese Journal of hemistry, 24, 22, Article Investigation on Molecular Non-covalent Interaction in the odium Dodecyl Benzene ulfonate-polychrome Blue B-protein Replacement Reaction GAO, Hong-Wen*,a (Y ) WU, Ji-Rong b (Û ) HEN, Rong c ( ) a Key Laboratory of hangjiang River Water Environment of Ministry of Education, ollege of Environmental cience and Engineering, Tongji University, hanghai 292, hina b Institute of New Material, Hangzhou Teacher s ollege, Hangzhou, Zhejiang 3136, hina c Anhui Professional Technology Institute, Hefei, Anhui 2351, hina The molecular non-covalent interaction often originates from the electrostatic attraction and accords with the Langmuir isothermal adsorption. The sodium dodecyl benzene sulfonate (DB)-polychrome blue B (PB)-protein [bovine serum albumin (BA), ovalbumin (OVA) and myoglobin (MB)] ternary reaction has been investigated at ph Protein to replace PB from the PB-DB binding product was used to characterize the assembly of an invisible-spectral compound, DB, on proteins by measuring the variation of PB light-absorption by the microsurface adsorption-spectral correction (MA) technique. The effect of ionic strength and temperature on the aggregation was studied. Results showed that the aggregates DB 92 BA, DB 58 OVA and DB 15 MB at 3 and DB 83 BA, DB 39 OVA and DB 1 MB at 5 are formed. Keywords MA technique, non-covalent interaction, sodium dodecyl benzene sulfonate, polychrome blue B, protein, invisible-spectral molecule, replacement reaction Introduction upramolecular assembly is always an important research direction for chemists and biochemists. 1-5 It is very helpful for us to accurately analyze the physicochemical action and process occurring inside biological cell, for example, function of a drug, damage of a toxicant, detection of oncogene and so on. Understanding the chemical, physical, biological and pharmacological activity of a complex system requires knowledge of the state of molecular aggregation of the system components. In fact, the molecular interaction is very complicated. A product may be formed by covalent bond, coordinate bond and non-covalent bond, e.g., electrostatic attraction, hydrogen bond, van der Waals force, hydrophobic bond, insertion, winding of long-chain molecules. Usually, the co-action of a number of kinds of them exists in aqueous medium of cell. The electrostatic attraction is always a source force to attract the oppositely charged or polar molecules to junction then to induce the co-action of the other non-covalent bonds. The non-covalent interaction is often weaker than a covalent bond in a diluted medium so it is destroyed easily by a high temperature or a high ionic strength. Because it accords with the Langmuir isothermal adsorption, the non-covalent interaction could be regarded as a microsurface adsorption reaction in only one monolayer in a diluted solution. Molecular spectrometry was extensively used 6-9 in study of biomacromolecular assembly and Pesavento, 1 catchard models 11, 12 and the microsurface adsorption-spectral correction (MA) are applied to characterization of the assembly. To characterize the interaction of an invisible spectral (IV) molecule, e.g., drug, surfactant, pesticide and toxicant with biomacromolecule, this paper established a fit approach which described as follows: at first, a positively charged dye reacted with an IV compound to form a product by non-covalent interaction and then a kind of protein was added into the product solution to replace the dye and to form the IV compound-protein assembly product. Thus, the assembly product was characterized by measuring the variation of the dye lightabsorption. Because the molecular non-covalent interaction often results in a little shift of the absorption spectra of solution, the MA technique must be applied to determination of the dye light-absorption in place of ordinary spectrophotometry. The sodium dodecyl benzene sulfonate (DB)-polychrome blue B (PB)- protein [bovine serum albumin (BA), ovalbumin (OVA) and myoglobin (MB)] ternary reaction has been investigated. PB formed monovalent cation in aqueous solution and then was adsorbed on DB. imilarly, the protonation of amino groups (NH 2 ) on protein chain made itself carry a number of positive charges in acidic solution and then DB was attracted. The break point * hwgao@mail.tongji.edu.cn; Tel. and Fax: Received November 11, 23; revised and accepted July 5, 24.

2 Molecular non-covalent interaction hin. J. hem., 24, Vol. 22, No approach 16 was used as an auxiliary approach to characterize the assembly product. Results showed that the aggregation of PB on DB and the assembly of DB on proteins obeyed the Langmuir isothermal adsorption. This approach is helpful for us to study the molecular interaction in cell in another way. Experimental Principle In Figure 1, charged surfactant () (1) can attract oppositely charged stain probe (L) (2) and also the aggregation of L on obeys the Langmuir isotherm adsorption. 17 Early, some synergism mechanisms, e.g., solubilization, stabilization and sensitivity enhancement were established and applied in, e.g., synergism perturbation, hydrogen bond formation, micelle catalysis and others. On the basis of these models, the microsurface adsorption mechanism has been developed. It units both the micelle catalysis and electrostatic field aggregate theories and also improves them. Its combination with spectral correction method named MA technique provided a very useful experimental strategy for study on the physicochemical interaction among molecules. In Figure 1, it is seen that the stain probe (L) can be attracted and bound on an oppositely charged surfactant () (1) by electrostatic attraction, hydrogen bond, van der Waals force, hydrophobic bond and winding of long-chain molecules to form the product L. The aggregation obeyed the Langmuir isotherm adsorption. 17 The L solution equilibrium occurs as follows (m.s. means microsurface phase): nl I (m.s.) L n (m.s.) Initial state L ( A, A') Equilibrium ( A ) L c Figure 1 The aggregation of spectral probe (L) on surfactant () (1, 2), substitution of L and assembly of on biomacromolecule (M) (3). The Langmuir isotherm equation is expressed as: I γ n Kn L (1) where K is the equilibrium constant, L the molarity of the excess L, and γ the molar ratio of the effective L adsorbed to. With increase in L concentration, γ approaches a maximal binding number, n. Both n and K may be calculated by plotting line γ 1 1 vs.. Both L and γ the same as n were calculated by the spectral correction equations: L γ [ n[ η ã (2) L L [ (1K η ) (3) A K c A η[ A where both and L are the initial molarities of and L and η indicates the effective fraction of L. A c, A and A are the real absorbance of the -L product, the absorbance of L against water and that of the -L solution against a reagent blank, respectively measured at the peak-absorption wavelength λ 2. A c was calculated by means of: A [ c A β A' 1Kαβ (4) (5) where A indicates the absorbance of the -L solution measured at the valley-absorption wavelength λ 1. Both α and β are the correction constants. In the reaction above, the addition of biomacromolecule (M) will result in a replacement of L and formation of a novel aggregate, M. This process is showed in Figure 1 (3) and expressed as follows: L I M (m.s.) M (m.s.) I n m m nl Initial state ( A c) M Equilibrium ( A') I n( ) c M L This was attributed to the protonation of amino groups (NH 2 ) in M. The assembly of happens on M by the electrostatic attraction, hydrogen bond, van der Waals force, hydrophobic bond, insertion and winding of long-chain molecules. The assembly obeyed the Langmuir isotherm adsorption, too. Thus, the characterization of M N' (N': maximal binding number) was made by measuring the variation of L n aggregate. The factors were calculated by the relations: K K γ [ m[ [ ã M M (6) [ ηã M (1 η ) (7) where L η A A A (8) ' c c c Ac Ac

3 8 hin. J. hem., 24, Vol. 22, No. 8 GAO, WU & HEN Materials and methods Absorption spectra were recorded on a TU191 spectrophotometer (P General, Beijing) and the individual absorbance was measured on a Model 722 spectrophotometer (hanghai 3rd Analytical Instruments). The drop shape analysis ystem (DA 1 MK 2, KRU GmbH Germany) was used to measure the surface tension of solutions. ph of solution was measured on a ph-2 acidity meter (Leici Instruments, hanghai). The temperature was adjusted and remained constant in an electronic heated thermostat bath, Model 116R (hangjiang Test Instruments of Tongjiang, hina). Protein standard solutions were prepared by dissolving the commercial bovine serum albumin (BA) (Dongfeng Biological Technological, hanghai), myoglobin (MB) (erva, Heidelberg, Germany) and ovalbumin (OVA) (hanghai hemical Reagents of hinese Medicine Group) in deionized water. The protein content (w, mg/ml) in the above solutions was determined and calculated by the relation: w (mg/ml) 1.45A 28 nm.74a nm by measuring their absorbances (A 26 nm and A 28 nm ) at 26 and 28 nm by UV spectrophotometry. The standard stock solution of DB (1. mmol/l) was prepared by dissolving sodium dodecyl benzene sulfonate (A.R., Hongguang hemical Reagents of Yixing, hina) in deionized water. PB solution (.5 mmol/l) was prepared by dissolving.242 g of polychrome blue B ( 16 H 18 N 3 l, igma hemicals) in 5 ml of deionized water. The Britton-Robinson buffer solutions between ph 1.73 and 9.93 were prepared to adjust the acidity of the solutions and 2. mol/l Nal was used to adjust the ionic strength of the solutions. The masking reagent solution, 5% Na 2 EDTA was prepared to mask metals in analysis of protein samples. Experimental procedures Aggregation of PB on DB Into a 25 ml of calibrated flask were added an appropriate working solution of 1. mmol/l DB, 2.5 ml of buffer solution (ph 3.88) and a known volume of.5 mmol/l PB. The mixture was diluted to 25 ml with deionized water and mixed thoroughly. After 5 min, the absorbances were measured at 488 and 6 nm, respectively against the blank treated in the same way without DB and then A c, η, L and γ were calculated by Eqs. (2) (5). Aggregation of DB in proteins Into a 25 ml of volumetric flask, 2.5 ml of ph 3.88 buffer solution and a known volume of 1. mmol/l DB were added. After mixing, a known volume of.5 mmol/l PB was added to keep a molarity as more as 1.2 times that of DB. The solution was diluted to about 2 ml and mixed. After 5 min, a known amount of a protein solution was added and then diluted to 25 ml. The resultant solution was mixed well. After 1 min, the absorbances were measured at 488 and 6 nm against the reagent blank treated in the same way without any protein and then A c, η, and γ were calculated by Eqs. (5) (8). Determination of protein in samples Two sample solutions were prepared. ample 1 was.1% milk powder liquid and sample 2 was 5% children drink. Into a 25 ml of volumetric flask, 2.5 ml of ph 3.88 buffer solution, 1. ml of 5% Na 2 EDTA, 1. ml of 1. mmol/l DB and 4. ml of.5 mmol/l PB were added. The mixture was diluted to 2 ml and mixed well. After 1 min,.5 ml of a sample solution was placed into the flask and the solution was diluted to 25 ml. After 1 min, the absorbance ( A) of the solution was measured at 6 nm against a reagent blank. Results and discussion Effect of ph and spectral analysis The absorption spectra of the PB and DB-PB solutions with ph are shown in Figure 2. By comparing curves 1 9, it can be known that the reaction is the most sensitive in acidic solution. It is attributed to the fact that PB was formed easily because of the protonation of NH 2. On the contrary from curves 8 and 9, PB cation will not be formed in basic medium. Among all of them, spectrum 2 gives the deepest valley and a highest peak. In this work, ph 3.88 was selected. The peak of PB is located at 6 nm in curve 1 and that of the PB-DB aggregate is located at 63 nm in curve 11. The spectral red shift of the aggregate is only 3 nm. This is just attributed to the weak non-covalent bond interaction in a diluted solution. In curve 2, the peak and valley of the DB-PB solution against PB Figure 2 Absorption spectra of PB, PB-DB and PB-DB-BA. olutions: from curves 1 to 9, at ph from 1.73, 3.85, 4.88, 5.3, 5.81, 6.8, 7., 8.27 and 9.93, where the solutions contained.4 mmol/l DB and.2 mmol/l PB, all measured against the reagent blanks without DB. urve 1,.2 mmol/l PB; urve 11,.2 mmol/l PB plus.1 mmol/l DB; urve 12,.2 mmol/l PB plus.1 mmol/l DB plus 1 mg/l BA. urves 1, 11 and 12 all at ph 3.88 measured against water.

4 Molecular non-covalent interaction hin. J. hem., 24, Vol. 22, No are located at 488 and 6 nm, so such two wavelengths were used. In an DB-PB solution, plenty of BA was added and the replacement of PB from its DB aggregate was observed from the coincidence of curve 1 with curve 12. urve 12 represents the absorption spectrum of the replacement solution where the PB was replaced completely by BA to form the ADB-BA aggregate. From curves 1 and 11, the correction coefficients were calculated to be β PB.9 and α DB-PB.672. The real absorbance of the DB-PB aggregate at 488 nm was calculated by A c 1.6 ( A.9 A ). Auxiliary characterization of the break point aproach to interaction of DB with PB and prteins urve A in Figure 3 indicates the change of the absorbance ratio, A 488 nm /A 6 nm of the solutions with DB. The absorbance ratio approaches to a minimum when DB is more than.4 mmol/l. Therefore, no free PB exists in the solutions where the molar ratio of DB to PB is more than 1. The break point (P) of curve A is located at about.2 mmol/l DB, so the maximal binding number of PB on DB was estimated to be 2. With the same method, from the variation of the surface tension of the DB-proteins solutions shown in Figure 3(B), their break points (P1, P2 and P3) were found easily to locate at P1 72 (BA), P2 6 (OVA) and P3 18 (MB). The binding numbers above will be still examined in the latter experiments. Effect of ionic strength and temperature on the DB-PB aggregation In order to investigate the effect of ionic strength on the aggregation of PB on DB, 2. mol/l Nal was added into the DB-PB solution. It was found that the binding ratio of PB to DB decreased by about 15% per increasing 1 mol/l of ionic strength. This was attributed to the fact that much more Na than PB would enclose DB to oppose the binding of PB. The effect of temperature has shown that γ of PB to DB decreased by about 1% 15% per increasing 1. This was attributed to the fact that a higher temperature would often cause a rapid desorption of PB from its DB aggregate. haracterization of the DB-PB aggregation By varying DB molar concentration, the absorbance of the DB-PB solutions was measured at three temperatures. γ of PB to DB and L of PB at equilibrium were calculated and plots γ 1 vs. 1 L are shown in Figure 4. All curves have good linearity. onsequently, the aggregation of PB on DB accords with the Langmuir isotherm adsorption. From the intercepts, the maximal binding number N of PB was calculated to be from n 2 at 3 to 1.5 at 7. The value at 3 is consistent with that above obtained by the break point approach. Therefore in the DB-PB solution at ph 3.88, only PB 2 DB at 3, both about 5% PB 2 DB and about 5% PB DB at 5 and both about 3% PB 2 DB and about 7% PB DB at 7 were formed. The transition of the PB-DB aggregate with temperature is sketched in Figure 4, too. From the slope of each curve, the binding constant of the aggregate was calculated to be K DB-PB, L/mol, K DB-PB, L/mol and K DB-PB, L/mol. Replacement of PB and characterization of the DB-protein aggregation In order to investigate the substitution of PB and the assembly of DB on protein, the solutions containing DB and PB at constant molar ratio of 1 2 were prepared. The solutions consisted of the colour compounds: PB and DB PB. They were used as a new chromogenic reagent to investigate the protein Figure 3 Application of the break point approach to auxiliary characterization of the binding number. (A) variation of A 6nm /A 488nm of the DB-PB solutions containing.4 mmol/l PB. (B) variation of the surface tension of the DB-protein solutions containing: 1, 38. mg/l BA; 2, 37. mg/l OVA; 3, 37.4 mg/l MB, all at ph 3.88

5 82 hin. J. hem., 24, Vol. 22, No. 8 GAO, WU & HEN Figure 4 Plots γ 1 vs. L 1 for the solutions containing.2 mmol/l DB and PB between.12 and.36 mmol/l. 1, 3 ; 2, 5 ; 3, 7 ; and sketch of transition of the DB-PB aggregate with temperature. assembly. By adding protein into the solution, it was observed that the solution approached to PB color from DB PB product color. Therefore, the following replacement reaction occurred: DB PB protein DB protein PB. The ternary solutions were measured at 3 and 5 according to the experimental procedure. γ of DB to protein and of DB existed in form of DB PB at equilibrium were calculated by Eqs. (6) (8). Plots γ 1 1 vs. are shown in Figure 5 and they are linear. Therefore, the aggregation of DB on BA, OVA and MB obeyed the Langmuir isotherm adsorption. From the intercepts, the maximal binding number of DB was calculated to be 92 in BA at 3 and 83 at 5, 58 at 3 in OVA and 39 at 5 15 at 3 in MB, and 1 at 5. The values at 3 are approximate to those above obtained by the break point approach. o, the effect of temperature on the assembly of small molecules on biomacromolecule is notable. The exact substitutions are expressed by the following reactions: at 3 92 DB PB BA DB 92 BA 92 PB 58 DB PB OVA DB 58 OVA 58 PB 15 DB PB MB DB 15 MB 15 PB at 5 83 DB PB BA DB 83 BA 83 PB 39 DB PB OVA DB 39 OVA 39 PB 1 DB PB MB DB 1 MB 1 PB The maximal binding number decreased with increase in temperature because of the same reason as that discussed in the last paragraph. From variation of the binding number, the adsorption of DB on OVA is the most notable at high temperature. It was attributed to the fact that the third grade structure of OVA is the loosest among them to weaken the electrostatic attraction. From the linear slopes, the binding constants of the DBprotein aggregates was calculated to be K DB-BA, , K DB-BA, , K DB-OVA, , K DB-OVA, , K DB-MB, and K DB-MB, L/mol. By comparing K at 5 with that at 3, the attraction of DB in proteins at 5 is greater than that at 3. The binding constant K DB-PB of the Figure 5 Plots of γ 1 1 vs. for the DB-protein solutions at ph (A) BA, (B) OVA and () MB. 1, 3 ; 2, 5. A-1 and A-2, B-1, and B-2, and -1 and -2 solutions contained 9.46 mg/l BA, 9.34 mg/l OVA and 8.97 mg/l MB, respectively, which all initially contained DB between.24 and.6 mmol/l and PB between.2 and.5 mmol/l.

6 Molecular non-covalent interaction hin. J. hem., 24, Vol. 22, No DB-PB at 5 is much less than that at 3 and PB is desorbed easily from the DB aggregate at higher temperatures. At a high temperature, the protein molecule will unfold. The binding area of the DB long chain on protein will be increased by hydrophobic bonds and winding. The three dimensional effect will hinder the binding of the other DB on protein to result in decrease of the binding number. However, the increase of the binding area will make the binding energy (K as indicator) of DB on proteins high. Application to detection of protein The DB-PB aggregation as the chromogenic reagent has been tried to apply to the quantitative analysis of protein in the presence of Na 2 EDTA at ph The standard series of various protein solutions were prepared and their absorbances were measured at 6 nm. The regression equations are followed: y.42x.152 in the range between and 1.5 mg of BA, y.741x.1336 in the range between and 7 mg of OVA and y.989x in the range between and.4 mg of MB (y: absorbance measured at 6 nm and x: mg of protein). The second equation should be used in the determination of protein content in foods, e.g., milk powder, beverage. By adding Na 2 EDTA in protein samples to mask metal ions possibly existing in the solution, none of the following ions or compounds affected the direct determination of 1 mg of OVA (less than 1% error): 1 mg of 2K vitamin, l and O 4,.5 mg of glucose, tyrosine, glutamic acid, lysine and a(ii),.2 mg of DNA and Fe(II) and.5 mg of u(ii), Pb(II), Zn(II), Mg(II), o(ii), d(ii), Fe(III) and Hg(II). Two samples, milk powder (1#) and children drink (2#) were analyzed with PB-DB aggregate as chromogenic reagent. The protein contents (1#: 16.6% and 2#:.84%) in the samples accorded with the index mark signed (1#: 17% and 2#: less than 1%) on the containers. The recovery of protein was between 97.2% and 18% and the relative standard deviation less than 3.4%. onclusion The investigation on the PB-DB-protein ternary reaction indicated that the aggregation of PB on DB and that of DB on proteins obeyed the Langmuir isothermal adsorption. The non-covalent interaction is easy to be destoryed by the concentrated eletrolyte and high temperature in a diluted medium. Also, the replacement reaction can be applied to the accurate determination of protein in foods. That proteins substitute the PB bound on DB provided a useful way to characterize the interaction of an IV compound with biomacromolecule. It will help to explain the molecular non-covalent interaction occurring in cell. References 1 Pasternack, R. F.; ollings, P. J. cience 1995, 269, Pasternack, R. F.; Gibbs, E. J.; Villafranca, J. J. Biochemistry 1983, 22, Jin, J.; Jiang, L.; hen, X.; Yang, W..; Li, T. J. hin. J. hem. 23, 21, Neault, J. F.; TajmirRiahi, H. A. J. Phys. hem. 1997, 11, arlisle, R..; Read, M. L.; Wolfert, M. A.; eymour, L. W. olloids urf., B 1999, 16, Muller,.; alsou, P.; Frit, P.; alles, B. Biochimie 1999, 81, Pasternack, F.; Bustamante,.; ollings, P. J. J. Am. hem. oc. 1993, 115, Huang,. Z.; Li, Y. F.; Liu, X. D. Anal. him. Acta 1998, 375, Fumlyo,.; hunsuke, K.; Nobuyuki, Y.; Isao, T.; Makoto, K. Biosci. Biotechnol. Biochem. 1999, 63, Pesavento, M.; Profumo, A. Talanta 1991, 38, catchard, G.; cheinerg, I. H.; Armstrong,. H. J. Am. hem. oc. 195, 72, Huang,. Z.; Li, Y. F.; Li, K. A. Anal. Lett. 1996, 3, Gao, H. W.; Xu, W. Q. Anal. him. Acta 22, 458, Gao, H. W.; Yang, J. X.; Jiang, J.; Yu, L. Q. upramol. hem. 22, 14, Gao, H. W.; Ye, Q..; Liu, W. G. Anal. ci. 22, 18, Gao, H. W.; Hu, Z. J.; Zhao, J. F. hem. Phys. Lett. 23, 376, Langmuir, I. J. Am. hem. oc. 1918, 4, Nishida, H. Bunseki 1977, 26, i, Y. X.; Yang, M. M. hin. ci. Bull. 1983, 16, 98 (in hinese). 2 Zheng, Y.; Li, L. D.; un,. Q. hem. Reag. 1994, 6, avvins, P. K.; hernova, P. K.; Kudpatseva, I. L. M. Zh. Anal. Khim. 1978, 33, Kohara, H. Bunseki Kagaku 1974, 23, Gao, H. W.; Yang, J. X. olloids urf., A 22, 25, Gao, H. W. hin. J. hem. 23, 21, Gao, H. W.; Yang, J. X.; Zhou, Z. Z.; Zhao, J. F. Phytochem. Anal. 23, 14, Murphy, J. B.; Kies, M. W. Biochem. Biophys. Acta 196, 45, 382. (E LU, Y. J.)