Comp. Biochem. Physiol. Vol. 101B,No. 1/2,pp. 185--188,1992 Printed in Great Britain
0305-0491/92$5.00+ 0.00 © 1992PergamonPresspie
STRUCTURE OF EXTRACELLULAR HEMOGLOBIN FROM THE BRINE SHRIMP ARTEMIA SALINA ABDUSSALAMAZEM and EZRA DANIEL Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel (Tel: 03-5459441) (Received 13 June 1991) A b s t r a c t - - l . Hemoglobin from the brine shrimp Artemia salina, purified by ultracentrifugation and
preparative gel electrophoresisin non-denaturing medium, gave in sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band corresponding to a polypeptide chain with M, 150,000. 2. Crosslinking by glutardialdehyde resulted in the appearance of a band corresponding to a molecular mass twice that of a polypeptide chain. 3. Limited trypsinolysis gave eight proteolytic bands corresponding to submultiples 8/9-1/9 of a polypeptide chain. 4. We conclude that a molecule of Artemia hemoglobin is composed of two single polypeptide chain subunits and that each subunit consists of nine structural units roughly equal in size.
INTRODUCTION
MATERIALS AND METHODS
Hemoglobin from the brine shrimp Artemia, anostraca, has attracted attention, probably due to the fact that the organism lives in water of high salt concentration. Over the years, data have been accumulated on the molecular parameters and the subunit structure of Artemia hemoglobin. For the native molecule, a sedimentation coefficient ~ 11.5 S (Wood et al., 1981) and a molecular mass ~240,000 (230,000, Bowen et al., 1976; 240,000, Moens and Kondo, 1978; 249,000, De Voeght and Clauwaert, 1981; 239,400, Wood et al., 1981) were determined. Dissociation of the molecule gave a ,,~6.4 S species (6.8 S, Bowen et al., 1976; 6.3 S, De Voeght and Clauwaert, 1981; 6.17 S, Wood et al., 1981) for which M , ~ 115,000 (Wood et aL, 1981~J was determined. Heme and iron content determinations gave a minimal mass per heme of 18,000 (Moens and Kondo, 1977) and 19,000 (Bowen et al., 1976), respectively. On the basis of the mol. wt determinations a model was proposed for Artemia hemoglobin according to which the molecule is composed of two polypeptide chains each containing seven (Moens and Kondo, 1977, 1978) or eight (Moens et al., 1990) heme groups. Krissansen et al. (1981) reported the occurrence in the hemolymph of Artemia of a latent protease which copurifies with hemoglobin in many of the procedures used for the preparation of the protein. The protease is activated by the sodium dodecyl sulfate (SDS) of the polyacrylamide gel system and readily degrades the hemoglobin subunit. From the background of their work, we made use of preparative electrophoresis to effectively remove the protein contaminant and obtain hemoglobin that yields intact polypeptide chains by SDS gel electrophoresis. We report here the results of experiments carried out on hemoglobin purified in this manner and on their implications on the structure of the molecule.
Preparation of hemoglobin Adult Artemia salina shrimps were obtained from the Underwater Observatory at Eilat. About 100 specimens were rinsed with distilled water and then suspended in 0.1 M Tris-HC1 buffer pH 7.7 containing 2.6 mM phenylmethanesulfonyl fluoride, 20,ug/ml soybean trypsin inhibitor and 1 mM EDTA, referred to hereafter as extraction buffer. The suspension was homogenized and the homogenate was repeatedly centrifuged for 20 min at low speed to remove particulate matter. The resulting clear fluid was centrifuged at 153,000g for 6hr. The precipitate was dissolved in the same buffer and centrifuged again at the same speed. The resulting red pellet was redissolved in extraction buffer. Samples containing 40/~g hemoglobin were applied to each of 15 wells in a slab of polyacrylamide gel gradient (2.6-28%) and electrophoresed in non-denaturing medium for 12 hr at 80 V. Staining with benzidine reagent was used to locate the hemoglobin band in one of the tracks. The hemoglobin bands in the remaining tracks were excised, finely sliced and eluted into a volume of extraction buffer. The eluate was centrifuged at 153,000g for 6 hr and the red pellet was dissolved in a minimal volume of 0.1 M phosphate buffer, pH 6.8. Electrophoresis Electrophoresis in non-denaturing medium was carried out in slab containing a polyacrylamide concentration gradient using the Laemmli buffer system (Laemmli, 1970) and in tubes containing uniform polyacrylamide concentration in 0.1 M sodium phosphate buffer, pH 7.2. SDS gel electrophoresis was performed in tubes according to Weber et al. (1972). Crosslinking Crosslinking of Artemia hemoglobin with glutardialdehyde was carried out in 0.01 M sodium phosphate buffer, pH7.5. The protein concentration was 0.25mg/ml and the glutardialdehyde concentration was 0.05% (v/v). At various times, aliquots were withdrawn from the reaction mixture and crosslinking was stopped by addition of SDS.
185
186
ABDUSSALAMAZEMand EZRADANIEL calibration curve of protein markers with known molecular masses, this band was identified as a polypeptide chain dimer (M, ~270,000). With progressively increasing times of reaction, the intensity of the faster moving monomeric band decreased, while that of the slower moving dimeric band increased. For the longest time shown, the monomeric band has virtually disappeared, and the dimeric band is the only band observed (Fig. 2).
Trypsinolysis Artemia hemoglobin was exposed to trypsin at pH 8.2 and the products were analysed by SDS gel electrophoresis. Before exposure to the enzyme, essentially one band was observed. At progressively increasing times of reaction, bands with higher mobility appeared. Eight bands, in addition to the monomeric band corresponding to the intact polypeptide chain, were observed after l hr of reaction (Fig. 3). Identification of the bands seen in the electrophoreric patterns was made on the basis of their mobilities, taking into consideration that a plot of log molecular mass vs mobility (not shown) is linear for the polyacrylamide concentration (5%) and protein molecular mass range (14,000-200,000) used. In order of increasing mobility, the eight bands obtained by
Fig. 1. Electrophoresis of purified Artemia hemoglobin. Hemoglobin was electrophoresed in 0.1 M phosphate buffer at pH 7.2 (a) in the absence of SDS and (b) in the presence of SDS and 2-mercaptoethanol. Polyacrylamide concentration was 3% in (a) and 3.3% in (b).
Trypsinolysis This was carried out by incubation of hemoglobin with TPCK-trypsin (enzyme treated with L-l-tosylamido2-phenyl-ethyl chloromethyl ketone) as described by Darawshe and Daniel (1991). RESULTS
Purity of Artemia hemoglobin Artemia hemoglobin prepared by the method described showed a typical oxyhemoglobin absorption spectrum with maxima at ~340, 415, 535 and 570 nm. Electrophoresis in non-denaturing medium at pH 7.2 yielded a single band (Fig. la). A single band was likewise obtained in SDS gel electrophoresis (Fig. lb). From the mobility of this band, and by reference to a linear calibration curve obtained with standard protein markers, a molecular mass of 150,000 Da was calculated for the polypeptide chain of Artemia hemoglobin.
Crosslinking Artemia hemoglobin was exposed to glutardialdehyde and the products of crosslinking were analysed by SDS gel electrophoresis. Before exposure to the crosslinking agent, the typical electrophoretic pattern showing one band was observed. Exposure to glutardialdehyde resulted in the appearance of an additional band of lower mobility. Using a
Fig. 2. Crosslinking of Artemia hemoglobin. Hemoglobin was exposed to glutardialdehyde and then electrophoresed in the presence of SDS and 2-mercaptoethanol in 3.3% polyacrylamide gels: (a) not exposed to glutardialdehyde; exposed for (b) 0.5min; (c) 1 min; (d) 15min; (e) 1 hr; (f) 18hr.
Structure of Artemia hemoglobin
187
g
i ÷
Fig. 3. Limited trypsinolysis of Artemia hemoglobin. Hemoglobin in 0.04 M borate buffer, pH 8.2, was exposed to trypsin and then electrophoresed in the presence of SDS and 2-mercaptoethanol in 5% polyacrylamide gels: (A) not exposed to trypsin; (B) exposed for 1 hr. (C) Densitometric scan of (B). (a)-(i) denote electrophoretic bands on gels. Upper scan is a magnification of the corresponding part in the lower scan. limited trypsinolysis of A r t e m i a hemoglobin were found to correspond to submulfiples 8/9-1/9 of a polypeptide chain (Fig. 4). DISCUSSION Mention has been made of the difficulties encountered in preparing intact A r t e m i a hemoglobin caused by the presence of a latent proteolytic enzyme in the hemolymph of the animal. The observation in this study of a single band in the electrophoresis of A r t e m i a hemoglobin in both non-denaturing and
I -8/9
719 619 519 419 :519
2/9
I / 9 ~0.2
04
0.6
08
u
Fig. 4. Identification of electrophoretic bands obtained in limited trypsinolysis of Artemia hemoglobin. Semi-logarithmic plot of the molecular mass associated with a given band, M, scaled relative to the molecular mass of a single polypeptide chain, M c, vs the corresponding relative electrophoretic mobility, u. Linearity of the plot (correlation coefficient 0.999) was achieved by setting M / M c = i/9, where i is an integer 1 ~
0305-0491/92$5.00+ 0.00 © 1992PergamonPresspie
STRUCTURE OF EXTRACELLULAR HEMOGLOBIN FROM THE BRINE SHRIMP ARTEMIA SALINA ABDUSSALAMAZEM and EZRA DANIEL Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel (Tel: 03-5459441) (Received 13 June 1991) A b s t r a c t - - l . Hemoglobin from the brine shrimp Artemia salina, purified by ultracentrifugation and
preparative gel electrophoresisin non-denaturing medium, gave in sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band corresponding to a polypeptide chain with M, 150,000. 2. Crosslinking by glutardialdehyde resulted in the appearance of a band corresponding to a molecular mass twice that of a polypeptide chain. 3. Limited trypsinolysis gave eight proteolytic bands corresponding to submultiples 8/9-1/9 of a polypeptide chain. 4. We conclude that a molecule of Artemia hemoglobin is composed of two single polypeptide chain subunits and that each subunit consists of nine structural units roughly equal in size.
INTRODUCTION
MATERIALS AND METHODS
Hemoglobin from the brine shrimp Artemia, anostraca, has attracted attention, probably due to the fact that the organism lives in water of high salt concentration. Over the years, data have been accumulated on the molecular parameters and the subunit structure of Artemia hemoglobin. For the native molecule, a sedimentation coefficient ~ 11.5 S (Wood et al., 1981) and a molecular mass ~240,000 (230,000, Bowen et al., 1976; 240,000, Moens and Kondo, 1978; 249,000, De Voeght and Clauwaert, 1981; 239,400, Wood et al., 1981) were determined. Dissociation of the molecule gave a ,,~6.4 S species (6.8 S, Bowen et al., 1976; 6.3 S, De Voeght and Clauwaert, 1981; 6.17 S, Wood et al., 1981) for which M , ~ 115,000 (Wood et aL, 1981~J was determined. Heme and iron content determinations gave a minimal mass per heme of 18,000 (Moens and Kondo, 1977) and 19,000 (Bowen et al., 1976), respectively. On the basis of the mol. wt determinations a model was proposed for Artemia hemoglobin according to which the molecule is composed of two polypeptide chains each containing seven (Moens and Kondo, 1977, 1978) or eight (Moens et al., 1990) heme groups. Krissansen et al. (1981) reported the occurrence in the hemolymph of Artemia of a latent protease which copurifies with hemoglobin in many of the procedures used for the preparation of the protein. The protease is activated by the sodium dodecyl sulfate (SDS) of the polyacrylamide gel system and readily degrades the hemoglobin subunit. From the background of their work, we made use of preparative electrophoresis to effectively remove the protein contaminant and obtain hemoglobin that yields intact polypeptide chains by SDS gel electrophoresis. We report here the results of experiments carried out on hemoglobin purified in this manner and on their implications on the structure of the molecule.
Preparation of hemoglobin Adult Artemia salina shrimps were obtained from the Underwater Observatory at Eilat. About 100 specimens were rinsed with distilled water and then suspended in 0.1 M Tris-HC1 buffer pH 7.7 containing 2.6 mM phenylmethanesulfonyl fluoride, 20,ug/ml soybean trypsin inhibitor and 1 mM EDTA, referred to hereafter as extraction buffer. The suspension was homogenized and the homogenate was repeatedly centrifuged for 20 min at low speed to remove particulate matter. The resulting clear fluid was centrifuged at 153,000g for 6hr. The precipitate was dissolved in the same buffer and centrifuged again at the same speed. The resulting red pellet was redissolved in extraction buffer. Samples containing 40/~g hemoglobin were applied to each of 15 wells in a slab of polyacrylamide gel gradient (2.6-28%) and electrophoresed in non-denaturing medium for 12 hr at 80 V. Staining with benzidine reagent was used to locate the hemoglobin band in one of the tracks. The hemoglobin bands in the remaining tracks were excised, finely sliced and eluted into a volume of extraction buffer. The eluate was centrifuged at 153,000g for 6 hr and the red pellet was dissolved in a minimal volume of 0.1 M phosphate buffer, pH 6.8. Electrophoresis Electrophoresis in non-denaturing medium was carried out in slab containing a polyacrylamide concentration gradient using the Laemmli buffer system (Laemmli, 1970) and in tubes containing uniform polyacrylamide concentration in 0.1 M sodium phosphate buffer, pH 7.2. SDS gel electrophoresis was performed in tubes according to Weber et al. (1972). Crosslinking Crosslinking of Artemia hemoglobin with glutardialdehyde was carried out in 0.01 M sodium phosphate buffer, pH7.5. The protein concentration was 0.25mg/ml and the glutardialdehyde concentration was 0.05% (v/v). At various times, aliquots were withdrawn from the reaction mixture and crosslinking was stopped by addition of SDS.
185
186
ABDUSSALAMAZEMand EZRADANIEL calibration curve of protein markers with known molecular masses, this band was identified as a polypeptide chain dimer (M, ~270,000). With progressively increasing times of reaction, the intensity of the faster moving monomeric band decreased, while that of the slower moving dimeric band increased. For the longest time shown, the monomeric band has virtually disappeared, and the dimeric band is the only band observed (Fig. 2).
Trypsinolysis Artemia hemoglobin was exposed to trypsin at pH 8.2 and the products were analysed by SDS gel electrophoresis. Before exposure to the enzyme, essentially one band was observed. At progressively increasing times of reaction, bands with higher mobility appeared. Eight bands, in addition to the monomeric band corresponding to the intact polypeptide chain, were observed after l hr of reaction (Fig. 3). Identification of the bands seen in the electrophoreric patterns was made on the basis of their mobilities, taking into consideration that a plot of log molecular mass vs mobility (not shown) is linear for the polyacrylamide concentration (5%) and protein molecular mass range (14,000-200,000) used. In order of increasing mobility, the eight bands obtained by
Fig. 1. Electrophoresis of purified Artemia hemoglobin. Hemoglobin was electrophoresed in 0.1 M phosphate buffer at pH 7.2 (a) in the absence of SDS and (b) in the presence of SDS and 2-mercaptoethanol. Polyacrylamide concentration was 3% in (a) and 3.3% in (b).
Trypsinolysis This was carried out by incubation of hemoglobin with TPCK-trypsin (enzyme treated with L-l-tosylamido2-phenyl-ethyl chloromethyl ketone) as described by Darawshe and Daniel (1991). RESULTS
Purity of Artemia hemoglobin Artemia hemoglobin prepared by the method described showed a typical oxyhemoglobin absorption spectrum with maxima at ~340, 415, 535 and 570 nm. Electrophoresis in non-denaturing medium at pH 7.2 yielded a single band (Fig. la). A single band was likewise obtained in SDS gel electrophoresis (Fig. lb). From the mobility of this band, and by reference to a linear calibration curve obtained with standard protein markers, a molecular mass of 150,000 Da was calculated for the polypeptide chain of Artemia hemoglobin.
Crosslinking Artemia hemoglobin was exposed to glutardialdehyde and the products of crosslinking were analysed by SDS gel electrophoresis. Before exposure to the crosslinking agent, the typical electrophoretic pattern showing one band was observed. Exposure to glutardialdehyde resulted in the appearance of an additional band of lower mobility. Using a
Fig. 2. Crosslinking of Artemia hemoglobin. Hemoglobin was exposed to glutardialdehyde and then electrophoresed in the presence of SDS and 2-mercaptoethanol in 3.3% polyacrylamide gels: (a) not exposed to glutardialdehyde; exposed for (b) 0.5min; (c) 1 min; (d) 15min; (e) 1 hr; (f) 18hr.
Structure of Artemia hemoglobin
187
g
i ÷
Fig. 3. Limited trypsinolysis of Artemia hemoglobin. Hemoglobin in 0.04 M borate buffer, pH 8.2, was exposed to trypsin and then electrophoresed in the presence of SDS and 2-mercaptoethanol in 5% polyacrylamide gels: (A) not exposed to trypsin; (B) exposed for 1 hr. (C) Densitometric scan of (B). (a)-(i) denote electrophoretic bands on gels. Upper scan is a magnification of the corresponding part in the lower scan. limited trypsinolysis of A r t e m i a hemoglobin were found to correspond to submulfiples 8/9-1/9 of a polypeptide chain (Fig. 4). DISCUSSION Mention has been made of the difficulties encountered in preparing intact A r t e m i a hemoglobin caused by the presence of a latent proteolytic enzyme in the hemolymph of the animal. The observation in this study of a single band in the electrophoresis of A r t e m i a hemoglobin in both non-denaturing and
I -8/9
719 619 519 419 :519
2/9
I / 9 ~0.2
04
0.6
08
u
Fig. 4. Identification of electrophoretic bands obtained in limited trypsinolysis of Artemia hemoglobin. Semi-logarithmic plot of the molecular mass associated with a given band, M, scaled relative to the molecular mass of a single polypeptide chain, M c, vs the corresponding relative electrophoretic mobility, u. Linearity of the plot (correlation coefficient 0.999) was achieved by setting M / M c = i/9, where i is an integer 1 ~
