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.
Keywords
- innate immunity
- phenolic substrates
- phenoloxidase
- hemocyanin
- kinetics
- inhibitors
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,
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
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
2. Materials and methods
2.1 Experimental freshwater prawns
Adult intermoult of the giant freshwater prawns,
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
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 Mr HC
To purify the
2.12 Determination of protein
The protein content in the plasma and purified
2.13 Electrophoretic analysis
The protein profiles of plasma and purified
The molecular masses of the purified
2.14 Oxidation of diphenolic substrates by purified Mr HC
We tested the oxidative activity of 40 μl purified
2.15 Phenoloxidase activity
The PO activity of plasma (0.1 ml) or purified
2.16 Effect of inhibitors on PO activity
In this experiment, 0.1 ml of plasma or 40 μl of purified
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 (**
3. Results
3.1 Effect PO activity with various substrates
The plasma separated from the hemolymph of the freshwater prawn
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.
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.
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).
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).
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).
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).
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
3.9 Phenoloxidase activity with diphenolic substrates in Mr HC
The purified
3.10 Effect of PO inhibitors on oxidation of L-DOPA by plasma and purified Mr HC
Pretreatment of plasma or purified
In summary, for the plasma or purified
4. Discussion
The hemocyanin showed phenoloxidase (PO) activity in
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
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
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
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
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
Comparative inhibition studies with the PTU and tropolone were made to confirm the PO activity in the plasma and purified
5. Conclusion
In the present study, we conclude that the immunological function of phenoloxidase observed in plasma and
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
Coomassie brilliant blue
3,4-dihydroxy-DL-phenylalanine
L-3,4-dihydroxyphenylalanine
Polyacrylamide gel electrophoresis
Phenoloxidase
prophenoloxidase
Phenylthiourea
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
Tris-buffered saline
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