Chem.-Biol. Interactions, 16 (1977) 107--114 © Elsevier/North-Holland Scientific Publishers, Ltd.
107
HEAVY ME T A L COMPLEXATION IN P O L L U T E D MOLLUSCS I. LIMPETS ( P A T E L L A
VULGA TA AND PATELLA
INTERMEDIA )
A.G. HOWARD and G. NICKLESS Department of Chemistry, University of Southampton, Southampton S09 5NH and Department of Inorganic Chemistry, University of Bristol, Cantock 's Close, Bristol, Avon (Great Britain)
(Received May 10th, 1976) (Accepted October 20th, 1976)
SUMMARY The accumulation of cadmium, zinc and copper by the marine gastropod molluscs Patella vulgata and Patella i n t e r m e d i a has been studied by gel permeatio n c h r o m a t o g r a p h y of water-soluble extracts of environmentally contaminated shellfish. A major p r o p o r t i o n of the water-soluble cadmium and copper in these molluscs is associated with a protein of molecular weight 10 800 daltons. Evidence is presented supporting the similarity of this protein with mammalian metallothionein. This protein contained only a small p r o p o r t i o n of the zinc f o u n d in the samples.
INTRODUCTION The ability o f molluscs to accumulate trace metals from their e n v i r o n m e n t has been the subject of considerable study in recent years. Investigations have been made bot h into the degree and rate of this p h e n o m e n o n but little e f f o r t has been applied to determining the biochemical m o d e of this accumulation. The concentrations of trace elements f o u n d in molluscs are d e p e n d e n t u p o n species examined. Comparison of report ed trace metal concentrations in molluscs [1--7] gives rise to the conclusion that, of the c o m m o n British molluscs, the highest levels of cadmium, zinc and copper are generally to be f o u n d in oysters, limpets, whelks and barnacles. The levels in these species are generally much larger than in o t h e r molluscs, such as the periwinkles, Abbreviation: XES, X-ray energy spectrum.
108 tellins, and cockles, obtained from identically polluted sites. It is generally accepted that oysters have a remarkable ability to accumulate cadmium, zinc and copper from their environment (concentration factors in the range 104--10 s relative to the concentration of these elements in the surrounding water [1]). It has been pointed out by Ayling [8] that if the concentration factor is measured relative to the metal concentrations found in the sediments, then the concentration factor of zinc by the oyster drops to 10--40. By similar arguments it can be suggested that the carnivorous molluscs accumulate from animal tissue and the filter-feeding species accumulate from the phytoplankton and suspended sediments. In this manner the efficiency of accumulation of trace elements is not required to be high, primary concentration being carried out via the food chain. Studies into the nature of cadmium and zinc present in the liver and kidneys of terrestrial mammals have shown that these metals are stored as a low molecular weight, high cysteine content protein, metallothionein [9--13]. Olafson and Thompson [14] have reported the isolation of low molecular weight, heavy metal binding proteins from the marine vertebrates, the Atlantic grey seal (Halichoerus grypus), the Pacific fur seal (Callorhinus ursinus) and the copper rock fish (Sebastodes caurinus). Similar studies have recently demonstrated the presence of proteins related to metallothionein in the goldfish (Carassius auratus) [15] and the edible mussel (Mytilus edulis) [16]. It has been suggested by Olafson and Thompson [14] "that metallothionein proteins may be ubiquitous in the living world". With this possibility in mind, the nature of water-soluble cadmium, zinc and copper complexes extracted from environmentally contaminated marine molluscs, has been studied. It was hoped that by this approach, the apparent differences between species might be explained and the creatures categorized according to the mode by which they accumulate trace metals. The results of investigations into the nature of cadmium, zinc and copper complexes in the limpets (Patella vulgata and Patella intermedia) are reported here. Similar work which has been carried out on whelks and the bivalve molluscs will be reported later. EXPERIMENTAL
Samples Specimens were obtained from three sites, providing different trace metal environments. From Sand Point in Somerset samples containing high levels of cadmium and zinc were obtained, reflecting the industrial activity of the Severn Estuary region. From Mylor Harbour in Cornwall, samples were obtained which were contaminated with copper and zinc originating from mining activity. Samples obtained from Aberystwyth, Cardiganshire and Newquay, Cornwall were relatively free from trace metal contamination.
Reagents Chemicals were of analytical reagent grade. Tap water was d o u b l y dis-
109 tilled, the second distillation being performed in an all,lass still. All glassware and plastic containers were soaked in 25% reagent grade nitric acid for at least 30 min, rinsed twice in singly distilled water, and dried overnight at 60°C.
Sample preparation The extraction procedure was chosen in order to avoid all organic and inorganic precipitating agents which might conceal information on the nature of metal complexation. Molluscs (number dependent u p o n their expected metal concentrations) were washed in distilled water and removed from their shells. The soft tissues were homogenized with an equal weight of phosphate buffer, pH 7 (0.025 M in Na2HPO4, 0.025 M in KH2PO4). A sample of this homogenate was taken for metal analysis and the remainder was stirred overnight at 4°C. The homogenate was centrifuged at 30 000 g for 3 h, after which time the supernatant and pellet were separated and weighed; a sample of each was taken for metal analysis, and the supernatant was taken to dryness b y lyophilization. Samples for metal analysis were digested in concentrated nitric acid and were analysed for cadmium, zinc and copper by atomic absorption spectrop h o t o m e t r y using a Varian-Techtron AA5 spectrophotometer.
Gel permeation chromatography Separation of mollusc extracts according to molecular size was performed on 1 m X 2.6 cm glass columns packed with Sephadex G75. Elution was carried out with 0.1 M Tris--HC1 buffer, pH 8.6, at a flow rate of approx. 15 ml/h. Samples of mollusc extract (generally 0.1 g) were dissolved in 1 ml of doubly distilled water and seprated on Sephadex G75. 5-ml fractions of the eluate were collected and analysed for cadmium, zinc and copper by atomic absorption s p e c t r o p h o t o m e t r y w i t h o u t further treatment. Ultra-violet absorption spectra of eluate fractions were obtained using a Pye Unicam SP1700 S p e c t r o p h o t o m e t e r fitted with 1 cm pathlength cells. Molecular weight estimations were performed on a 1 m × 2.6 cm column of Sephadex G75, calibrated by the elution of standard polypeptides and proteins after the m e t h o d described by Andrews [17], using 0.1 M Tris--HC1 buffer (pH 8.6) eluent.
Sulphydryl group determination Sulphydryl groups were colourimetrically determined by reaction with 4,4'-bis-dimethylaminodiphenylcarbinol [18].
X-Ray spectrometry The XES of dried protein samples was obtained using a Phillips transmission electron microscope fitted with a Kevex-ray X-ray Energy Spectrometer system. Approx. 1 mg of protein sample was pressed onto a nylon grid which was then placed in the sample chamber of the electron microscope. The XES
110 spectrum was recorded over the range 0 to 40 keV for a period of 1000 sec. A background spectrum was obtained by repeating the e x p e r i m e n t w i t h o u t the p r o tein sample. A m i n o acid analysis
A m i n o acids were det er m i ned after performic acid oxidation [19], using a T e c h n i c o n Amino Acid Analyser TSM1 fitted with a single 22 cm column of Tech n ico n C3 resin. Ultra filtration
Protein samples were desalted by ultrafiltration through a 4.3 cm diameter Amicon UM2 m e m b r a n e filter. RESULTS Th e concentrations of cadmium, zinc and copper in samples of P. vulgata from Sand Point, Mylor H a r b o u r and A b e r y s t w y t h , t oget her with calculated ex tr actio n efficiencies, are shown in Table I. Extracts o f these three groups of limpets were separated on Sephadex G75; the resulting chromatograms are shown in Figs. l a , l b and 2a. Three resolved cadmium peaks are evident from Fig. l a , the first is centred around the void volume o f the column and is believed to be comprised of high molecular weight material and cellular debris. The second and third peaks, eluted after 1.4 (Protein M2) and 1.9 {Protein M1) void volumes of buffer had passed through the column, were shown by gel permeation chromatogr ap h y on a calibrated column of Sephadex G75 t o correspond to globular proteins o f molecular weights 22 000 and 10 800 daltons, respectively. Proteins M1 and M2 also appeared to be responsible for the binding of a significant p r o p o r t i o n of the copper present in the specimens (Fig. l b ) . Copper in P. i n t e r m e d i a f r om A b e r y s t w y t h was f o u n d to be associated with Protein M1. To investigate the metal c o n t e n t of Protein M1 obtained from Sand Point
TABLE I DETAILS OF SAMPLES (Tide zones: U, unknown; MT, mid tide) Sample
A B C D
Species
P. P. P. P.
vulgata vulgata vulgata intermedia
Origin
Aberystwyth (U) Sand Point (MT) Mylor (MT) Newquay (MT)
Metal concentration ppm (wet weight)
Extraction efficiency (%)
Cd
Zn
Cu
Cd
Zn
Cu
3.14 62.7
107
HEAVY ME T A L COMPLEXATION IN P O L L U T E D MOLLUSCS I. LIMPETS ( P A T E L L A
VULGA TA AND PATELLA
INTERMEDIA )
A.G. HOWARD and G. NICKLESS Department of Chemistry, University of Southampton, Southampton S09 5NH and Department of Inorganic Chemistry, University of Bristol, Cantock 's Close, Bristol, Avon (Great Britain)
(Received May 10th, 1976) (Accepted October 20th, 1976)
SUMMARY The accumulation of cadmium, zinc and copper by the marine gastropod molluscs Patella vulgata and Patella i n t e r m e d i a has been studied by gel permeatio n c h r o m a t o g r a p h y of water-soluble extracts of environmentally contaminated shellfish. A major p r o p o r t i o n of the water-soluble cadmium and copper in these molluscs is associated with a protein of molecular weight 10 800 daltons. Evidence is presented supporting the similarity of this protein with mammalian metallothionein. This protein contained only a small p r o p o r t i o n of the zinc f o u n d in the samples.
INTRODUCTION The ability o f molluscs to accumulate trace metals from their e n v i r o n m e n t has been the subject of considerable study in recent years. Investigations have been made bot h into the degree and rate of this p h e n o m e n o n but little e f f o r t has been applied to determining the biochemical m o d e of this accumulation. The concentrations of trace elements f o u n d in molluscs are d e p e n d e n t u p o n species examined. Comparison of report ed trace metal concentrations in molluscs [1--7] gives rise to the conclusion that, of the c o m m o n British molluscs, the highest levels of cadmium, zinc and copper are generally to be f o u n d in oysters, limpets, whelks and barnacles. The levels in these species are generally much larger than in o t h e r molluscs, such as the periwinkles, Abbreviation: XES, X-ray energy spectrum.
108 tellins, and cockles, obtained from identically polluted sites. It is generally accepted that oysters have a remarkable ability to accumulate cadmium, zinc and copper from their environment (concentration factors in the range 104--10 s relative to the concentration of these elements in the surrounding water [1]). It has been pointed out by Ayling [8] that if the concentration factor is measured relative to the metal concentrations found in the sediments, then the concentration factor of zinc by the oyster drops to 10--40. By similar arguments it can be suggested that the carnivorous molluscs accumulate from animal tissue and the filter-feeding species accumulate from the phytoplankton and suspended sediments. In this manner the efficiency of accumulation of trace elements is not required to be high, primary concentration being carried out via the food chain. Studies into the nature of cadmium and zinc present in the liver and kidneys of terrestrial mammals have shown that these metals are stored as a low molecular weight, high cysteine content protein, metallothionein [9--13]. Olafson and Thompson [14] have reported the isolation of low molecular weight, heavy metal binding proteins from the marine vertebrates, the Atlantic grey seal (Halichoerus grypus), the Pacific fur seal (Callorhinus ursinus) and the copper rock fish (Sebastodes caurinus). Similar studies have recently demonstrated the presence of proteins related to metallothionein in the goldfish (Carassius auratus) [15] and the edible mussel (Mytilus edulis) [16]. It has been suggested by Olafson and Thompson [14] "that metallothionein proteins may be ubiquitous in the living world". With this possibility in mind, the nature of water-soluble cadmium, zinc and copper complexes extracted from environmentally contaminated marine molluscs, has been studied. It was hoped that by this approach, the apparent differences between species might be explained and the creatures categorized according to the mode by which they accumulate trace metals. The results of investigations into the nature of cadmium, zinc and copper complexes in the limpets (Patella vulgata and Patella intermedia) are reported here. Similar work which has been carried out on whelks and the bivalve molluscs will be reported later. EXPERIMENTAL
Samples Specimens were obtained from three sites, providing different trace metal environments. From Sand Point in Somerset samples containing high levels of cadmium and zinc were obtained, reflecting the industrial activity of the Severn Estuary region. From Mylor Harbour in Cornwall, samples were obtained which were contaminated with copper and zinc originating from mining activity. Samples obtained from Aberystwyth, Cardiganshire and Newquay, Cornwall were relatively free from trace metal contamination.
Reagents Chemicals were of analytical reagent grade. Tap water was d o u b l y dis-
109 tilled, the second distillation being performed in an all,lass still. All glassware and plastic containers were soaked in 25% reagent grade nitric acid for at least 30 min, rinsed twice in singly distilled water, and dried overnight at 60°C.
Sample preparation The extraction procedure was chosen in order to avoid all organic and inorganic precipitating agents which might conceal information on the nature of metal complexation. Molluscs (number dependent u p o n their expected metal concentrations) were washed in distilled water and removed from their shells. The soft tissues were homogenized with an equal weight of phosphate buffer, pH 7 (0.025 M in Na2HPO4, 0.025 M in KH2PO4). A sample of this homogenate was taken for metal analysis and the remainder was stirred overnight at 4°C. The homogenate was centrifuged at 30 000 g for 3 h, after which time the supernatant and pellet were separated and weighed; a sample of each was taken for metal analysis, and the supernatant was taken to dryness b y lyophilization. Samples for metal analysis were digested in concentrated nitric acid and were analysed for cadmium, zinc and copper by atomic absorption spectrop h o t o m e t r y using a Varian-Techtron AA5 spectrophotometer.
Gel permeation chromatography Separation of mollusc extracts according to molecular size was performed on 1 m X 2.6 cm glass columns packed with Sephadex G75. Elution was carried out with 0.1 M Tris--HC1 buffer, pH 8.6, at a flow rate of approx. 15 ml/h. Samples of mollusc extract (generally 0.1 g) were dissolved in 1 ml of doubly distilled water and seprated on Sephadex G75. 5-ml fractions of the eluate were collected and analysed for cadmium, zinc and copper by atomic absorption s p e c t r o p h o t o m e t r y w i t h o u t further treatment. Ultra-violet absorption spectra of eluate fractions were obtained using a Pye Unicam SP1700 S p e c t r o p h o t o m e t e r fitted with 1 cm pathlength cells. Molecular weight estimations were performed on a 1 m × 2.6 cm column of Sephadex G75, calibrated by the elution of standard polypeptides and proteins after the m e t h o d described by Andrews [17], using 0.1 M Tris--HC1 buffer (pH 8.6) eluent.
Sulphydryl group determination Sulphydryl groups were colourimetrically determined by reaction with 4,4'-bis-dimethylaminodiphenylcarbinol [18].
X-Ray spectrometry The XES of dried protein samples was obtained using a Phillips transmission electron microscope fitted with a Kevex-ray X-ray Energy Spectrometer system. Approx. 1 mg of protein sample was pressed onto a nylon grid which was then placed in the sample chamber of the electron microscope. The XES
110 spectrum was recorded over the range 0 to 40 keV for a period of 1000 sec. A background spectrum was obtained by repeating the e x p e r i m e n t w i t h o u t the p r o tein sample. A m i n o acid analysis
A m i n o acids were det er m i ned after performic acid oxidation [19], using a T e c h n i c o n Amino Acid Analyser TSM1 fitted with a single 22 cm column of Tech n ico n C3 resin. Ultra filtration
Protein samples were desalted by ultrafiltration through a 4.3 cm diameter Amicon UM2 m e m b r a n e filter. RESULTS Th e concentrations of cadmium, zinc and copper in samples of P. vulgata from Sand Point, Mylor H a r b o u r and A b e r y s t w y t h , t oget her with calculated ex tr actio n efficiencies, are shown in Table I. Extracts o f these three groups of limpets were separated on Sephadex G75; the resulting chromatograms are shown in Figs. l a , l b and 2a. Three resolved cadmium peaks are evident from Fig. l a , the first is centred around the void volume o f the column and is believed to be comprised of high molecular weight material and cellular debris. The second and third peaks, eluted after 1.4 (Protein M2) and 1.9 {Protein M1) void volumes of buffer had passed through the column, were shown by gel permeation chromatogr ap h y on a calibrated column of Sephadex G75 t o correspond to globular proteins o f molecular weights 22 000 and 10 800 daltons, respectively. Proteins M1 and M2 also appeared to be responsible for the binding of a significant p r o p o r t i o n of the copper present in the specimens (Fig. l b ) . Copper in P. i n t e r m e d i a f r om A b e r y s t w y t h was f o u n d to be associated with Protein M1. To investigate the metal c o n t e n t of Protein M1 obtained from Sand Point
TABLE I DETAILS OF SAMPLES (Tide zones: U, unknown; MT, mid tide) Sample
A B C D
Species
P. P. P. P.
vulgata vulgata vulgata intermedia
Origin
Aberystwyth (U) Sand Point (MT) Mylor (MT) Newquay (MT)
Metal concentration ppm (wet weight)
Extraction efficiency (%)
Cd
Zn
Cu
Cd
Zn
Cu
3.14 62.7
