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Stanka Stoeva a, Pavlina Dolashka b, Banko Bankov b, Wolfgang Voeltef,. Benedeto Salvato , Nicolay Genov b'*. °Department of Physical Biochemistry at the ...



Spectrochimica Acta Part A 51 (1995) 1965-1974

Spectroscopic properties of Callinectes sapidus hemocyanin subunits Stanka Stoeva a, Pavlina Dolashka b, Banko Bankov b, Wolfgang Voeltef, Benedeto Salvato ~, Nicolay Genov b'* °Department of Physical Biochemistry at the Institute of Physiological Chemistry, University of Tiibingen, Hoppe-Seyler Strafle 4, 72076 Tiibingen, German)' hlnstitute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia 1040, Bulgaria "Department of Biology, Unit,ersity of Padova and CNR Center, Via Trieste 75, 1-35131 Padova, Italy Received 1 May 1995; in final form 25 July 1995


The two major subunits of the Callinectes sapidus hemocyanin were isolated and characterized by spectroscopic techniques. They consist of 641 and 652 residues, respectively. Circular dichroism spectra showed that the structural integrity of the isolated polypeptide chains is preserved. Tryptophan fluorescence parameters were determined for the hemocyanin aggregates and for the subunits Csl and Cs2. The emitting tryptophyl fluorophores in the native hemocyanin are deeply buried in hydrophobic regions and are shielded from the solvent by the quaternary structure of the protein aggregates. In two subunits, obtained after dissociation of the aggregates, these residues become "exposed". It is concluded that the tryptophyl side chains in Csl and Cs2 are located in subunit interfaces (contact regions) in a negatively charged environment when the polypeptide chains are aggregated. Most probably they participate in hydrophobic protein-protein interactions. The environment of these fluorophores is more negatively charged after the dissociation of the aggregates to subunits.

I. Introduction

Hemocyanins (Hcs) are oxygen-transporting proteins present in many species of arthropods and molluscs. The quaternary and subunit structure and the arrangement of the oxygen-binding units are quite different in the respiratory proteins from the two phyla. Arthropod Hcs consist of one, two, four, six or eight hexameric aggregates of 67-90 kDa structural subunits [1]. Investigations on the structural diversity of the constituent polypeptide chains revealed the existence of eight immunologically discernible subunit types [2]. Crustacean hemocyanin subunits were classified into three categories on the basis of their immunological properties [2,3]. Complete or partial amino acid sequences of seven subunits from arthropod Hcs have been reported [4-8]. The X-ray structures of two arthropod deoxy-Hcs from Panulirus interruptus [9] and from Limulus polyphemus [10] have been solved at 3.20 and 2.18 A,, respectively.

* Corresponding author.

SSDI 0584-8539(95)01538-8


s. Stoeva et al./Spectrochirnica Acta Part A 51 (1995) 1965-1974

Molluscan hemocyanins have a molecular mass in the range (8.7- 43.4 x 106 daltons, and are composed of 10, 20 or more large subunits arranged to form cylinders ~, 300 A in diameter and of variable height [1,11]. Each subunit contains seven or eight folded domains and each domain contains a dinuclear oxygen binding site. The molecular architecture of arthropod and molluscan Hcs has been described in reviews [1,12,13]. The blue crab Callinectes sapidus is a representative of brachyuran crabs which appeared 200 million years ago and are the latest appearing decapod group [14]. The hemolymph of this invertebrate contains a mixture of hexamer and dodecamer hemocyanin aggregates. The ratio of the two oligomers varies in natural populations. The aggregates are built of five or six different subunits. The oligomer composition and the respiratory properties of this Hc have been intensively studied [15]. The native aggregates dissociate at pH 10 to form subunit species. Sodium dodecyl sulfate (SDS) gel electrophoresis showed molecular masses of 74 x l03 and 79 x 103 daltons for the subunits [16]. The dissociation behavior of the C. sapidus Hc has been investigated under different conditions (ref. [1] and citations therein). Immunological studies showed that this respiratory protein is composed of two immunologically discernible subunit fractions designated as alpha and beta [2]. The study of the arthropod hemocyanin subunit structure and properties is of fundamental interest. Data in this field will enlarge knowledge on the relationships between structural organization and function of the respiratory proteins in invertebrates. The purpose of the present paper is to report data about the structure in solution of the native Callinectes sapidus hemocyanin and its major subunits, as studied by spectroscopic methods.

2. Experimental 2.1. Purification of Callinectes sapidus hemocyanin and isolation of subunits Callinectes sapidus hemolymph was withdrawn with a syringe injected into the dorsal lacuna, and hemocyanin was purified as described in [17]. For dissociation, the native hemocyanin was dialyzed overnight against 0.1 M NaHCO3 buffer, pH 10.0, containing 20 mM EDTA. Fractionation of the subunits was performed by ion-exchange FPLC on a Mono-Q HR 5/5 column (Pharmacia, Uppsala, Sweden). Elution was performed under the following conditions: eluent A, 0.1 M NaHCO3, pH 10.0, containing 20 mM EDTA and 2 M urea; eluent B, 1 M NaC1 in A; gradient program: 0% B for 5 rain then 0-100% B in 30 min at a flow rate of 1 ml min-1. Samples were desalted by reversed-phase HPLC on a 2.1 x 30 mm Aquapore RP-300 column using a trifluoroacetic acid (TFA)/ acetonitrile/water solvent system. The elution was performed under the following conditions: eluent A, 0.1% TFA; eluent B, 0.085% TFA, 80% acetonitrile and 20% water. Gradient program: 0% B for 5 min, then 0-100% B in 10 min and after that 100% B for 15 min; flow rate 0.2 ml min2.2. Spectroscopic measurements Absorption spectra were recorded with a Shimadzu recording spectrophotometer, model 3000. Circular dichroism was measured with a Roussel Jouan Dichrographe III instrument. The data are expressed in terms of mean ellipticity. The concentration of C. sapidus Hc was determined spectrophotometrically at 278 nm using the coefficient E = 1.24 ml mg-1 cm-~. Molar concentrations were referred to 75 x 103 daltons as molecular mass for the species containing one dinuclear copper site [17]. Fluorescence measurements were performed with a Perkin-Elmer model LS 5 spectrofluorimeter, equipped with a thermostatted assembly and a Data Station, model 3600. The optical absorbance of the solutions was < 0.05 at the excitation wavelength to avoid inner filter effects. The measurements were performed at 25"C.

s. Stoeva et al.lSpectrochimica Acta Part A 51 (1995) 1965-1974


Fluorescence quantum yields were determined by the equation [18]


Q , t ( F x l A x ) ( A U F , t)(2xl2,t)

where Ox, Fx and A, are the emission quantum yield, the emission intensity at wavelength 2 and the optical density at the excitation wavelength, respectively, for the protein sample, and Qst, Fst and Ast are the same parameters for the reference standard. N-Acetyltryptophan amide (Ac-Trp-NH2) with a quantum yield of 0.13 [19] was chosen as standard. The results of the quenching reactions between the excited indole rings of tryptophans and acrylamide, Cs ÷ or I - were analyzed according to the Stern-Volmer equation [19] Fo/F= 1 + Ksv[X]

where F0 and F are the fluorescence intensities at an appropriate emission wavelength in the absence and in the presence of quencher; Ksv is the collisional quenching constant, and [X] is the quencher concentration. The inner filter effect due to acrylamide was corrected by the factor Y= antilog(dA + dE)/2, where dA and dE are the absorbances at the excitation and emission wavelength, respectively. In the Cs + and I - quenching experiments the ionic strength was kept constant by adding KCI. A small amount of Na2S203 was added to the iodide solution to prevent 3 - formation. The efficiency e of the tyrosine-to-tryptophan energy transfer was calculated using the relationship [20] Q = QT~pDCT,p(2)+ efTyr(~)] where Q is the fluorescence quantum yield of the protein sample at the respective excitation wavelength 2, Q'r~p is the fluorescence quantum yield of the tryptophyl residues in the protein molecule after excitation at 300 nm, and fT,p(2) and fTyr(2) are the fractional absorptions of tryptophan and tyrosine, respectively, at the excitation wavelength 2, calculated from their molar ratio in the protein. Numerical data for fTq, andfTyr are tabulated (see below). 2.3. SDS polyacrylamide gel electrophoresis

SDS polyacrylamide gel electrophoresis was carried out as described by Laemmli [21], using a 10% gel. 2.4. Amino acid analysis

Amino acid compositions of the protein samples were determined after hydrolysis in 6 M HC1 or in 5% thioglycolic acid in evacuated sealed tubes for 24, 48 and 72 h at 110°C. A BIOTRONIK model LC 3000 automatic amino acid analyzer was used. The values for the hemocyanin aggregates are expressed as number of residues per structural subunit of 75 x 10 3 daltons. Cysteine was determined by the method of Ellmann [22] after reduction of the samples with dithiothreitol under a nitrogen atmosphere. The copper ions were completely removed from the samples by precipitation of the apoprotein with trichloroacetic acid and washing the pellet with several portions of 0.1 M HCI.

3. Results and discussion

The dissociation of the Callinectes sapidus Hc was performed in the presence of 2 M urea, in which essentially no denaturation occurs, to avoid re-association. The polypeptide mixture obtained was fractionated by ion-exchange FPLC and eluted in six peaks, as is demonstrated in Fig. 1. Two of the fractions, designated Csl and Cs2, were in far greater quantities than the others. Callinectes sapidus Hc is made up of five or six different polypeptide chains, four of which exhibit considerable quantitative variability in

S. Stoeva et al./Spectrochimica Acta Part A 51 (1995) 1965-1974


natural populations. Some of the chains are present in very low concentration. Two invariant chains are present in sufficient quantities to be represented in each hexamer [15]. We have collected the two main fractions which seem to contain the major components of the native protein aggregates. These fractions were desalted by HPLC (Fig. 2). The electrophoretic mobilities of Csl and Cs2 corresponded to a molecular mass of 74 + 2 kDa. Similar values have been obtained by Hamlin and Fish [16] for the SDS-polypeptide complex derived from the 25,7 S Callinectes sapidus Hc species. Amino acid compositions of Csl and Cs2 are very similar to one another and also to that of the native Hc (Table 1), which is indicative of sequence similarities in the polypeptide chains of the two subunits. Our data for the composition of the native C. sapidus Hc are similar to those published in [23] with several exceptions: the values for glutamic acid, methionine, isoleucine and histidine are somewhat different. Csl and Cs2 consist of 641 and 652 residues, respectively, and their molecular masses calculated from the compositions are 73345 and 74797 daltons. The ultraviolet (UV) absorption spectrum of the oxygenated native Hc showed bands at 280 nm due to the aromatic chromophores and at 342 nm, the "copper band", characteristic of oxyhemocyanins. The last band disappeared in the spectra of the two subunits in the absence of oxygen. Possible conformational changes in the two subunits Csl and Cs2 as a result of the dissociation of the aggregates were checked by circular dichroism (CD) and fluorescence spectroscopy. The far UV CD spectra of the subunits (not shown) were almost identical to those of the native Hc, which was remarkably similar to the spectrum published in [16]. It can be concluded that little conformational change (if any) occurs upon dissociation and the structural integrity of the two polypeptide chains is preserved.



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