Influence of organIc solvents on the furIn actIvIty

Furin belongs to a family of calcium-dependent serine proprotein convertases, which transform the inactive protein precursors into mature polypeptides. In model experiments, we studied the effect of organic solvents such as acetone, dimethyl sulfoxide (dmSO), dioxane, isopropanol and ethanol on the furin activity. Furin was found to retain up to 88% of its initial activity in the presence of dmSO, whereas in the presence of acetone only 30%. Organic solvents formed the following decreasing sequence of their effects on furin: acetone> isopropanol> ethanol> dioxane> dimethyl sulfoxide. The relationship between the residual furin activity and solvent parameters such as relative polarity, dipole moment and log P were investigated. The effect of the organic solvent appeared not to correlate with any of the listed characteristics. Laidler-Sсatchard’s graphs, which according to a theory must be linear, demostrated non-linearity. These results indicate that not only electrostatic interactions play an important role in the studied enzymatic reaction but also other factors, e.g. hydrophobic contacts, hydrogen bonds can influence furin catalysis. This seems relevant for further research in this area.


F
urin is a member of the Ca 2+ -dependent serine proprotein convertases (PCs) family, which is involved in the maturation of precursors in the secretory pathway.It transforms both soluble and membrane-bound proproteines into biologically active products: hormones, growth factors, receptors, enzymes, adhesion molecules, etc. [1][2][3].In addition to normal physiological functions furin can participate in an array of different pathological states including cancer, various bacterial and viral infections, atherosclerosis, endocrinopathies, neurodegene rative diseases [4][5][6].Therefore, furin is considered as an attractive target for the synthesis of specific inhibitors, which could find biochemical, clinical and thera peutic applications [3][4][5][6].
Furin is accumulated in the trans-Golgi network (TGN) and then recycles along TGN the cell membrane, and the endosomal compartmets [3], where it interacts with its protein substrates activating them.Since the polarity, pH values and ion concentration in cell compartments differ [7], we decided to study in the model experiments the effect of various components of the incubation medium (for example, metal ions, organic solvents, etc.) on furin.
In the literature, there are data on the effect of metal cations such as sodium and potassium [8][9][10], calcium and magnesium [1,10], zinc, mercury and manganese [11] and other ions on the catalytic proper ties of furin.Previously, we also investigated the effect of some heavy metal ions: cesium, cobalt, and cadmium on the enzyme [12].
This report focused on the studying of the effect of some organic solvents added to the reaction medium on the furin catalytic activity.The influence of organic solvents on various enzymes has been studied in many laboratories [13][14][15][16][17][18], however there are no much relevant data on furin.Thus, it is crucial not only to create specific inhibitors of furin, but also to study the specific effects of various factors of the incubation medium on it.That can provide valuab le information on the molecular mechanisms of furin action.
The purpose of our work was to carry out a comparative study of the effect of organic solvents on furin.To obtain data on the sensitivity and stability of the enzyme to the action of different solvents, we investigated their effect on the furin activity in the range of solvent concentrations from 0 to 30% (v/v).
Polar dimethyl sulfoxide (DMSO) is often used as a standard solvent for studying the effects of various synthetic and natural compounds on enzymatic processes.It is usually used to dissolve test compounds and then to screen them in model experiments.The addition of nonpolar dioxane to the incubation medium allows changing its dielectric constant in the wide range from about 80 to 2. Ethanol and isopropanol, in addition to changing the permittivity, can compete with water in the acylenzyme cleavage stage, thereby inhibiting the enzymatic reaction.

Materials and Methods
reagents and preparations.The following organic solvents were used: 1,4-dioxane, DMSO, acetone, ethanol and isopropanol (Sigma, USA).Commercial products were: EDTA (Serva, Germany), HEPES (Sigma, USA), β-mercaptoethanol, Triton (Fluka, Switzerland).The fluorogenic substrate Boc-Arg-Val-Arg-Arg-AMC was purchased from Bachem (Switzerland), and the shorter recombinant human furin (2000 U/ml) was purchased from New England BioLabs (USA).The amount of enzyme that under standard conditions cleaves 1 pmol of 7-amino-4-methylcoumarin (AMC) from the fluorogenic substrate per min was taken as the unit of furin activity.Before work, the solution of the commercial preparation of the enzyme was diluted 20-fold with a working HEPES buffer (pH 7.3) and the resulting solution was used to carry out the enzymatic reaction.
The inhibition coefficients of furin activity were calculated by the formula: А = (V 0 -V)/V 0 , where V 0 -is the initial maximum rate of the enzymatic reaction, and V -is the maximum reaction rate in the presence of an organic solvent in the incubation medium.
determination of furin activity.An aliquot of the furin solution, containing 1 unit of the enzyme activity was incubated in pH 7.3 buffer (100 mM HEPES, 1 mM CaCl 2 , 0.5% Triton X-100 and 1 mM β-mercaptoethanol) for 1 h at 37 ° C with fluorogenic substrate (75-250 μM) in a 150 μl sample.The reaction was terminated by adding 2 ml EDTA (initial concentration 5 mM) and the relative fluorescence was measured on a PTI Quanta Master 40 spectrofluorometer (Canada) at an excitation wavelength of 380 nm and an emission of 460 nm.The fluorescence readings were recorded for 60 sec.The values of the Michaelis constants were determined by plotting the Lineweaver-Burk graphs in three independent experiments.
Determination of the influence of organic solvents on furin.The concentrations of organic solvents in the mixture with buffer ranged from 0 to 30% (v/v).The furin solution (10 μl) containing 1 unit of enzyme activity, the required amount of organic solvent and 10 μl of the fluorogenic substrate were successively added to HEPES buffer (total volu me of the sample was 150 μl).The reaction was terminated by adding 2 ml EDTA, and further processing of the samples was conducted as described above.The fluorescence values for the mixtures which did not contain organic solvents, were taken as 100%

results and Discussion
In the presence of organic solvents, enzymes usually, undergo denaturation and inactivation [13,14].This is due to, for example, changes in pK values of acidic and basic groups not only of the active center or surface of the enzyme, but also dissociating groups of buffer solutions [13].
Organic solvents can affect not only the stability and activity of enzymes but also change their specificity [13].To date, examples of proteinases resistant to the action of organic solvents are well known [15][16][17][18].
To obtain data on the furin stability and sensitivity to various solvents, we tested following organic solvents: acetone (aprotic polar solvent), 1,4-dioxane (aprotic nonpolar solvent), ethanol and isopropanol (proton-containing polar solvents).Their effects on furin activity are shown in Fig. 1.
It is seen that the effect of the solvent depends on its chemical nature.Thus, the presence of dioxane (5%) or isopropanol (10%) in the incubation medium caused an increase in the enzyme activity by about 30% and 60%, respectively.A further increase in the concentration of these solvents led to a dose-dependent decrease in the furin activity.In the presence of ethyl alcohol or acetone (concentration 10%), there was practically no increase in the enzyme activity, but an increase in the solvent concentration in the reaction medium was found to inhibit furin activity.Acetone appeared to exibit the greatest inhibitory effect on the enzyme.Thus, at concentration of 25% a decrease in the initial enzyme activity by 70% was observed.The least effect on furin was observed in the presence of DMSO: furin remained active (~88%) at the solvent concentration up to ~25%.Thus, among the studied solvents, DMSO was found to be the best solvent in terms of furin stability.The tested organic solvents (at a concentration of 25%) can be arranged in the descending order of their effect on the enzyme: acetone > isopropanol > ethanol > dioxane > DMSO.
The important physico-chemical characteristics of organic solvents are known to be dielectric permeability, polarity and hydrophobicity.Thus, the influence of the solvent on the chemical reaction is determined by its donor-acceptor properties and dielec tric permittivity [13].
We studied how the enzyme activity changed (at a solvent concentration of 25% in the incubation medium) depending on the following characteristics of the solvents: dipole moment, the calculated log P value, and relative polarity.These characteristics were found on the Internet and are given in Table 1.
The inhibitory effect of the studied solvents was found to not correlate with any of the above characteristics, although the best approximation to the linear dependence was observed for the solvent relative polarity values (Fig. 2).
In this graph, the two points corresponding to dioxane and DMSO were outside the straight line.
According to the theory [21], Laidler-Scatchard's plots reflecting the dependence of the enzymatic reaction efficiency (ln A) on the dielectric constant of the medium (1/ε) must be linear.To make sure, we decided to construct an appropriate graph for furin.Using the initial data (Table 2), the values of the dielectric permittivity of buffer-solvents mixtures (pH 7.3) were calculated (see the Materials and Methods section) at their concentrations 0, 5, 10, 15, 20, 25 and 30%, and changes in the furin activity depending on 1/ε of the incubation medium were traced (Fig. 3).
Fig. 3 shows the corresponding plots in the Laidler-Scatchard's coordinates.
The presented plots show a non-linear dependen ce, and the greatest deviations from linearity were observed at sites corresponding to an or-   ganic solvent concentration of 0-15%.When ethyl alcohol was used at concentration 0-20% the linear dependence of the Laidler-Scatchard graph was reali zed in this part of the curve (Fig. 3, B, 2).For isopropanol and dioxane (Fig. 3, a, 1 and 2), as well as for acetone (Fig. 3, B, 3), the rectilinear section corresponds to the solvent concentration range of 15-30%.When using DMSO, there is no linear dependence on the Laidler-Scatchard graph (Fig. 3, B,  1).These features of the plots may indicate that in catalysis by furin, an important role play not only electrostatic interactions, but other factors such as hydrophobic contacts and hydrogen bonds between the enzyme binding site and the substrate/inhibitor also have significant values for reaction [13].The viscosity of the medium or its ionic strength, etc., can also be of great importance for catalysis [13].To assess their contribution to the enzymatic reaction, further study is needed.