Analytical and semipreparative HPLC analysis and isolation of hemocyanin from the American lobster Homarus americanus.
KEY WORDS: lobster, Homarus americanus, refractometry, hemolymph, hemocyanin, absorbance, lobster health
Hemocyanin from molluscs and arthropods has antiviral and antimicrobial properties proved to be beneficial across a broad spectrum of scientific applications (Stiefel 1993, Cuthbertson 2001, Zhang et al. 2004, Dolashka & Voelter 2013). Hemocyanin has reduced the size of malignant tumors in rat and human hosts (Stiefel 1993). In addition, hemocyanin is commonly used as a carrier during antibody formation. Small antigens must be conjugated chemically with immunogenic carrier proteins. Widely used immunological carrier proteins currently include ovalbumin, bovine serum albumin, and the current industry standard, giant keyhole limpet (Megathura crenulata) hemocyanin (KLH). As a result of its high molecular weight (approximately 400 kDa per cylindrical subunit) (Gaykema et al. 1984), KLH exhibits poor solubility, producing turbid aqueous suspensions that result in a reduced immunological response. Yet, KLH is preferred over the albumin-based alternatives in production of assays for human applications, because the phylogenetic separation between mammals and molluscs multiplies the immunogenicity of KLH and prevents cross-reactivity between the carrier antibodies and naturally occurring mammalian proteins (Musselli et al. 2001, Plitnick & Herzyk 2010).
Lobsters, like most crustacean arthropods, have hemocyanin that is composed predominately of hexamer and dodecamer aggregates, with small concentrations of 24-mers and 48-mers (Mangum et al. 1991). It is theorized that lobster blood hemocyanin, with a molecular weight of 75 kDa per hexameric subunit, can be substituted for KLH in immunoassay development and antibody production, yielding more sensitive diagnostic tests that produce consistent and accurate results using current laboratory protocols.
The American lobster (Homarus americanus) fishery is economically important in New England and Canada. In 2009, American lobster landings exceeded 200 million pounds, with a value of nearly $1 billion (Fisheries and Oceans Canada 2012, NOAA 2012). The lobster catch from these regions is distributed between two markets: live and processed. Processed products include frozen or fresh tails, claws, or meat. During processing, hemolymph, which comprises as much as 20% of a lobster's body weight (Bayer 2005), is discarded as waste. Hemocyanin constitutes roughly 90% of the total hemolymph protein (Engel et al. 1993). Thus, lobster hemolymph is, potentially, a ready source of hemocyanin. Based on the market value of KLH and horseshoe crab hemocyanins, harvesting just a portion of the hemocyanin available in the waste stream could generate a significant increase in lobster value and processing revenues.
The current hemocyanin purification protocol requires centrifuging hemolymph for 9-10 h at 165,000g (Markl et al. 1979). This laborious process drastically increases the cost of hemocyanin. In addition, this method does not support reliable separation of the oligomeric components of lobster hemolymph.
Other separation and purification techniques investigated include high-performance liquid chromatography (HPLC). The structure of hemocyanin in lobster blood hemocyanin varies widely, ranging from single hemocyanin molecules to an aggregation of as many as 48 oligomers. In blue crabs (Callinectes sapidus), hemocyanin is found most commonly in hexameric and dodecameric forms, with the majority being dodecameric (Mangum et al. 1991, Mangum 1992). Others have used size-exclusion HPLC to separate hexamers from dodecamers in blue crab hemolymph with a diode array detector set at 280 nm to monitor for aromatic proteins (Van Kuik et al. 1987, Makino & Kimura 1988, Greaves et al. 1992). Because the earlier studies demonstrated that hemocyanin is relatively resistant to dissociation, hemolymph samples were frozen and thawed several times to examine the effects of freezing and thawing on quaternary structure and dissociation of dodecamers into hexamers (Bolton 2008). Size-exclusion columns under specific HPLC conditions provided good separation and yielded a comprehensive overview of the proteins contained in hemolymph, including the hemocyanin oligomers (Bolton 2008), but require a 120-min runtime. Brouwer and Brouwer-Hoexum (1992) also used size-exclusion HPLC to separate hemocyanin oligomers from hemolymph in studies focused on the transfer and binding of copper in lobster apohemocyanin.
Our work included development of HPLC methods to analyze and purify American lobster hemocyanin. including an analytical method for quality profiling of hemocyanin oligomers and a scaled-up semipreparative HPLC method for extracting hemocyanin hexamer and dodecamer oligomers from lobster hemolymph. Following the successful development of a semipreparative method, the shelf-life and stability of the purified hemocyanin was tested.
MATERIALS AND METHODS
Hemocyanin Purification with Ultracentrifugation
Eight lobsters were donated by Sorrento Lobster Pound, (Sorrento, ME). Thirty milliliters of hemolymph was extracted on the ventral side of each lobster tail at the third segment from the telson using 2 22-gauge needles with 10-mL syringes. Samples were drawn 3 times from each lobster, for a total of 30 mL. All hemolymph samples from all 8 lobsters were combined in a 500-mL centrifuge tube and held at 5[degrees]C for 48 h to allow clotting. Clotted hemolymph was homogenized using a Tissue Tearor 398 (Biospec Products Inc., Bartlesville, OK) for 30 sec with the centrifuge tube submerged in an ice water bath. Samples were spun at 12,000g for 20 min at 5[degrees]C in a Beckman Coulter Avanti J-E centrifuge equipped with a JA 25.50 Rotor to remove insoluble clotted material. The supernatant was pipetted into preweighed ([+ or -] 0.001 g) 10-mL polycarbonate ultracentrifuge tubes and centrifuged at 163,000g for 7 h at 5[degrees]C using a Beckman Coulter L8-70M ultracentrifuge with a 75Ti rotor (Brea, CA). Pellets formed during ultracentrifugation (bluish gray hemocyanin (HC) pellet) were saved; the supernatant was discarded. Total mass of purified HC was determined by weighing ([+ or -] 0.001 g) each tube containing a pellet. Pellets were subsequently combined, then dissolved in 7 mL salt solution consisting of 37.5 mmol/L NaCl, 300 mmol/L Mg[Cl.sub.2], and 10 mmol/L Ca[Cl.sub.2] in HPLC-grade purified water. The salt mobile phase was sonicated at 22[degrees]C under vacuum for 5 min before use. The composition of this mobile phase provided protein stability and facilitated column flow under heavy use. Samples were placed in storage at 5[degrees]C for 24 h, or until the pellets dissolved completely. After the pellets dissolved, 3 mL glycerin was added to ensure protein stability and to preserve samples for long-term storage.
Hemolymph Sample Preparation for HPLC Analytical and Preparative Methods
Hemolymph (30 mL) was extracted on the ventral side of 8 lobsters at the third segment from the telson using two 22-gauge needles with 10-mL syringes, as described previously. Total hemolymph protein (measured in grams per 100 mL) was determined for each sample using a Reichert VET 360 analog blood animal protein refractometer (Depew, NY) (Bayer 1986). The hemolymph from all 8 lobsters was combined and placed into a 500-mL centrifuge tube and held at 5[degrees]C for at least 12 h to allow clotting. The hemolymph was subsequently frozen at -20[degrees]C for 24 h and then thawed at 5[degrees]C for 12 h. These temperature treatments were designed to simulate common storage and handling conditions for hemolymph collected in a lobster processing facility. A 40-mL working hemolymph sample was poured into a 50-mL centrifuge tube and was spun at 7,750g for 10 min in an Eppendorf Centrifuge 5430 with an F-35-6-50 rotor (Hamburg, Germany) to separate insoluble clotted material. Before HPLC analysis, each sample was filtered using a 0.22-[micro]m Phenomenex polyethersulfone membrane (nonprotein binding) filter (Torrance, CA). Filtered samples were analyzed immediately using a single-wavelength detector (SWD) HPLC.
HPLC-SWD Analytical Parameters
Hemocyanin subunit separation was performed using a Dionex UltiMate 3000 Series system with Chromeleon 6.7 software (Germering, Germany), equipped with an automatic sampler, SWD, and Dionex analytical SST 11-[micro]L, 0.4-mm flow cell (Germering, Germany). The system was equipped with 2 (coupled) Phenomenex Biosep-SEC S3000 (7.8 x 300 mm) size-exclusion columns in tandem with matching guard column. One milliliter of purified hemocyanin or 1 mL of the hemolymph sample was placed in 1.5-mL glass autosampler vials (National Scientific, TN) with the sample vial holder temperature set to 5[degrees]C. The UV signal was monitored at 280 nm. This protocol was developed based on previous work by Bolton (2008), and Mangum et al. (1991). The salt mobile phase, which was prepared as described earlier, was sonicated under vacuum for 5 min, and was used to provide protein stability and to facilitate column flow under heavy use. Fifty microliters from each vial (purified hemocyanin standard or hemolymph sample) was injected into the HPLC system with a flow rate of 0.5 mL/min.
HPLC-SWD Semipreparative Parameters
HPLC-SWD separation was performed using a Dionex UltiMate 3000 system with Chromeleon 6.7 software (Germering, Germany), equipped with an automatic sampler, SWD, and Dionex semipreparative Peek 0.7-[micro]L, 0.4-mm flow cell (Germering, Germany). This system was equipped with a Phenomenex Biospec-SEC S3000 (21.1 x 600 mm; Torrance, CA) size-exclusion column with matching guard column. The mobile phase, 37.5 mmol/L NaCl, 3.125 mmol/L Mg[Cl.sub.2], and 1.25 mmol/L Ca[Cl.sub.2] in HPLC-grade purified water, was sonicated under vacuum for 5 min before use. One milliliter of the hemolymph sample was placed into 1.5-mL autosampler vials (National Scientific). The temperature of the HPLC autosampler vial holder was set to 5[degrees]C, and the UV signal was monitored at 280 nm.
HPLC-SWD Semipreparative Purification Parameters
The parameters for this analysis were the same as those specified earlier in the semipreparative section, with the addition of a Froxy, Jr Teledyne Isco (Lincoln, NE) fraction collector coupled to the SWD. Fractions were stored at 5[degrees]C after collection until the subunit confirmation reanalysis was performed using the analytical method described earlier.
HPLC-SWD Purified Hemocyanin Shelf-Life Study
Hemolymph samples were analyzed using the HPLC-SWD semipreparative parameters and HPLC-SWD semipreparative purification parameters. Hemolymph samples were injected into the HPLC system 4 times, with the F2 hemocyanin fractions collected and combined into a sterile 100-mL glass tube, and the F3 hemocyanin fractions combined in a separate sterile 100-mL glass tube. Fractions were stored at 5[degrees]C for 30 days. Every day for the first 2 wk, including day 0, the samples were analyzed in duplicate on the HPLC-SWD using the prep method described earlier. After the first 2 wk, the samples were run in duplicate every 5 days. The HPLC column was cleaned with 10% DMSO and distilled water mobile phase after the first 7 runs and then after every subsequent run to ensure a clean assay and to prevent bacterial growth in the column.
Ultracentrifuge Hemocyanin Purification Procedure
Hemocyanin was purified using the centrifugation technique described in Materials and Methods. The weights of the final pure hemocyanin pellets were 0.101 g and 0.095 g.
Analytical Method Development for Hemocyanin Quality Control
Hemocyanin concentrated using the ultracentrifugation method was used as a standard before injecting hemolymph under the assumption that this standard contained several hemocyanin oligomers. The analysis time was reduced from 120 min to 45 min per sample run. A typical chromatogram of this accelerated method is shown in Figure 1.
Three separate protein oligomers eluted at 26.593 min, 28.293 min, and 31.120 min, with a shoulder at approximately 25 min, and the most intensely absorbing peak at 28.3 min. Elution times, from slowest to fastest, are correlated to the 4 quaternary structures of hemocyanin: 16s (hexamer), 24s (dodecamer), 37s (24-mer), and 60s (48-mer). The 48-mers eluted first because they have the greatest molecular weight. The 24-mers and 48-mers eluted from the column before dodecamers and hexamers, but both occurred in lower concentrations, with the 60s occurring in the lowest concentration.
The accelerated HPLC analysis was repeated with hemolymph samples after determination of protein concentration using a digital refractometer. These results are shown in Table 1. Our study determined that minimal processing of the hemolymph samples was required to prevent fouling of the analytical guard column. Hemolymph samples were centrifuged to remove clotted material, passed through a 0.22-[micro]m nonprotein binding syringe filter, and then added directly to the HPLC sample vials, a method that alleviated guard column fouling issues.
As with the ultracentrifugation hemocyanin samples, 3 separate protein subunits eluted at 26.687 min, 28.373 min, and 31.153 min, respectively, with a shoulder at approximately 25 min and the most intense absorption peak at 28 min (Fig. 2). Peak elution times were the same as those of the ultracentrifugation hemocyanin samples, with nearly identical ratios of the peak areas between the 2 sample types. The peak areas were approximately 45.2 mAU/min, 871.4 mAU/min, and 90.9 mAU/min for 24-mers, dodecamers, and hexamers, respectively.
Semipreparative Method Development for Hemocyanin Collection
Design of this method required a new high-flow column with a dynamic range similar to the analytical column. The Phenomenex Company manufactures a large-diameter (21.1 mm), 600-mm-long semipreparative column that we expected could provide similar separation with a higher flow rate and larger injection volume than more conventional analytical columns. To define the scale-up procedure for the semipreparative column, generic flow rate and injection volume calculations were performed. Eqs (1)-(4) were used to calculate the scale-up of the flow rate and injection volume for the semipreparative column.
[F.sub.2] = [F.sub.1] ([d.sup.2.sub.2]/[d.sup.2.sub.1]) to adjust the surface area to flow rate (1)
[IV.sub.2] = [IV.sub.1] [([d.sub.2]/[d.sub.1]).sup.2(L2/L1)], (2)
where F is flow, d is the diameter of the column, IV is the injection volume, and L is the column length.
[F.sub.2] [(0.5 mL/min)[(21.2 mm).sup.2]/[(7.8 mm).sup.2]] = 3.69 mL/min (3)
50 [micro]L [(21.2 mm/7.8).sup.2(600/600)] = 369 [micro]L (4)
The hemolymph samples were allowed to clot and were then centrifuged to collect the clotted material, and were subsequently passed through a 0.22-[micro]m syringe filter before HPLC analysis. Based on the calculations completed using the previous equations, a flow rate of 3.69 mL/min and an injection volume of 369 [micro]L was designated as a starting point. Trials revealed that using a flow rate of 3 mL/min and an injection volume of 500 [micro]L created optimal conditions for hemocyanin subunit separation. Figure 3 illustrates a typical chromatogram of hemolymph separation with 3 major protein peaks at 31.413 min, 33.348 min, and 35.807 min, and a shoulder at approximately 29 min. Peak areas were 120.5 mAU/min, 1,892.1 mAU/min, and 255.6 mAU/min for 24-mers, dodecamers, and hexamers, respectively.
A method was also developed to collect fractions of separated dodecamers and hexamers. Fractions collected were injected into the HPLC system to determine purity. Figure 4 is a chromatogram reflecting separation of two components of hemolymph. The gray shading over the peaks represents the two separate fractions collected and the corresponding collection times for dodecamers and hexamers. Dodecamer and hexamer fractions were collected several times and reanalyzed using the analytical HPLC method. Figures 5 and 6 show typical reanalysis chromatograms of the respective dodecamer and hexamer fractions.
The dodecamer and hexamer fractions depicted in Figures 5 and 6 correlate to lobster hemolymph fractions 2 (F2), and 1 (FI), respectively. The dodecamer peak area is 38.946 mAU/ min whereas the area of the peak representing the hexamers is 15.837 mAU/min.
Hemocyanin Dodecamer and Hexamer Fraction Shelf-Life Study
The peak areas of the hemocyanin dodecamer and hexamer fractions over time are recorded in Table 2, with results graphed in Figure 7. Our study shows that at time 0, peak areas were 17.14 mAU/min and 36.44 mAU/min. After 30 days, peak areas were 18.88 mAU/min and 37.8 mAU/min for hexamers and dodecamers, respectively. After 30 days, elution times for dodecamers and hexamers were 33.1 [+ or -] 0.213 min and 36.1 [+ or -] 0.091 min, respectively. Peak shape was uniform as seen in Figures 8 and 9 (day 0 vs. day 30 dodecamer and hexamer peaks).
Analytical Method Development for Hemocyanin Quality Control
Currently, the Bolton (Bolton 2008) method is the only published procedure for quantifying American lobster hemocyanin oligomers. In the that study (Bolton et al. 2008), hemocyanin and hemolymph samples were run for 120 min at 0.2 mL/min to ensure proper separation time and for the columns to clear before the next sample was injected. Results from analysis of the concentrated HC standard yielded four distinct peaks. The major limitation of this method was an excessive per-sample runtime. To be commercially viable, new protocols must reduce runtime while maintaining separation quality and resolution. New analytical HPLC parameters were established based on work from Greaves et al. (1992), Mangum (1992), Brouwer & Brouwer-Hoexum (1992), and Bolton (2008), which included a shorter runtime of 45 min.
In the chromatographs included in this article, the 48-mer display shouldering that was a result of the estimated molecular weight of 3.6 million Da. This large protein exceeds the separating capabilities of many size-exclusion columns. The manufacturer specifications of the Bio-Sec 3000 column identifies a dynamic range of 5,000-1,000,000 Da. Hemocyanin hexamers and dodecamers, with molecular weights of 450,000 Da and 900,000 Da, respectively, are in the normal operating range for most size-exclusion columns. Larger hemocyanin components such as the 24-mers (37s) and 48-mers (60s) exceed these specifications, and therefore show some loss of separation with a size-exclusion column.
The ratios of the peaks found in Figure 1 and Figure 2 are consistent with data for many crustacean hemocyanins, in that the dodecamers are the most prevalent hemolymph subunits. Greaves et al. (1992) reported that the dodecamer occurred in greater concentration than the hexamer, which is consistent with our results. The 24-mers and 48-mers eluted from the column before dodecamers and hexamers in slightly lower concentrations, with the 48-mers comprising the lowest concentration. Based on preliminary results and calculations from Fan (pers. comm., 2006), the typical hexamer and dodecamer subunits should provide the most intense immune response and trigger the greatest antibody production, and therefore would be the most useful for commercialization.
One prominent difference between the hemolymph sample chromatographs (Fig. 1) and the ultracentrifugation hemocyanin chromatographs (Fig. 2) is the number of peaks. The hemolymph chromatograph shows several peaks that appear well before and after the HC subunits. As with the concentrated hemocyanin runs, peaks that eluted quickly likely represent low concentrations of proteins with molecular weights that exceed the column's separation range; peaks that eluted after the HC components represent smaller proteins.
Semipreparative Method Development for Hemocyanin Collection
Development of a new method for rapid, high-volume separation of hemocyanin from hemolymph also supports separation and purification of hemocyanin subunits. Optimizing initial scale-up calculations yielded a flow rate 6 times faster than the analytical method and an injection volume 10 times greater than the analytical method. When compared with the analytical method, the semipreparative method produced a similar chromatographic fingerprint, with the exception of a higher baseline in the semipreparative peaks. On average, peak areas were 2 times greater than those generated with analytical methods. The differences in the ratios of subunit peak areas derived using the analytical and semipreparative methods were most likely a result of inherent variations among hemolymph collected from individual lobsters. Viable new methods must also support separation and quantification of the hemocyanin subunit fractions and collection of the individual hemocyanin oligomers. Based on information provided by Fan (2006) and the size of KF1L HC, it was determined that the hemocyanin oligomers of particular interest are the hexamers and dodecamers. The new method outlined here allows for separation, collection, and purification of these commercially important hemocyanin subunits.
Figures 5 and 6 reflect lower dodecamer and hexamer concentrations than in the hemolymph samples. This is a result of the mobile phase dilution occurring during sample analysis. This fraction is also void of any other peaks, which translates to high-quality sample. Peak elution times and approximate peak area ratios were consistent with those of the hemolymph samples, as depicted in Figures 3 and 4. The dodecamer fractions were more concentrated than the hexamer fractions, which aligns with the results shown in the hemolymph and ultracentrifugation FIC chromatographs.
Hemocyanin Dodecamer and Hexamer Fraction Shelf-Life Study
Testing showed that the hemocyanin oligomers isolated using the new semipreparative F1PLC method were viable for at least 30 days. Peak shape and area, and elution time were consistent for hemocyanin hexamer and dodecamer oligomers during the 30-day study period. The increased peak area at the end of the 30-day study (Fig. 7) can be attributed to evaporation that occurred during storage under refrigeration. Hemocyanin hexamers and dodecamers were stable at refrigeration temperature and would likely be more stable when frozen. The peak conformation, peak area, and elution time confirm the stability of the subunit structures, which remained stable during the 30-day study. This is an important finding as the current literature indicates that hemocyanin subunits may degrade over time (Dolashka-Angelova et al. 2003, Dolashka-Angelova et al. 2005). Shelf-life would likely be extended further by adding glycerol and storing at freezer temperatures. In addition, samples could be concentrated further using lyophilization.
Hemolymph, a by-product discarded by lobster processors, is an abundant source of hemocyanin. However, developing efficient methods of separating and purifying hemocyanin from lobster hemolymph is required to establish the economic viability of this potential source. Rapid analytical protocols for separating, quantifying, and confirming the quality of purified hemocyanin subunits were developed by scaling up a 2008 HPLC method for quantification of hemocyanin oligomers in lobster hemolymph.
Bolton's 2008 analytical method was refined by reducing sample runtime and increasing injection volume to support development of commercial hemocyanin production. The oligomeric components were separated with semipreparative size-exclusion columns and subsequently purified with a scaled-up method. After successful development of analytical, preparative, and purification methods, the shelf-life of the purified oligomers was evaluated. The peak conformation, peak area, and elution time of samples analyzed in this study confirm that the hemocyanin subunit structures remained stable during storage at 5[degrees]C for 30 days. These studies will facilitate the development of a new source of an economically important product with diverse therapeutic, biotechnology, and research applications.
This work was supported by the Lobster Institute, the University of Maine Department of Food Science and Human Nutrition, and the University of Maine Cooperative Extension. We acknowledge and thank Joan Perkins for reviewing this manuscript.
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JASON C. BOLTON, (1,2) ROBERT BAYER, (1) ROD BUSHWAY, (1) SCOTT COLLINS (3,4) AND BRIAN PERKINS (1)
(1) School of Food and Agriculture, 5735 Hitchner Flail, University of Maine, Orono, ME 04469; (2) Cooperative Extension, 5735 Hitchner Hall, University of Maine, Orono, ME 04469; (3) Department of Chemistry, University of Maine, Orono, ME 04469; (4) Laboratory of Surface Science and Technology, University of Maine, Orono, ME 04469
* Corresponding author. E-mail: email@example.com
Maine Agricultural and Forest Experiments Station publication no. 3366.
TABLE 1. Protein concentrations of 5 lobsters determined using a refractometer. Lobster no. Refractometer analog designation ([RI.sub.a]) (g/100 mL) L1 6.5 L2 7.2 L3 7.5 L4 5.9 L5 6.0 TABLE 2. Hemocyanin hexamer and dodecamer shelf-life study. Peak area Peak area hexamer (n = 2) dodecamer (n = 2) average average Day (mAU/min) (mAU/min) 0 17.14 36.44 1 16.77 37.01 2 16.91 37.17 3 16.48 36.98 4 16.37 35.28 5 15.41 35.79 6 15.65 34.96 7 15.81 34.89 9 18.72 36.66 11 17.70 36.65 13 17.73 36.60 15 17.55 37.13 20 19.01 37.07 25 18.45 37.04 30 18.88 37.80
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|Author:||Bolton, Jason C.; Bayer, Robert; Bushway, Rod; Collins, Scott; Perkins, Brian|
|Publication:||Journal of Shellfish Research|
|Date:||Apr 1, 2014|
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