Plasma and Hemocyanin Phenoloxidase Derived from the Hemolymph of Giant Freshwater Prawn Macrobrachium rosenbergii (De Man, 1879)

Abstract

We attempted to study the immune response in M. rosenbergii by melanization reaction produced by plasma phenoloxidase (PO) activity. The substrate affinity of the PO enzyme was determined using different phenolic substrates, and it was found that the diphenols were only oxidized. The enzyme was characterized as catechol oxidase type of PO and L-3,4 dihydroxyphenylalanine (L-DOPA) showed the highest substrate affinity to the enzyme. The biochemical parameters that determined optimum enzyme activity were found to be 2.5 mM L-DOPA at an absorbance of 470 nm, 10 mM Tris–HCl buffer at pH 7.5, temperature at 25°C, and 15 min incubation. Kinetic characteristics of plasma were studied from the M. rosenbergii. The hemocyanin was isolated by gel filtration chromatographic technique using Sephadex G-100. The M. rosenbergii hemocyanin (MrHC) showed only one band with a molecular weight of 325 kDa on native polyacrylamide gel electrophoresis (PAGE) when stained with Coomassie Brilliant Blue (CBB) and bathocuproine sulfonic acid. The reduction of MrHC protein in SDS-PAGE displayed three subunits with a molecular weight of 74, 76, and 78 kDa, respectively. Determination of optimal condition for PO activity of plasma has also been attempted. The plasma optimal condition taken for the MrHC was tested for its ability to oxidize diphenols such as L-DOPA was shown only PO activity. These results showed that in the presence of PO and peroxidase inhibitors, phenylthiourea (PTU) and tropolone respectively have decreased plasma and MrHC PO activity. This indicates that hemocyanin triggers innate immunity probably through one of its subunits that function as the active moiety.

1. Introduction

Aquaculture is the fastest-growing, food-producing, profitable, and one of the major employment generating sectors in coastal areas and is expected to quintuple in the coming 50 years [1]. The giant freshwater prawn, Macrobrachium rosenbergiiknown as scampi, is an important aquaculture species in tropical and subtropical regions with the immense commercial export market value; however, the production of the prawns is curtailed by diseases caused by opportunistic pathogens in the rearing environment [2]. Resistance to diseases is based on the strategic improvement of the immune system of the animal and requires intensive research on immune components and its function [3]. As for any invertebrate, the innate immune mechanism in prawns includes cellular [4, 5] and humoral components [6, 7, 8, 9].

Invertebrates lack adaptive immunity; therefore, they completely depend on innate immune systems for host defense. Melanization, which is a major innate defense system in invertebrates, is controlled by the enzyme phenoloxidase (PO) [10, 11, 12]. The active PO is a bifunctional enzyme that catalyzes the o-hydroxylation of monophenols to catechols and the oxidation of o-diphenols into o-quinones [13]. The first reaction involves monophenolase activity, which converts tyrosine to L-DOPA, which is then oxidized to quinone by diphenols activity of PO [13, 14]. The resulting quinones are converted to melanin by a series of intermediate steps involving enzymatic and nonenzymatic reactions [15, 16]. In one of these enzymatic reactions, dopachrome is decarboxylated by dopachrome isomerase (also called dopachrome tautomerase or Dopachrome Conversion Factor) to form dihydroxy indole, which is then converted to melanin [17, 18, 19].

The immune response in crustaceans mainly depends on nonspecific immunity, involving the cellular immunity of hemocytes [20] and humoral immunity through phenoloxidase [8] and agglutination [9]. In insects and crustaceans, phenoloxidase usually exists as a nonactive zymogen, prophenoloxidase (proPO), whose activation to the PO form is tightly regulated via an enzymatic cascade because the melanization reaction generates toxic compounds such as quinone species. This cascade is triggered by the presence of several microbial cell wall components such as β-1,3-glucan, lipopolysaccharides, and peptidoglycan [10]. There is a detectable or high amount of PO activity in crustacean plasma [8, 21, 22] that could be derived from proPO released from hemocytes [22] or from hemocyanin [8, 23, 24] for melanization activity, which remains unclear.

The present study attempted to characterize plasma PO activity in terms of substrate specificity, optimum ionic strength, pH, temperature, and incubation time to determine the biochemical and physiological conditions that support enzyme activity. Furthermore, to understand the substrate affinity of the plasma PO enzyme activity, the kinetics of the enzyme’s rate of reaction was determined in the Lineweaver-Burk plot. There is evidence to show that the kinetics of the crustaceans phenoloxidases vary among the different components of the hemolymph as well as species [25, 26, 27, 28]. Hence, an attempt has been made to optimize the conditions for determining PO activity of plasma including Km and Vmax value of freshwater prawn M. rosenbergii. Based on the determination of optimal condition, PO activity in MrHC (325 kDa) has also been attempted.

2. Materials and methods

2.1 Experimental freshwater prawns

Adult intermoult of the giant freshwater prawns, M. rosenbergiiweighing around 25–30 ± 1.58 g were purchased from Aqua Farm, Kalpakkam, Kanchipuram District, Tamil Nadu, India. The prawns were carefully transported to the laboratory maintained in 500 L FRP tanks (10 no of tanks, per tank 25 animals) containing at room temperature (28°C + 1°C) continuously aerated freshwater, which was changed thrice a week. The prawns were fed with egg white ad libitumand were acclimatized to the laboratory conditions for at least 4–5 days before use. Twenty-five percent of water was renewed daily to remove the unfed and fecal materials. The uninjured, intermoult animals were used throughout this study.

2.2 Hemolymph collection and preparation of plasma

Hemolymph (100 μl) was collected by cardiac puncture using a 23G needle attached to a clean, sterile plastic syringe containing 1.9 ml of ice cold iso-osmotic buffer, TBS-I (Tris 50 mM, NaCl 210 mM, KCl 5 mM, MgCl2 2.5 mM, pH 7.5) mixed and centrifuged in a pre-chilled polypropylene tube (161 x g, 8 min, 4°C) to obtain 1.5 ml of the supernatant as plasma. The exclusion of hemocytes was verified in the collected plasma by observation under phase-contrast microscope. About 50 prawns (each determination, N = 50) were required for collection of 100 μl acellular plasma, following Sivakumar et al. [8].

2.3 Oxidation of phenolic substrates

We tested the oxidative activity of 0.1 ml plasma was tested by incubating with 1.9 ml of different phenolic substrate solutions (5 mM tyrosine, tyramine, L-DOPA, DL-DOPA, dopamine, catechol, hydroquinone, and pyrogallol) for 20 min at 25°C. The color developed was measured spectrophotometrically (Shimadzu UV-160A spectrophotometer, Kyoto, Japan) at 300–700 nm against a reagent blank in which suitable substrates were substituted for plasma.

2.4 Effect of different concentrations of L-DOPA

To 0.1 ml of plasma was mixed 1.9 ml of L-DOPA at different concentrations (1–10 mM) and incubated for 20 min at 25°C. The color developed was measured spectrophotometrically at 470 nm against a reagent blank (L-DOPA).

2.5 Effect of ionic strength on oxidation of L-DOPA

The effect of buffer ionic strength on oxidation of L–DOPA by plasma was assessed by incubating 0.1 ml plasma with 1.9 ml of 2.5 mM L–DOPA prepared in different ionic strength (5–100 mM) at 25°C. After 20 min, the optical density of each of these reaction mixtures was determined spectrophotometrically at 470 nm against a reagent blank (L-DOPA).

2.6 Effect of pH on oxidation of L-DOPA

The ability of plasma to oxidize L-DOPA at different pH was tested by incubating 0.1 ml of plasma with 1.9 ml of a substrate of (2.5 mM L-DOPA) solutions prepared in 10 mM Tris–HCl buffer at different pH (6.0–9.0) for 20 min at 25°C. The color developed was measured spectrophotometrically at 470 nm against a reagent blank (L-DOPA).

2.7 Oxidation of L-DOPA exposed to different temperature

Effect of different temperature was tested by incubating 0.1 ml of plasma with 1.9 ml of substrate (2.5 mM L-DOPA) solutions prepared in 10 mM Tris–HCl (pH 7.5) buffer at a different temperature ranging from 10 to 90°C for 20 min. The color developed was measured spectrophotometrically at 470 nm against a reagent blank (L-DOPA).

2.8 Effect of various time intervals on L-DOPA

To 0.1 ml of plasma was mixed 1.9 ml of 2.5 mM L-DOPA (10 mM Tris–HCl; pH 7.5) and incubated for different time intervals (5–30 min) at 25°C. The color developed was measured spectrophotometrically at 470 nm against a reagent blank (L-DOPA).

2.9 Kinetic parameters, km, and Vmax of plasma phenoloxidase enzyme

To measure the kinetic parameters of plasma PO enzyme, different concentrations of L-DOPA (1.0–10.0 mM) were mixed with 0.1 ml of plasma and incubated for 15 min and absorbance was read at 470 nm. Michaelis–Menten constant was estimated by plotting substrate concentrations [S] and rate of PO activity [V]. Lineweaver-Burk plot was plotted as reciprocal of substrate concentration [1/S] and rate of PO activity [1/V]. The resultant plot is given a line that intercepted X-axis to give −1/Km value and intercepted the Y-axis to give 1/Vmax. The slope Km/Vmax was determined, and the resultant plot was rechecked using Eq. Y = mx + c.

2.10 Partial purification of hemocyanin

To 50 ml of plasma was centrifuged and dialyzed (MW exclusion limit <14,000 kDa and > 12,000 kDa) extensively against TBS-II (Tris 10 mM, NaCl 200 mM, CaCl₂ 10 mM; pH 7.5). Then the dialyzed plasma was ultracentrifugation at 200,000 xg for 180 min at 4°C (Beckman LE-80; Beckman Coulter, Brea, CA, USA). After ultracentrifugation, the supernatant was decanted and the pellet, which is made of hemocyanin, was collected and dissolved in TBS-II and used freshly for further purification.

2.11 Purification of MrHC

To purify the MrHC, a Sephadex G-100 (Sigma-Aldrich; bead diameter: 40–120 μm) column (36 x 1.6 cm; XK 16, Pharmacia, Uppsala, Sweden) was prepared using gel filtration chromatography technique and thoroughly equilibrated with TBS-II. The hemocyanin collected by ultracentrifugation from the plasma was passed through the Sephadex G-100 column at a flow rate15 ml.h⁻¹. The purified fractions were continuously monitored for absorbance at 280 nm and 1 ml fractions were collected. The collected protein samples were stored at −80°C for further analyses.

2.12 Determination of protein

The protein content in the plasma and purified MrHC (325 kDa) samples were determined according to Bradford [29] using bovine serum albumin as the standard. All chemicals used in the study were purchased from Sigma-Aldrich, St. Louis, MO, USA.

2.13 Electrophoretic analysis

The protein profiles of plasma and purified MrHc were analyzed in discontinuous polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions [30]. This was performed using 3% stacking gel (pH 6.7) and a 7% separating gel in Tris-glycine buffer (pH 8.9). Electrophoresis was performed at a constant current of 3 mA/sample at 10°C for 2 h on a slab gel measuring 8 x 8 cm. The gels were stained with Coomassie brilliant blue (CBB) R-250 (GE Health Care Biosciences, Tamil Nadu, India) or bathocuproine sulfonic acid following the methods of Maurer [30] and Bruyninckx et al. [31].

The molecular masses of the purified MrHc were estimated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with 5 and 10% polyacrylamide as the stacking and separating gel, respectively, following the method of Laemmli [32]. The purified MrHc subunits were visualized by staining the gel with CBB R-250. The molecular weight of the purified MrHc subunits was calculated using molecular marker proteins (GE Health Care Biosciences, Tamil Nadu, India).

2.14 Oxidation of diphenolic substrates by purified MrHC

We tested the oxidative activity of 40 μl purified MrHC by incubating with 160 μl of different phenolic substrate solutions (2.5 mM L-DOPA, DL-DOPA, dopamine, and catechol) for 15 min at 25°C. The color developed was measured spectrophotometrically at 300–700 nm against a reagent blank in which Tris–HCl buffer (10 mM, pH 7.5) was substituted for purified MrHC.

2.15 Phenoloxidase activity

The PO activity of plasma (0.1 ml) or purified MrHC (40 μl) was incubating with 1.9 ml or 160 μl of phenolic substrate solution (2.5 mM L–DOPA) for 15 min at 25°C. After incubation, the color developed was measured spectrophotometrically (Shimadzu UV-160A spectrophotometer, Japan) or using ELISA plate reader (BioTek, PowerWave XS, USA) at an absorbance of 470 nm against a reagent blank of substrate solution (L–DOPA).

2.16 Effect of inhibitors on PO activity

In this experiment, 0.1 ml of plasma or 40 μl of purified MrHC was mixed with an equal volume of inhibitors. 3 mM Phenylthiourea (PTU) or 16 mM tropolone containing 16 mM H₂O₂ was preincubated for 10 min at 25°C. An aliquot of 0.2 ml or 80 μl of these reaction mixtures from control or experiments was incubated with 1.8 ml or 120 μl of 2.5 mM L-DOPA for 15 min at 25°C. After incubation, the color developed was measured spectrophotometrically or ELISA plate reader at an absorbance of 470 nm against a reagent blank of substrate solution.

2.17 Statistical analysis

The data were expressed as mean ± SD of triplicate experiments from five determinations. Statistical analyses were done using SPSS software (version 20; SPSS, New York, USA). The variation between experimental and control was evaluated by one-way analysis of variance (ANOVA) and significance was assessed at 0.01 probability (^**p < 0.01).

3. Results

3.1 Effect PO activity with various substrates

The plasma separated from the hemolymph of the freshwater prawn M. rosenbergiiwas tested for PO activity with various phenolic substrates. Among the diphenolic substrates, the plasma showed the highest activity with L–DOPA (470 nm) when compared with DL-DOPA (440 nm), dopamine (440 nm), and catechol (470 nm) as shown in Figure 1. However, the monophenols including tyramine and L-tyrosine or polyphenols such as hydroquinone and pyrogallol failed to show any oxidation by plasma. Since the highest oxidative activity was obtained with L-DOPA, this substrate was used to detect PO activity in all subsequent experiments performed in this study.

Figure 1.

PO activity of plasma with different phenolic substrates (5 mM) in Tris buffer (Tris–HCl 50 mM, pH 7.5) incubated at 25°C for 20 min and absorbance at 300–700 nm. The PO activity in optical density obtained at absorbancy maxima of respective substrates. Data represents mean of triplicate repeats of five determinations (mean ± SD) in the same way in all further experiments.

3.2 Effect of substrate concentration on PO activity

The plasma PO activity was tested with different concentrations of L-DOPA (1.0–10.0 mM), and the PO activity was found to be higher with L-DOPA at a concentration of 2.5 mM than that of 1 mM or higher concentrations (5.0, 7.5, and 10.0 mM) as shown in Figure 2. This experiment clearly suggested that the optimum concentration for testing PO activity in plasma was 2.5 mM of L-DOPA.

Figure 2.

Effect of different concentrations of substrate (L-DOPA) by plasma phenoloxidase activity of freshwater prawnM. rosenbergii.

3.3 Effect of ionic strength

The PO activity of plasma was tested with Tris–HCl buffer (pH 7.5) of different ionic strengths (5–100 mM), and the highest PO activity was found with 10 mM Tris–HCl buffer when compared with other ionic strengths tested as shown in Figure 3. This result recommended that the optimum concentration for testing PO activity in plasma was 10 mM of Tris–HCl buffer.

Figure 3.

Effect of ionic strength of Tris–HCl buffer on oxidation of L-DOPA (2.5 mM) by plasma phenoloxidase activity of freshwater prawnM. rosenbergii.

3.4 Effect of optimum pH

The PO activity of plasma was assessed by oxidation of L-DOPA at various pH values ranging from 6.0 to 9.0, pH above 7.5 showed the brown color formation of dopachrome. The PO activity was decreased at pH 6.0–7.0 and 8.0–9.0; thus, pH 7.5 was taken as the optimum pH for the study of plasma PO activity (Figure 4).

Figure 4.

Effect of pH on oxidation of L-DOPA (2.5 mM), Tris–HCl buffer (10 mM) by plasma phenoloxidase activity of freshwater prawnM. rosenbergii.

3.5 Effect of optimum temperature

The PO activity of plasma was demonstrated by performing oxidation of 2.5 mM L-DOPA in the presence of 10 mM Tris–HCl at a pH 7.5. The reaction mixture was incubated for 20 min at different temperatures ranging from 10 to 90°C. The PO activity was stable and attained a peak at 25°C, which was taken as an optimum temperature for PO activity. At temperature below or above 25°C, a decline in PO activity was observed (Figure 5).

Figure 5.

Effect of temperature on oxidation of L-DOPA (2.5 mM), Tris–HCl buffer (10 mM), pH 7.5 by plasma phenoloxidase activity of freshwater prawnM. rosenbergii.

3.6 Effect of time intervals

The PO activity of plasma was evaluated by performing oxidation of 2.5 mM L-DOPA in the presence of 10 mM Tris–HCl at a pH 7.5 and temperature 25°C at various incubation periods ranging from 5 to 30 min. The maximum PO activity was at 15 min, which was determined as the optimum incubation time (Figure 6).

Figure 6.

Effect of incubation time on oxidation of L-DOPA (2.5 mM), Tris–HCl buffer (10 mM, pH 7.5) by plasma phenoloxidase activity of freshwater prawnM. rosenbergii.

3.7 Kinetic behavior

The kinetic characteristics of plasma PO activity were determined from the rate of the reaction, which was calculated from the oxidation of L-DOPA at different concentrations (1.0–10.0 mM) in 15 min. The Michaelis–Menten constant Km was calculated to be 0.75, and maximum velocity (Vmax) was found to be 0.58 as shown in Figure 7A. Application of Km and Vmax yielded Lineweaver-Burk plot with a line slope of 1.2, which on extrapolation intercepted at −1.3 that was plotted as −1/Km and on Y-axis 1/Vmax was derived at 1.7 on X-axis (Figure 7B).

Figure 7.

(A) Kinetic properties of PO activity in plasma ofM. rosenbergiiat different substrate concentrations of L-DOPA as shown in Michaelis–Menten curve. (B) The K_m and V_max values are calculated using Lineweaver-Burk plot of PO activity in plasma ofM. rosnbergiiwith L-DOPA as substrate.

3.8 Purification of hemocyanin from the plasma of M. rosenbergii

The hemocyanin was loaded on the Sephadex G-100 column for gel filtration chromatographic separation, and the purified MrHC peak fractions were collected at an absorbance of 280 nm (Fiure 8A). Hemocyanin protein was identified on PAGE–7% by CBB staining as distinct single bands of molecular weight 325 kDa (Figure 8B; lane 2). Staining with BCSA affirmed that the proteins contained copper and represented the copper containing proteins of hemocyanin (Figure 8B; lane 3). The chromatographic separation with electrophoretic observations of the separated proteins clearly indicated the occurrence of hemocyanin in M. rosenbergiias single separate copper-containing protein. As shown in Figure 8C, the purified MrHC protein after reduction in SDS-PAGE (10%) cleaves into three subunits of 74, 76, and 78 kDa molecular mass, respectively (lane 2).

Figure 8.

(A) Gel filtration chromatographic profile of hemocyanin sample was applied on to the pre-equilibrated column of Sephadex G-100. The elution was performed at a flow rate 15 ml.h⁻¹. The fractions were continuously monitored for absorbance at 280 nm. (B) Electrophoretic analysis (PAGE—7%) of purifiedMrHC stained with CBB and BCSA after gel filtration chromatography from freshwater prawnM. rosenbergii. Lane 1: Molecular weight markers; lane 2: PurifiedMrHC (325 kDa) CBB stained; lane 3: PurifiedMrHC (325 kDa) bathocuproine sulfonic acid stained under UV light for copper-protein. (C) Electrophoretic profile of purifiedMrHC (325 kDa) protein was run under reduced conditions of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE 10%) and stained with CBB. Lane 1 molecular weight protein markers; lane 2 purifiedMrHC (74, 76 and 78 kDa).

3.9 Phenoloxidase activity with diphenolic substrates in MrHC

The purified MrHC (325 kDa) was tested for PO activity with diphenolic substrates. Among the substrates, the purified MrHC (325 kDa) showed activity only with L-DOPA while with the other diphenols, such as DL-DOPA, dopamine, and catechol, it failed to show any oxidation activity. Since the highest oxidative activity was detected with L-DOPA (Figure 9), this substrate was used for the determination of phenoloxidase activity and inhibition study.

Figure 9.

PO activity of purifiedMrHC (325 kDa) with diphenolic substrates (2.5 mM) in Tris buffer (10 mM, pH 7.5) incubated at 25°C for 15 min and absorbance at 300–700 nm. The PO activity in optical density obtained at absorbancy maxima of respective substrates. Data represents mean ± SD of 5 determinations using purifiedMrHC (325 kDa) sample from different preparations.

3.10 Effect of PO inhibitors on oxidation of L-DOPA by plasma and purified MrHC

Pretreatment of plasma or purified MrHC with PTU (3 mM) decreased the oxidation of 2.5 mM L-DOPA compared with control, and the reduction was found to be about 91.55% and 55.80%, respectively. However, pretreatment of plasma or purified MrHC with tropolone (16 mM), another strong inhibitor of PO activity, also showed a significant reduction in the oxidation of L-DOPA when compared with control, and the reduction in the phenoloxidase with plasma and MrHc (77.88% and 88.95%) was statistically significant (p < 0.01) as shown in Figures 10A and B.

Figure 10.

(A) Phenoloxidase activity in plasma and (B) purifiedMrHc (325 kDa) ofM. rosenbergiiand conformation of PO activity using inhibitors (PTU and tropolone). Asterisk indicates significant variation determined from the value obtained for a treatment to untreated control by one way ANOVA at ^**p < 0.01. Data represents mean of triplicate repeats of five determinations (mean ± SD).

In summary, for the plasma or purified MrHC (325 kDa), the optimal conditions for measuring PO activity on L-DOPA (2.5 mM) were 10 mM Tris–HCl, pH 7.5 at 25°C for 15 min at 470 nm.

4. Discussion

The hemocyanin showed phenoloxidase (PO) activity in M. rosenbergiiand appears to be enhanced with activators such as proteases, SDS, and microbes [8] and agglutination activity [9]. The PO enzymes and hemocyanin molecules belong to the same class of copper proteins, and this explains its PO function [33]. Considering the complexity of crustacean immune defenses, our study attempted to explain the variance in the immune function of PO activity in the plasma and hemocyanin of M. rosenbergii. In most crustacean species active PO is a bifunctional enzyme that catalyzes o-hydroxylation of monophenols to diphenols and then oxidizes o-diphenols into o-quinone [15, 26]. The substrate affinity of plasma PO activity was attempted with monophenols including tyrosine and tyramine, diphenols such as L-DOPA, DL-DOPA, dopamine, catechol, or polyphenols such as hydroquinone and pyrogallol. The results clearly suggested that the plasma showed the highest substrate affinity with diphenols, and among the diphenols, L-DOPA was found to show the highest PO activity suggesting catechol oxidase activity.

Biochemical studies were undertaken to describe the optimum condition of the plasma PO activity. The enzyme reaction was observed with different concentrations of L-DOPA. There was a steady increase in the enzyme activity from 1 mM to 2.5 mM concentration of L-DOPA after which an increase in substrate concentration did not enhance the enzyme activity proving substrate inhibition as the cause of the decline in enzyme activity. The previous reported substrate-specific phenoloxidase activity of hemocytes derived from Penaeus monodonand M. rosenbergiiusing 1.6 mg/ml of L-DOPA [4] and M. rosenbergiiinjected with Gram-positive Lactococcus garvieaeand Gram-negative Aeromonas veroniiwas monitored for changes in phenoloxidase (PO) activity using 10 mM L–DOPA [34].

Since PO is an enzyme, its activity depends on the steady state of the active sites, which are necessary for substrate binding and subsequent activity. The optimum ionic interactions were studied by taking the plasma in different ionic strength of Tris–HCl buffer, and PO activity was determined. The optimum ionic strength of 10 mM Tris–HCl that showed highest PO activity was used as a buffer for the study. To continue on ionic interactions, the optimum pH of the buffer required for plasma PO activity was also determined. The optimum pH was observed at pH 7.5 (brown color formation of dopachrome), which was same as that of purified Charybdis japonicaPO [27] against L-DOPA and Penaeus chinensis[35], but different from that of brown shrimp Penaeus californiensisthat showed optimum pH at 8.0 [36], Penaeus setiferusat pH 7.5 [37]. The differences in optimum pH may be correlated with the species specificity.

Temperature is an important factor that can either enhance enzyme activity or decline it. As the enzyme is a protein catalyst, a steady state of an active site binding to substrate depends on the intactness of the active site, which can be disrupted by temperature. In the present study, the optimum temperature of plasma PO activity in M. rosenbergiiwas determined by incubating plasma at various temperatures ranging from 10 to 90°C. The optimum temperature was found to be 25°C. The hemocyte of M. rosenbergiishowed optimum PO activity at 37°C [4]. The differences in temperature optima in plasma and hemocytes suggest a difference in PO characteristics.

However, in different crustaceans, several authors found maximum activities of PO activity in a temperature range of 40–45°C [27, 35, 37, 38, 39, 40, 41] while reported maxima at 30°C and 55°C for shrimp P. paulensis, lobster Homarus americanus,and tiger prawn P. monodon, respectively [42, 43, 44]. The difference in temperature optima among different species of crustacea can be attributed to species specificity and existing physiological conditions. Time is an important factor that can either enhance PO enzyme activity or decline it. In the present study, the oxidation of the substrate (L-DOPA) was tested at various time intervals from 5 to 30 minutes, and the activity was observed to be high at 15 min of incubation. The hemocyte of M. rosenbergiishowed optimum PO activity at 1 minute [4] while 40 minutes was recorded in C. japonica[27]. The differences in time interval optima in plasma and hemocytes suggest a difference in PO characteristics.

The enzyme kinetics of the plasma PO activity was determined using Michaelis–Menten curve by plotting various concentrations of L-DOPA (1–10 mM), and the rate of reaction was determined in 15 min (1/V). The initial rate of reaction increased up to a maximum reaction velocity after which it stabilized and then declined. The Km value determined for substrate enzyme affinity was 0.75 mM, and this suggested a strong affinity between the enzyme and L-DOPA and the Vmax was calculated as 0.58. Lineweaver-Burk plot showed a slope of 1.2 with a correlation coefficient of R² = 0.996. This indicated that the enzyme had active sites to maintain a steady increase in the rate of reaction. The kinetic and biochemical characteristics of the plasma PO activity demonstrate a distinct PO activity among the crustaceans [27, 45].

Our study also included the determination of PO activity concerning substrate affinity and inhibition using the optimized conditions as determined in plasma for hemocyanin (325 kDa) separated from the hemolymph of M. rosenbergii.This study on substrate affinity of purified MrHC was undertaken with diphenols such as L-DOPA, DL-DOPA, dopamine, and catechol and was specifically PO activity with L-DOPA only. It failed to show any binding affinity with any other diphenolic groups, and this indicated its distinct catechol oxidase nature [46].

Comparative inhibition studies with the PTU and tropolone were made to confirm the PO activity in the plasma and purified MrHC. The typical o-diphenoloxidase inhibitor, phenylthiourea, inhibited the enzyme activity drastically in the plasma and the tropolone inhibited the phenoloxidase activity in purified MrHC. The inhibition studies revealed that the plasma and the purified MrHC showed phenoloxidase activity. These results are following phenoloxidase from P. californiensis[36], P. chinensis[35], C. japonica(Liu et al. 2006) [27], and Limulus polyphemus[47]. Phenylthiourea (PTU), known as a chelating reagent of copper [47, 48], effectively inhibited the activity of plasma phenoloxidase activity and also that of purified MrHC, suggesting that phenoloxidase from M. rosenbergiiprawn has copper in its active site. Furthermore, the observed oxidation of L-DOPA was not due to peroxidase since tropolone that inhibited PO activity in the plasma and purified MrHC did not act as a substrate for peroxidase in the presence of H₂O₂ [49, 50].

5. Conclusion

In the present study, we conclude that the immunological function of phenoloxidase observed in plasma and MrHC (326 kDa) of freshwater prawn M. rosenbergiiappears to enhance resistance against various diseases, and investigation of PO activity in plasma and hemocyanin protein revealed catechol oxidase type. However, for the plasma and purified MrHC (325 kDa), the optimal conditions for measuring PO activity on L-DOPA (2.5 mM) were 10 mM Tris–HCl, pH 7.5 at 25°C for 15 min at 470 nm. This clearly indicates the significance of humoral immune components in boosting immune response. This finding provides evidence that the plasma and MrHC of M. rosenbergiiare a potent immune system with an ability to enzymatically function as humoral PO.

Conflict of interest

The authors declare no conflict of interest.

Funding

Not applicable.

Declarations

I confirm that the manuscript, or its contents in some other form, has not been published previously by any of the authors and/or is not under consideration for publication in another journal at the time of submission.

Abbreviations

CBB

Coomassie brilliant blue

DL–DOPA

3,4-dihydroxy-DL-phenylalanine

L–DOPA

L-3,4-dihydroxyphenylalanine

MrHC

M. rosenbergiihemocyanin

PAGE

Polyacrylamide gel electrophoresis

PO

Phenoloxidase

proPO

prophenoloxidase

PTU

Phenylthiourea

SDS–PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

TBS

Tris-buffered saline