Volumetric and viscometric behaviour of soya bean and gram proteins in aqueous methotrexate ( anticancer drug ) solution at 298 . 15 to 308 . 15 K

Apparent molar volumes (Vφ) and viscosities (η) for 0.00005 to 0.0004 kg.mol -1 (50 to 400 μmol kg) aqueous methotrexate (MTX), gram (Gp) and soya bean (SBp) proteins, and similar compositions of Gp and SBp each in 50 to 400 μmol kg aqueous MTX were determined at 298.15, 303.15 and 308.15K. The Vφ values are positive except aqueous MTX and listed as SBp > Gp > MTX in aqueous and SBp > Gp in MTX solutions, respectively. It infers weaker hydrophobic heteromolecular interaction of SBp in binary and ternary systems. The higher η values of SBp and Gp in MTX than those of aqueous MTX prove strengthening of hydrophobic interactions of proteins by MTX. It illustrates the conformational changes of proteins; slightly higher Vφ values of SBp than of Gp. The MTX confirm higher structural activity in biological process. The Vφ 0 of MTX increases with K and decrease with composition. But with compositions the values continuously decrease with lower magnitude.


INTRODUCTION
Currently thermodynamics and transport functions of naturally occurring proteins in aqueous solution are of biophysical significance.Due to dominance of spectroscopic techniques, only limited physico-chemical studies are available in literature; soya and gram proteins have never been focused for such studies.Majority of proteins responds to aqueous and mixed solvent due to polyionic nature.Thus solvation has become an interesting tool for solute-solvent interactions, which gives some insights of structural interactions useful for biological activities due to conformational states.Therefore protein-drugs interactions have become the nucleus of biotechnological and pharmaceutical innovations.The physico-chemical characterizations of proteins in drug solutions have not been paid adequate attention and scarcity of such data with protein-drug solutions is noted.However such studies *Corresponding author.E-mail: rajeshkr_yadav2003@yahoo.co.in.Tel: 91-011-26217579.could render substantial help for drug design and understanding the physical basis of their structural interactions.Thereby in the present work the model studies with methotrexate (MTX), an anticancer drug was chosen for the studies of the ρ, V φ , η and B data which depict internal state of the molecule in solution.This approximately calculates the state of intermolecular forces, hydrophilic and hydrophobic interaction.Additionally Several biological effects and action of MTX are given elsewhere (Farber et al., 1956;Weinblatt et al., 1985;Bookbinder et al., 1984;Baggott et al., 1993;Cronstein et al., 1991Cronstein et al., , 1993;;Morabito et al., 1998;), and biochemical mechanism (Gao et al., 1998;Dolhain et al., 1998), influence on production in lymphocytes (Morgan et al., 1998;Nesher et al., 1997;Genestier et al., 1998) have dealt with, with biological aspects where no cognizance of physicochemical studies is made.However HPLC studies of MTX with reverse phase are quoted elsewhere (Smolenski et al., 1990(Smolenski et al., , 1993(Smolenski et al., , 1991)).Thus a brief literature review on studies of MTX with respect to biological relevance has found no physico-chemical reference yet, along with Table1 a .Experimental and literatures density values along with apparent molal volumes and viscosities at 303.15k.

MATERIAL AND METHODS
Soya bean and gram were extracted from raw seed powder of soya and gram, respectively, and their purity was checked (David et al., 2000).The MTX (purity 98%, Aldrich), Gp and SBp solutions were prepared, while cleaned bicapillary pyknmometer and viscometer were calibrated with water with ±0.00003/10 3 kg mol -1 and ±0.00004/0.1 kg m -1 s -1 accuracies, respectively, agreeing with reported values.The solutions were filled in them for density and viscosity, with special attention to prevent an evaporation of solutions.The aqueous BSA, egg albumin and lysozyme were run as controlled system and an agreement with our data given in Table 1.The densities, apparent molar volumes and viscosities of systems are measured and found as a linear function of temperature.The measurements were carried out in a thermo-statically controlled and well-stirred water bath with a temperature accuracy of ± 0.01°C, read on Beckmann thermometer.

Measurements
Calibration of Pyknometer and viscometer was checked with aqueous NaCl solutions (Mohammad et al., 2002) at 298.15K at a thermal stability of bath better than ± 0.01°C.A Hewlett-Packard quartz thermometer calibrated with gallium temperature standard measures the bath temperature and an accuracy of concentration of solutions was better than 1 x 10 -5 m.The 0.99705, 0.99565 and 0.99404/10 3 kg m -3 density of water (Alexander and Manzurola. 1999) at 298.15, 303.15 and 308.15K, respectively, were used.The calibration was repeated immediately before and after each measurement of density and reproducibility was better than 1 x 10 -3 kg m -3 .The kinetic correction to energy of viscometer was calculated and found to be 1.8977 x 10 -4 , 2.32647 x 10 -5 and 2.32647 x 10 -6 at The Vpyk is pyknometer volume calculated from (W0-We)/ρ0 and Vφ data are computed with ρ from following equation: The M is molar mass of solute.An uncertainty in Vφ is computed from the equation Vφ = (1000/m)∆ρ/ρ The ∆ρ = ρ-ρ0, the viscosity η is calculated from relation η = η0(ρ.t)/(ρ0.t0) The t and t0 flow times and η and η0 are viscosity of solution and solvent and the ηrel (relative viscosity) is calculated from η/η0.Like ρ, the errors in η data were obtained.

RESULTS AND DISCUSSION
The ρ, V φ and η data are least square fitted against m for values at infinite dilution referred to as limiting values) from the following equation: The ρ 0 is limiting density and S d slopes.The V φ is fitted in equation The V φ 0 is limiting constant, S v and S v * are slopes.The V φ 0 focuses solute-solvent and the S v and S v * the solutesolute and charge-charge interactions, respectively.The η rel is fitted to extended Jones-Dole equation (Singh et al., 2005)  ) 3 are slopes measuring heteromolecular interactions.Tables 2-4 and 7-12 contains primary data,    13-15 the regression constants, 5 and 6 regression constants of systems.MTX > Gp > SBp sequence of ρ 0 values for binary systems at each K with 0.00162 and 0.00184 decreases for MTX, the 0.00157 and 0.00459 for Gp, and 0.00459 and 0.00175/10 3 kg.m -3 for SBp from 298.15 to 303.15K and from latter to 308.15K (Table 5), respectively.It depicts a generation of higher internal pressure on the solutions due to stronger heteromolecular forces in sequence of MTX > Gp > SBp.The higher ρ 0 values than of water at each K conclude their stronger hydrogen bond formation with water.Firstly, the hydrogen bonded water is broken and interact strongly with MTX and proteins; thus are referred to as water structure breaker.It reveals stronger MTX structural interactions than those of Gp and weaker of the SBp with water, due to its one -CONH-    -3 kg mol -1 decrease in B values with K is noted for SBp proving a larger hydrodynamic volume than of others due to an effective conformational structure.It shows larger dependence of the stability of hydrodynamic size of their hydrated complex on thermal energy.The B values for MTX are lower by 2701.2083, 1916.5833, 273.0417 and 1519.5000, 263.4584, 104.2917/10 -3 kg mol -1 than those of SBp and Gp at three temperatures.It proves weaker Newtonian force on vis-cous flow due to weakly stable water-MTX hydrogen bonding.But the B values of SBp are higher than of Gp by 1519.5000,263.4584 and 104.2917/10 -3 kg mol -1 at each K (Table 6) with reverse behavior of MTX.

Ternary systems
The ρ 0 values of proteins for aqueous MTX are found higher than those of the aqueous (Table 13) by 0.00079 to 0.00255 and 0.00090 to 0.00182/10 3 kg m -3 at each K.The MTX asserts an additional internal pressure in solutions, credited to stronger protein-MTX interaction rather than water-proteins (Figure 1a).Here, the MTX with polar parts, foster the water structure breaking action of proteins weakening hydrogen bonds of water facilitating a cage formation of it and MTX around the proteins (Figure 1b).The hydrated MTX may be fitted into the interstitiary spaces of the β pleated sheets and α helix of the protein to channe-  Secondly MTX might be causing stronger hydrophilic interaction and hydrophobic with proteins making sandwiched between them or their hydrated complex.In either way it is reorienting and modifying structures and activity of the proteins hampering the hydrophobic force.   lowering in values (Table 13), reveals a comparatively stronger Gp-MTX interactions with larger activity of the Gp along with stronger concentration effect.Thus water with MTX favors cage formation around Gp with stronger molecular forces due to hydrophobic interactions of Gp with MTX and weaker with SBp.The Gp causes stronger cohesive forces that increase with composition with higher ρ 0 values in MTX.The ρ 0 values of Gp and SBp systems decrease with composition and K.It elucidates an increase in residual forces attributed to solute-solute interac-Table 13.The regression constants of ρ of proteins in aqueous Methotrexate system.The limiting density, ρ 0 /g cm - 3 , slope constants Sd/10 3 g 2 cm -3 mol -1 at 298.15, 303.15 and 308.15K.bonding with water against water-water molecules.Such interac-tions play key role in chemical denaturation and conform-ational state of proteins and contribution of MTX as is estimated with the present set of thermodynamic and transport properties.The SBp>Gp order of V φ 0 values in MTX at each K indicates that the SBp is comparatively less packed.Thus its packing might be associated with the number of amino group and nature of amino acids of proteins.The ρ 0 values in MTX could be rationalized to strengthening of the protein-water interactions by MTX due to their stronger binding with each other exerting higher internal pressure.With MTX concentration, the ρ 0 of proteins decreases (Tables 7 and 10) weakening electrostatic forces between water and proteins.For higher MTX concentrations the ρ 0 values remains almost same depicting stabilizing effect on protein conformational states that overcomes the influence of protein concentrations.Thus stronger water-MTX-protein interactions exist facilitating their solvation, where MTX concentrations cause unrest increasing entropy leading to the unfolding of proteins.For SBp, an increase in MTX composition seems to weaken the intermolecular forces between protein and water as MTX decreases the electrorestriction of water by increasing MTX-water interactions (Figure 1c).The van der Waals forces operating among MTX, protein and water (Figure 1b) does monitor hydrophilic as well as hydrophobic interactions.The action of MTX for concentrations and temperature remains similar for Gp (Table 13).Their V φ 0 (Marshall, 1993;Pandey et al., 1987) in MTX with concentrations and temperature decrease while the S v and S v * increase.It infers generation of slightly stronger intermolecular forces with stabilization of hydrophobic forces amounting to their restriction of rearrangement of bonds around higher concentrations.Their V φ 0 values with MTX concentration decrease (Table 14) with a close agreement with the observations of (Marsh et al., 1995).Thus MTX develops higher internal pressure by strengthening heteromolecular forces shrinking the volume.Such action mechanism of MTX matches the exertion effect caused by nonsteroidal anti-inflammatory drugs reported (Dewan et al., 1998).Molecular ion of MTX causes stronger dipole-dipole and dipole-induced-dipole with polyions (proteins) interactions due to hydrogen bonding.Due to the larger sizes of the molecular and polyions, a weak London or dispersive molecular forces operate in solutions.The compositions enhance the magnitude of their contributions due to various types of interactions and temperatures.Critically these effects derived from the S v and S v * values are positive and negative, respectively and show similarity with organic molecules in ethanol systems (Parmar et al., 2004;Fort, 1966).

0.0004m Regression constants of Gram protein
The SBp>Gp sequence of B values in MTX (Table 15) with 7.9167 to 79.2917/kg.mol - decreases at each K is lower than of aqueous proteins by 37.0000 to 4024.5833/kg.mol - and predicts stronger influence of MTX on their hydrodynamic sphere.In this context the Gp is seen to be mild water structure breaker while SBp slightly strengthen the intermolecular forces.Such behaviors of them could be attributed to the torsional forces in complexes due to their geometry.Notably the η values for SBp and Gp are higher in MTX than in water proving protein-protein interaction as hindrance to the laminar flow causing stronger tortional forces (Tables 4, 9  and 12).The stronger intermolecular force on viscous flow causes stronger protein interactions with larger cohesive forces.This depicts that the cage model remains very effective and polyvalent polyions of proteins develop the hydration sphere of larger size.The B values are found higher (Table 15).The B values of SBp and Gp are lower in aqueous MTX than those of in water, it prove disruption of the cage model developing smaller sized hydration sphere due to the weakening of solutecosolute-solvent interactions by Newtonian flow.Due to a large hydrophobic part in proteins and the possible dominance of the hydrophobic and hydrohpilic groups, their interactions in MTX are expected to produce lower B values as compared to those in water.The positive B values denote a net structure-promoting tendency of MTX, also the MTX, SBp and Gp produce positive dB/dT values which are reported to be structure breakers in MTX, while in aqueous behave as structure maker.For aqueous proteins a slight decrease in the B values with K, denotes the thermal denaturation and a negligible decrease in B values in MTX, shows less denaturation.Singh et al. 1819 Thus the MTX bears with the thermal changes proteins structure at higher temperatures as is reported elsewhere (Nishida et al., 2002(Nishida et al., , 2001;;Ramesh et al., 1992;Shitara et al., 2003).

Conclusion
The studies substantiate the naturally occuring proteinsanticancer drug interactions focusing a drug phamacokinetics and dynamics.Also the impact of composition and thermal changes on the solute-solvent and solutecosolute-solvent interactions denoted with ρ 0 , V φ 0 and B functions.The V φ 0 and slope values of proteins demonstrate that Gp casts stronger intra and intermolecular forces shrinking the molecule to a lower volume.The higher and lower S v and S v * values for SBp in MTX prove that SBp-SBp interaction becomes stronger than that of Gp-Gp and higher value of slope proves stronger pairwise interaction.
MTX), gram (Gp) and soya bean (SBp) proteins, and similar compositions of Gp and SBp each in 50 to 400 µ µ aqueous and SBp > Gp in MTX solutions, respectively.It infers weaker hydrophobic heteromolecular interaction of SBp in binary and ternary systems.The higher η η η η values of SBp and Gp in MTX than those of aqueous MTX prove strengthening of hydrophobic interactions of proteins by MTX.It illustrates the conformational changes of proteins; slightly higher V φ φ φ φ values of SBp than of Gp.The MTX confirm higher structural activity in biological process.The V φ

Table 7 .
Densities (ρ) of Soya Bean protein in aqueous Methotrexate system with uncertainty in each data given ±σ sign at298.15,303.15 and 308.15K.

Table 9 .
Viscosity (η)of Soya Bean protein in aqueous Methotrexate system with uncertainty in each data given ±σ sign at298.15,303.15 and 308.15K.
lize the residual force that shrinks the molecules.

Table 14 .
The regression constants of Vφ of proteins in aqueous Methotrexate system.

Table 15 .
The regression constants of (ηr -1)/m of proteins in aqueous Methotrexate system.