ADSORPTION BEHAVIOUR OF 5-METHYLRESORCINOL AND ITS MIXTURES WITH LYSOZYME AT AIR-WATER INTERFACE - Biochemistry and Biotechnology: Research and Development - page 97

M ATERIALS AND M ETHODS
A sample of hen egg white lysozyme (Sigma, USA) with activity 20,000 U/mg and
molecular mass 14,445 Da was used.
Alkyl-substituted hydroxybenzenes, 5-Methylresorcinol (5-Methylbenzene-1,3-diol)
(Sigma, USA) with molecular mass 124,14 g/mol (anhydr) was taken.
All reagents for PBS preparation in Milli-Q water were of analytical grade.
Surface Tension Measurement
Dynamic surface tension was measured with a Tracker tensiometer (ITC, France),
connected to thermostatic bath to maintain the temperature constant at 25 O C during the
measurements. The principle of tensiometer is to determine the surface tension of the studied
solution from the axisymmetric shape of a rising bubble analysis [Loglio et al., 2001]. Due to
the active control loop, the instrument allows long-time experiments with a constant
drop/bubble volume or surface area.
Surface tension, σ (mN/m), was measured in 7 mL samples at constant lysozyme (0.05
mg/mL) and varying 5-methylresorcinol (0.16-128.2 mM) concentrations and their mixed
solutions in 0.05M PBS, pH 7.4 at 25 O C.
The solutions of pure MR were taken for surface tension measurement after 2 or 24 hours
of storage at 25 O C in darkness. The mixed solutions LYS-MR were prepared with using of
lysozyme and MR after 24 hours incubated separately. After mixing of these solutions 1:1
they were stored 3 hours at 25 O C away from light. The dynamic surface tension was
measured over 30 000 s to guarantee steady-state of the adsorption layer. A t→∞ asymptotic
extrapolation was used to find the steady-state surface tension values. Standard deviations
were always less, than 0.5 mN/m, and duplicate measurements were made for each MR
concentration. Finally phosphate buffer were confirmed not to present surface activity by
measuring separately the surface tension of a PBS buffer solution, obtaining values
practically equal to those of pure water.
From the kinetic curves (surface tension versus time) for the solutions of different
composition, the steady-state surface tension isotherms (surface tension versus concentration)
were obtained. As the concentration of the protein in the mixture was fixed, the abscissa in all
graphs represents the concentration of the surfactant in the mixed solution. From surface
tension data the MR critical concentration of self-organization (micelle-formation) in the
absence and in presence of lysozyme were estimated. For this purpose surface tension values
were fitted with the logarithm of the MR concentration.
R ESULTS AND D ISCUSSION
Adsorption of Methylresorcinol
Surface tension is a phenomenon which arises due to the existence of intermolecular
forces in solution. The molecules at the interface experience an inward pull from bulk