As mentioned above, milk XO exists in molybdenum-free form and is localized in the inner layer of MFGM. Therefore, the activation of milk XO requires the incorporation of exogenous molybdenum into its active center. It is generally known that XO belongs to heat-stable enzymes – it remains active at a 75 – 80 °C temperature in several minutes (Alikulov et al., 1980). However, at this temperature, the enzyme molecules undergo partially reversible denaturation. Therefore, one of the possible ways for the availability of XO molecules for exogenous molybdenum is the disintegration of milk fat globule membranes and partial denaturation of enzyme molecules. Thus, during the heat treatment:

Fresh milk samples were obtained from healthy female animals at a local farm near Astana city.

Chemicals were purchased from Sigma-Aldrich Chemical Co. All common chemicals and solvents used were of analytical grade.

Ethylenediaminetetraacetic acid (EDTA) to bind heavy metals was used for binding heavy metals from milk. Milk was boiled in narrow conical tubes in a volume of 2 ml. Special reagents were used dor detecting different activities of XO. Egg yolk lecithin from AppliChem (Panreac, ITW Companies, Darmstadt, Germany) was used in the experiments.

Method for determination of the intrinsic activity of xanthine oxidase (XO). To determine the intrinsic activity of XO 200 μL aliquot of milk was mixed with 700 μL 0.1 M sodium phosphate buffer containing 10 μM EDTA, 5 μL phenylmethylsulfonylfluor (for inhibition of protease activity). To this mixture was added 100 μM 10 mM hypoxanthine. The mixture was incubated at 30 °C for 10 min under aerobic conditions. The proteins in the mixture are precipitated with trichloroacetic acid. After centrifugation, the amount of uric acid (under the influence of XO converted hypoxanthine to uric acid) in the supernatant was determined by measuring the absorbance of the reaction mixture in a spectrophotometer at 295 nm (Suzuki et al., 2015). The amount of uric acid also is determined by its chemical oxidation with potassium permanganate (K₂MnO₄) or hydrogen peroxide (H₂O₂), thereby forming allantoin (Poje and Sokolić-Maravić, 1986). Allantoin in biological liquids is easy to very accurately determined by a known method (Vogels and Van der Drift, 1970).

Method for the determination of nitrate- and nitrite-reducing activity of XО. The nitrate-reducing activity of XO is determined by the disappearance of the added nitrate (NO₃^-) or by the appearance of nitrite (NO₂^-) in the reaction medium (Alikulov et al., 1980). The nitrite-reducing activity of XO is determined by the disappearance of the nitrite to the reaction medium or by the appearance of nitric oxide (NO) (Kuo et al., 2003).

Preparation of the milk for the detection of the various enzymatic activities. Before treatment in the milk of domestic animals added 10 μM ethylenediaminetetraacetic acid (EDTA) to bind heavy metals. For boiling fresh milk is poured into the narrow conical tubes in a volume of 2 mL. Then, for further determination of the enzymatic activity the tubes are placed in the water bath at 35 °C temperature and kept for 10 min, then using the special reagents different activities of XO are determined.

All determinations were conducted in triplicate or more and all results were calculated as mean ± standard deviation (SD). In this study Statistical analysis was performed using Student’s t-test for significance and analysis of variance (ANOVA). The p-values less than 0.05 were considered statistically significant. The statistical analysis data were produced by Microsoft Excel and Statistica 15.

Our preliminary experiments showed that the optimal heating temperature and its duration for detection of all activities in fresh milk XO were 80 °C and 7 min (data not shown). Besides, the presence of 0.1 mM concentrations of cysteine and exogenous molybdenum was also necessary for the detection. Thus, to detect the activity of MFGM-bound enzyme is required heat treatment and the addition of cysteine as an antioxidant and molybdenum to fresh sheep milk. In the next experiments, we examined various natural and artificial antioxidants for the detection of milk XO activity. Natural antioxidants – cysteine, glutathione, lipoic acid, and ascorbic acid, and artificial antioxidants – dithiothreitol, unithiol and mercaptoethanol (2,3-dimercapto-1-propane sulfonic acid) were used in different concentrations (Figures 2, 3, 4).

Results present in the Figures 2, 3, 4 show that among the natural antioxidants L-cysteine was more effective in the stabilization of XO in heat-treated milk. In the same concentrations the effectiveness of cysteine-containing antioxidant – glutathione was considerably lower. This is because the tripeptide molecule of glutathione contains only one cysteine residue. Although both the oxidized (disulfide) and reduced (dihydric) forms of lipoic acid (dithiol) show antioxidant properties (Milkowski et al., 2010; Rochette et al., 2013), its stabilizing effect for milk XO was lower than glutathione. Among the artificial antioxidants, monothiol mercaptoethanol was more effective in the stabilization of heated milk XO. Although dithiothreitol and unithiol contain two sulfhydryl groups they don’t stabilize heat-treated milk XO. Recently we found that dithiothreitol in the presence of sulfanilamide forms a stable blue complex with molybdate ion (based on this finding we developed a new fast method for molybdenum determination in the biological materials).

Results in the Figures 5, 6, 7 show that the highest levels of the associated activities of milk XO observed with artificial electron donor – methyl viologen reduced by dithionite (Na₂S₂O₄). Among the physiological electron donors, the highest associated activities were obtained using reduced NADH. The natural substrate of XO – hypoxanthine was also effective in the donating of electrons for NR and NiR activities of the enzyme in fresh sheep milk. Thus, the electron transfer may also occur from the hypoxanthine (the reducing substrate) to nitrite and nitrate (the oxidizing substrates) (Abadeh et al., 1992; Godber et al., 1997). Because in the presence of tungstate in the reaction mixture the associated activities were not detected, during nitrate reduction by hypoxanthine, the XO molybdenum center may participate directly in both the oxidative (hypoxanthine oxidation) and the reductive (nitrate and nitrite reduction) half-reactions.

It is found that animal XO converts NO₃^- and NO₂^- to physiologically important gas – nitric oxide (NO) (Millar et al., 1998; Zhang et al., 1998; Hord et al., 2011). NO effectively reacts with L-cysteine or reduced glutathione (GSH) at pH 7.0 and 7.4, to form orange-pink products of S-nitrosocysteine (CySNO) or S-nitrosoglutathione (GSNO). These products exhibited a peak absorbance at around 340 and 540 nm (Kuo et al., 2003).

It is known that NADH is one of the potential physiological electron donors for XO and it also has an absorbance at 340 nm (reduced NADH exhibits strong UV absorption at 340 nm whilst the oxidized form has virtually no absorption at this wavelength).

Therefore, to avoid mutual interference between the optical density of NADH and CysNO at 340 nm, instead of NADH we used hypoxanthine as an electron donor for NR and NiR activities of milk XO. It was demonstrated that heat treatment (80oC, 10 min) of homogeny XO resulted in the release of molybdenum cofactor (Mo-co) from the active center of the denatured enzyme molecule. During the heat treatment of XO ascorbic acid was the potential protector against the oxidation of released Mo-co. However, in the absence of ascorbic acid, it is quickly inactivated by oxygen (even in anaerobic conditions) (Alikulov et al., 1980; Vogels and Van der Drift, 1970). Later we showed that glutathione and cysteine were the more powerful protectors for isolated Mo-co (Alikulov and Mendel, 1984; Poje and Sokolić-Maravić, 1986).

However ascorbic acid decomposes S-nitrosocysteine (Stsiapura et al., 2018) and, therefore, we used L-cysteine as the protector against the oxidation of the cofactor in the active center of XO localized in MFGM. Thus, in our experiments L-cysteine was used for double purposes: as the protector of enzyme Mo-co against the oxidation during heat treatment of milk and as a reagent for S-nitrosothiol formation. For the construction of the calibration curve, we used nitroprusside as a donor of NO. Increasing concentrations of nitroprusside from 10 nM to 1.0 μM mixed with constant 0.1 mM concentration of cysteine in milk serum. Micromolar concentrations of nitroprusside release Nanomolar concentrations of NO (Porras et al., 1981; Kramer et al., 1987). The serum was obtained by heating fresh milk, precipitating its proteins with acetic acid, and centrifuging. To obtain a transparent milk serum, its filtration was carried out through a membrane (33 mm Millex Filter).

For NO determination sheep milk mixed in the ratio of 1:1 with 0.2 M chlorinated phosphate buffer (PBS), pH 6.5, containing 10mM NEM, 2.5 mM EDTA (Kisker et al., 1997; Marley et al., 2001), 0.2 mM Na₂MoO₄ or Na₂WO₄ and 0.1 mM cysteine. After incubation in 15 minutes at a temperature of 36 °C, the milk proteins were precipitated by diluted acetic acid added to the reaction mixture until pH 4.0. After centrifugation in the supernatant absorbance at 340 nm and 540 nm were measured. For many years tungsten was considered to be a biological antagonist of molybdenum and was used for the study of the properties and functions of molybdenum in Mo-enzymes. This was because tungsten can replace molybdenum in Mo-enzymes, forming catalytically inactive analogs (Kletzin and Adams, 1996; Bray et al., 1999; Beedham, 2001; Taibi et al., 2001). Therefore, to make sure that it is the molybdenum enzyme that catalyzes the formation of NO, instead of molybdenum we incubated the milk in the presence of tungsten, and the results obtained (Tables 1, 2) convincingly show that the heat treatment of fresh sheep milk in the presence of exogenous molybdenum activates XO and the enzyme becomes capable of converting nitrate and nitrite to nitric oxide. However, when nitrates were used as a substrate NO formation was very low. At the same time, using nitrites as substrate resulted in a 10 times higher amount of formed NO (i.e. CySNO) in comparison with nitrate substrates. The levels of CysNO determined by absorbance at 340 and 540 nm were completely different. This was likely due to the difference in the sensitivity of the absorption at the ultraviolet and visible wavelengths of the spectrophotometer.

Phospholipids (PLs) are formed by glycerol, phosphoric acid, fatty acids, and a hydroxyl compound (e.g., choline, ethanolamine, serine, inositol). It is well known that milk fat globule membranes (MFGM) is composed of four main species of PLs: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS). The most abundant PLs in milk fat expressed as a percentage of the total, are PE (26.4% – 72.3% of total PLs), PC (8.0% – 45.5%), PI (1.4% – 14.1%), and PS (2.0% – 16.1%). Of the lipids, phosphatidylcholine is largely located on the outside while the neutral lipids, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol are concentrated on the inner surface (Garcia et al., 2012; Suzuki et al., 2015). Amphiphilic properties of PLs derive from the presence of both a hydrophobic tail and a hydrophilic head make them natural detergents. Thus, under high-temperature phospholipids may effectively disrupt hydrophobic bonds between fatty acids, e.g., the integrity of all membrane layers of MFGM (Godber et al., 2000; Gladwin et al., 2005). Therefore, the destruction of MFGM makes the enzyme molecules available for exogenous molybdenum and its substrates (hypoxanthine, nitrate, and nitrite).

In the next experiments, we examined the effect of lecithin on NR and NiR activities of heat-treated fresh sheep milk. Egg yolk lecithin was used in the experiments. The main phospholipids of this lecithin are phosphatidylcholine and phosphatidylethanolamine, i.e. they are components of the outer and inner layers of the membrane. The lecithin was dissolved in isopropyl alcohol and then added to the milk before heat treatment. The other components required for the heat treatment of milk have already been written above. Hypoxanthine was used as an electron donor for NR and NiR activities.