The Inhibitory Effects of L-Adrenaline on Lactoperoxidase Enzyme Purified from Bovine Milk

Abstract

L-Adrenaline belongs to a group of compounds known as catecholamines, which play an important role in the regulation of the physiological process in living organisms. In the present study, the inhibitory effect of L-adrenaline on lactoperoxidase was examined. Lactoperoxidase (E.C.1.11.1.7) was purified from bovine milk with three consecutive steps: Amberlite CG-50 resin, CM-Sephadex C-50 ion-exchange, and Sephadex G-100 gel filtration chromatography. Lactoperoxidase was purified with a yield of 42.18%, a specific activity of 30.33 EU/mg proteins, and 20.77 purification fold. Enzyme purity was determined with SDS-PAGE, where a single band was observed. The R_z (A₄₁₂/A₂₈₀) value for lactoperoxidase was 0.9. The effect of L-adrenaline on lactoperoxidase was determined using ABTS as a chromogenic substrate. The half maximal inhibitory concentration (IC₅₀) value and an inhibition constant (K_i) values for L-adrenaline were 34.5 and 2.26 μM, respectively. L-Adrenaline was found to be a non-competitive inhibitor.

Keywords:

• L-Adrenaline
• Lactoperoxidase
• Enzyme purification
• Inhibition

INTRODUCTION

L-Adrenaline (L-epinephrine) is one of the neurotransmitter catecholamines that are released by the sympathetic nervous system and adrenal medulla in response to a range of stresses in order to regulate the host physiological functions in living systems. These physiological functions are involved in regulation of blood pressure, vasoconstriction, cardiac stimulation, relaxation of the smooth muscles, as well as in several metabolic processes.[1,2] L-Adrenaline has a strong antioxidant and antiradical activity.[2] Recently, antioxidant properties of pure molecules or extracts has gained great importance.[3–6] Adrenaline has a variety of clinical applications; for example, it is included in relieving respiratory distress in asthma, in treating hypersensitivity reactions due to various allergens, cardiac arrest, or it is used as a topical hemostatic agent, etc.[7–9] It is responsible for metabolic actions like raising the blood glucose levels. Hypoglycemia causes the elevation of plasma adrenaline.[10] In the adrenal medulla, adrenaline is localized in separate populations of chromaffin cells and in adrenaline-containing a cells.[11] Adrenaline serves as a carrier for the nervous system, influencing the constriction of blood vessels and controlling tissue metabolism by increasing the glucose and lactic acid levels.[12] It plays a central role in the short-term stress reaction, the physiological response to the conditions that threaten the physical integrity of the body. The medical treatment of chronic heart failure has undergone a remarkable transition over the past 10 years. But the exact mechanism of adrenaline and antagonist recognition remains unknown. In addition, the signal-transduction path from the initial adrenaline binding across the membrane into the interior of the cell has not been fully elucidated to date.[2,13]

Milk is an opaque white liquid produced by the mammary glands of mammals. It provides the primary source of nutrition for young mammals. Milk also contains a variety of compounds that protect the neonate as well as the milk itself from a host of deleterious microorganisms. One of those compounds is the enzyme lactoperoxidase.[14] Lactoperoxidase (LPO; EC 1.11.1.7) is a heme-containing glycoprotein with a single chain of 612 residues. LPO contains about 10% carbohydrate and its molecular mass is approximately 78 kDa.[15] LPO catalyzes the oxidation of halides and pseudohalides at the expense of hydrogen peroxide and it generates antimicrobial products, and as a result, catalyzes the inactivation of a wide range of microorganisms.[16–18] The other members of the mammalian peroxidase family include eosinophil peroxidase (EPO), thyroid peroxidase (TPO), and myeloperoxidase (MPO). LPO, EPO, and MPO contribute to the nonimmune host defense system by oxidizing halide and pseudohalide ions to produce potent antimicrobial agents.[19] LPO is also present and active in many more secretory fluids in various parts of the body, including tears and saliva.[17] It was reported that LPO carries out this function in exocrine secretions, including milk, tears, and saliva, while EPO and MPO play similar roles in the phagosomes of eosinophils and neutrophils, respectively, during engulfment of microorganisms. On the other hand, TPO is an intracellular membrane-bound protein, which is involved in the catalysis of the iodination and coupling of thyroglobulin moieties in the biosynthesis of the thyroid hormones thyroxine and triiodothyronine.[19] Bovine milk LPO has higher activities, more than other types of LPO; therefore, most research works are focused on bovine LPO.[20] The objective of this study was to evaluate in vitro effects of L-adrenaline on LPO, which was purified from bovine milk.

MATERIALS AND METHODS

Chemicals

Adrenalin, Sephadex G-100, CM-Sephadex C-50, 2,2′-azino-bis(3-ethylbenzthiazoline-6 sulfonic acid) (ABTS), Coomassie brilliant blue R-250, and standard proteins (egg albumin, bovine albumin, and β-galactosidase) were obtained from Sigma Aldrich Chemie Gmbh. Co. (Steinheim, Germany) Amberlite CG-50 resin was purchased from Fluka Chemie (Switzerland).

Determination of Lactoperoxidase Activity

Lactoperoxidase activity was determined by the procedure of Shindler and Bardsley[18] with a slight modification.[21] This method is based on oxidation of ABTS as a chromogenic substrate with hydrogen peroxide. The formed, colored compound gives an absorbance at 412 nm. As typical procedure, 2.8 mL of 1 mM ABTS in phosphate buffer (0.1 M, pH: 6.0) was mixed with 0.1 mL of enzyme in phosphate buffer (1 mM, pH 6.0) and 0.1 mL of H₂O₂ solution (3.2 mM) and the absorbance was taken at 412 nm as a function of time in every 15 s during 3 min.[21]

Lactoperoxidase Activity Unit

One unit of LPO is defined as the amount of enzyme catalyzing the oxidation of 1 μmol of ABTS min^-1 at 298°K (molar absorption coefficient; 32,400 M^-1 cm^-1).[22,22]

Protein Determination

Protein concentration was determined according to the method of Lowry and co-workers.[23] Bovine serum albumin (BSA) was used as standard protein.[24–26]

Purification of Lactoperoxidase

Bovine milk fat was removed by centrifugation at 5000× g for 15 min at 48°C. Afterwards, fresh raw skimmed milk, in the proportion of 22 g/L, was added on Amberlite CG-50 resin, which was prior equilibrated with sodium phosphate buffer (5 mM, pH 6.8).[20,21,27] The supernatant was decanted and the resin was washed with distilled water, then equilibrated with 20 mM sodium phosphate buffer (pH 6.8). The bound protein was eluted with 500 mM sodium phosphate buffer (pH 6.8). Solid ammonium sulphate was gradually added into eluted green-colored mixture (I Precipitation; saturation 90%), over a period of 30 min while it was being stirred continuously and the enzyme solution was dialyzed overnight against 5 mM sodium phosphate buffer (pH: 6.8).

The clear greenish supernatant obtained above was loaded onto a column of CM-Sephadex C-50 (3 × 10 cm) previously equilibrated with 10 mM sodium phosphate buffer (pH: 6.8). The column bounded enzyme was washed with 100 mL of 10 mM phosphate buffer (pH: 6.8) containing 100 mM NaCl. The enzyme was eluted with a linear gradient by using 100–200 mM NaCl in 10 mM phosphate buffer (pH: 6.8) and subjected to ammonium sulphate precipitation (II Precipitation, saturation 90%). Thereafter, the enzyme solution was dialyzed overnight against phosphate buffer (5 mM, pH: 6.8).[21,22]

LPO was obtained from the CM-Sephadex C-50 column, which was applied to a column of Sephadex G-100 (2.5 × 100 cm). The column-bound enzyme was eluted with 100 mM phosphate buffer (pH: 6.8), and salted out with ammonium sulphate precipitation (III Precipitation, 90% saturation). The enzyme solution was dialyzed overnight against phosphate buffer (500 mM, pH 6.0). Fractions were lyophilized and checked for purity by SDS-PAGE gel.[28–30]

Ammonium Sulphate Precipitation

Ammonium sulphate precipitation is a putative method used to purify proteins by altering their solubility. It is a specific case of a more general technique. Since proteins differ markedly in their solubilities at high ionic strength, salting-out is a very useful procedure to assist in the purification of a given protein. Ammonium sulphate is the most commonly used salt. It is very water soluble and has no adverse effects upon enzyme activity. In the preliminary test, the ammonium sulphate concentration is increased stepwise, and the precipitated protein is recovered at each stage. The precipitated protein is then removed by centrifugation and then the ammonium sulphate concentration is increased to a value that will precipitate most of the protein of interest while leaving the maximum amount of protein contaminants still in the solution. The precipitated protein of interest is recovered by centrifugation and dissolved in fresh buffer for the next stage of purification. LPO obtained from the CM-Sephadex C-50 column was subjected to ammonium sulphate fractionation and the precipitate in the 0–90% saturation range was collected by centrifugation 60 min at 15.000× g. The precipitate was suspended in about 2 mL of phosphate buffers (0.5 M, pH 6.0). This technique is useful as a first step in many purification schemes to quickly remove large amounts of contaminant proteins. It is also often employed during the later stages of purification to concentrate protein from dilute solution following procedures, such as gel filtration.[31,32]

SDS-PAGE

SDS-PAGE was performed under denaturing conditions after LPO purification, according to Laemmli's procedure.[33] The stacking and running gels comprised 3 and 10% acrylamide, respectively, and SDS (0.1%). The electrode buffer was 25 mM Tris/200 mM glycine (pH 8.3). The sample buffer was prepared by mixing Tris-HCl (0.65 mL; 1 M, pH: 6.8), SDS (3 mL 10%), neat glycerol (1 mL), bromphenol blue (1 mL, 0.1%), β-mercaptoethanol (0.5 mL), and water (3.85 mL). A 20-μg aliquot of enzyme (50 μL) was added into the sample buffer (50 μL) and the mixture was heated in a boiling water bath for 3 min and then cooled at room temperature.[34–37]

LPO samples were loaded into each space of the stacking gel. LPO was analyzed separately by polyacrylamide gel electrophoresis. Initially, an electric potential of 80 V was (Hoefer Scientific Instruments SE 600, Belgium) applied until the bromphenol dye reached the running gel. Then it was increased to 200 V for 3–4 h. Gels were stained for 1.5 h in 0.1% (w/v) Coommassie Brilliant Blue R-250 in (50%) methanol and (10%) acetic acid, and destained with methanol/acetic acid.[38–40]

RESULTS AND DISCUSSION

LPO is a member of the mammalian peroxidase family. It catalyzes the oxidation of thiocyanate and halides. As reported in the literature, LPO, EPO, and TPO are monomeric proteins while MPO is a covalently linked dimer of two identical halves each consisting of two polypeptide chains of 108 and 466 amino acid residues as a result of a posttranslational deletion of 6 amino acid residues.[41] LPO has been intensely studied over the years, and it is also present and active in many more secretory fluids in various parts of the body. The effect of increasing concentrations of L-adrenaline (8.33–55.0 μM) on LPO enzyme activity was determined.

Firstly, LPO was purified from bovine milk using CM-Sephadex C-50 ion exchange chromatography. Then the enzyme obtained from CM-Sephadex C-50 ion exchange chromatography was applied to a Sephadex G-100 gel filtration chromatography (Table 1). Specific activity was determined for each of the purification steps. Kinetic parameters such as K_m and V _max were calculated by a Lineweaver-Burk graph for ABTS substrate.

Table 1 Purification scheme of LPO obtained after the application of different purification steps

Purification steps	Total volume (mL)	Enzyme activity (EU/mL)	Total enzyme activity (ABTS Unit)	Protein (mg/mL)	Total protein (mg)	Specific activity (EU/mg)	Recovery (%)	Purification fold
Homogenate	185	2.53	468.05	1.73	320.05	1.46	100	1.00
Ammonium sulphate precipitation	35	12.86	450.10	2.09	73.15	6.15	96.19	4.21
CM-Sephadex C-50 column chromatography	302	1.35	407.70	0.095	28.69	14.21	87.1	9.73
Ammonium sulphate precipitation	31	11.43	354.33	0.66	20.46	17.32	75.7	11.86
Sephadex G-100 column chromatography	143	1.96	280.28	0.10	14.30	19.60	59.9	13.42
Ammonium sulphate precipitation and dialyze	31	6.37	197.47	0.21	6.51	30.33	42.18	20.77
LPO: Lactoperoxidase enzyme purified from bovine milk.

The K_m value was found to be 0.358 mM; on the other hand, V _max values were calculated as 18.86 μmol/(mL × min). The purification of LPO was controlled by SDS-PAGE. Bovine serum albumin (66 kDa) and ovalbumin (45 kDa) were used as standards proteins. LPO purified from bovine milk when subjected to SDS-PAGE electrophoresis exhibited only one band of LPO, as shown in Fig. 1, column 1.

Figure 1 Column 1: SDS-PAGE bands of LPO purified from bovine milk. Column 2: standard proteins. Line a: bovine serum albumine (66 kDa). Line b: ovalbumine (45 kDa). LPO: lactoperoxidase; SDS-PAGE: sodium dodecylsulphate-polyacrylamide gel electrophoresis. (Color figure available online.)

There is no detailed study regarding the effect of L-adrenaline on LPO activity. In the present study, L-adrenaline was investigated for its inhibitory effect on LPO and kinetic constants, such as K_i and IC₅₀, were evaluated (Table 2). The results obtained from the present study clearly showed that L-adrenaline had a strong inhibitory effect on LPO activity.

Table 2 IC₅₀ values, K _i constant, and inhibition type of L-adrenaline as inhibitor of LPO

	L-Adrenaline
IC50 value (mM)	34.5
Ki constant (μM)	2.26
Inhibition type	Non-competitive
LPO: Lactoperoxidase enzyme purified from bovine milk.

The concentration required for 50% inhibition (IC₅₀) and of inhibition constants (K_i ) values are often reported in the literature, but direct comparison of these values is not possible. The concentration of L-adrenaline that required inhibiting LPO activity by 50% (IC₅₀) and inhibition constant K_i were determined. The IC₅₀ and K_i values were used to compare the inhibitory potential of L-adrenaline. To determine K_i value as well as the inhibition type, at least three different L-adrenaline concentrations were selected. At each of the L-adrenaline concentrations, enzyme activity was measured in the presence of various substrate concentrations. The relationship of K_i and IC₅₀ for a given compound varies depending on the assay conditions and the compound's mechanism of inhibition. In this study, K_i and IC₅₀ parameters for L-adrenaline as an inhibitor of bovine LPO was determined. The inhibitor concentrations causing up to 50% inhibition were determined from activity (%)-[L-adrenaline] graphs. As seen from Fig. 2, the IC₅₀ value for L-adrenaline was determined to be 34.5 μM. In addition, K_i values were calculated from Lineweaver-Burk graphs (Fig. 3). The K_i constant for L-adrenaline was 2.26 μM. L-Adrenaline had exhibited non-competitive inhibition. These results showed that LPO had an affinity to L-adrenaline. On the other hand, in a previous study, in vitro effects of ketamine and bupivacaine as analgesic agents were determined on LPO activity.[20,21] The K_i constants for both anesthetic drugs were found to be 19 and 15 μM. Moreover, IC₅₀ values were found to be 290 and 155 μM, respectively. Similarly, the inhibitor effect of melatonin and serotonin on LPO activity was determined by the same group.^[21] K_i values for both hormones were found to be 0.82 and 0.26 μM.

Figure 2 The effect of different concentrations of L-adrenaline (8.33–55.0 mM) on lactoperoxidase obtained from bovine milk (R ²: 0.9984).

Figure 3 Lineweaver-Burk graph for different ABTS concentrations and three different L-adrenaline concentrations for determination of K_i constant.

The binding and structural studies of bovine lactoperoxidase with three aromatic ligands, such as acetylsalicylic acid (ASA), salicylhydoxamic acid (SHA), and benzylhydroxamic acid (BHA), were studied by Singh and co-workers.[41] Their study demonstrated that all three compounds bind to lactoperoxidase at the substrate binding site on the distal heme side. The binding of ASA occurs without perturbing the position of conserved heme water molecule W-1, whereas both SHA and BHA displace it by the hydroxyl group on hydroxamic acid moieties. The acetyl group carbonyl oxygen atom of ASA forms a hydrogen bond with W-1, which in turn makes three other hydrogen bonds, one each with heme iron, His-109 N2, and Gln-105 N2. In contrast, in the complexes of SHA and BHA, the OH group of hydroxamic acid moiety in both complexes interacts with heme iron directly. The OH is also hydrogen bonded to His-109 N2 and Gln-105 N2. The plane of benzene ring of ASA is inclined at 70.7° from the plane of heme moiety, whereas the aromatic planes of SHA and BHA are nearly parallel to the heme plane. The mode of ASA binding provides the information about the mechanism of action of aromatic substrates, whereas the binding characteristics of SHA and BHA indicate the mode of inhibitor binding.^[41] This molecule can bound the heme group of lactoperoxidase. As a conclusion, these results showed that L-adrenaline had greater inhibition on LPO. It showed in vitro inhibition of LPO activity as non-competitive. According to the results obtained from the present study, L-adrenaline was found to be a marked LPO inhibitor.