Marine polychaetes, collected from the Vellar Estuary exhibited arylsulfatase activity. Lumbriconeries sp. Polydora sp. Monojis sp. and Heteromastus sp. were selected for this study. Of these, Heteromastus sp. showed maximum enzymatic activity and it has been chosen for the enzyme kinetic studies such as pH, optimal temperature, period of incubation and the effect of DDT. Enzyme activity showed single peak at pH 6.2 possibly indicating the presence of one type of arylsulfatase. Maximum activity was attained after 12h of incubation at 29°C. DDT has an inhibiting effect on the arylsulfatase activity even at the concentration of 10 p.p.m. and the activity was completely lost at 100 p.p.m

Dhevendaran, K.

Aquatic Commons

Arylsulfa'tase Ac'tivi'ty in Marine Polycha.e'tes 
K. DHEVENDARAN 
Schoql of Marine Sciences, University of Cochin, Cochin- 682 016 
Marine polychaetes, collected from the Vellar Estuary exhibited arylsulfatase acti-
vity. Lumbriconeries sp. Polydora sp. M onojis sp. and H eteromastus sp. were selected 
for this study. Of these, Heteromastus sp. showed maximum enzymatic activity and it 
has been chosen for the enzyme kinetic studies such as pH, optimal temperature, period 
of incubation and the effect of DDT. Enzyme activity showed single peak at pH 6.2 
possibly indicating the presence of one type of arylsulfatase. Maxim.'Jm activity was 
attained after 12 h of incubation at 29°C. DDT has an inhibiting effect on the aryl-
sulfatase activity even at the concentration of 10 p.p.m. and the activity was completely 
lost at 100 p.p.m. 
Arylsufatase ( arylsulfate sulfohydrolase 
E. C. 3.1.6.1) is a lysozyme that partcipates 
in the hydrolysis of sulfuric acid esters of 
aromatic compounds. Since the natural 
substrate is still obscure, it stimulates the 
detailed investigation of this enzyme in both 
vertebrates and invertebrates (Dodgson & 
Spencer, 1957; Wortman &Schneider, 1960; 
Conner, eta!., 1960; Jarrige, 1963; Agogbua 
et a!., (1978) suggested that molluscs were 
the potential sources and in continuance of 
this line various other invertebrates were 
considered. Quite interestingly, the 
marine environment is a vast untapped 
resource and this has been considered 
to be the best suitable biotope for the easy 
accessible exploitation of this enzyme. 
The bacterium, Alcaligenes metalcaligenes 
exhibited arylsulfatase activity and is inha-
biting in the marine intertidal mud (Dodgson, 
et al., 1953). Shimony & Nigrelli (1972) 
correlated this enzyme activity with the 
cyclic formation and hardening of the ,exo-
skeleton of Balanus eburneus, whereas Cor-
net & Jangoux (1974) noted the functional 
relationship between this enzyme activity 
and the feeding habits in some echinoderms. 
The enzyme kinetics of the arylsulfatase 
in madne sediments and salt marshes showed 
variety of the components of this enzyme 
(Dhevendaran, ·1978; Oshrain & Wei be, 
1979). Dhevendaran. eta!. (1980) have found 
variations in the activity of this enzyme in foot, 
visceral hump and crystalline style of some 
marine gastropods, inhabiting ecologically 
different biotopes. Corner et a!. (1960) 
Vol. 21, 1984 
reported the occurrence of this enzyme in 
the polychaete Chaetopterus variopedatus. 
The present investigation is the first attempt 
of enzyme kinetics of arylsulfatase in marine 
polychaetes and gives an added information 
about its functional significance. 
.Materials aml Methods 
Luinbriconeries sp. and Monijis sp. were 
collected from sandy beaches, whereas Poly-
dora sp. and Heteromastus sp. were taken 
from the station, situated in the Vellar Estuary, 
just opposite to the Biological Station (Fig. 1). 
Fig. 1. Vellar estuary showing sampling stations 
The sandy beach is directly under the influence 
of seawater and the sediment is mainly coarse 
and also the organic matter is negligible. 
But the sediment nature in the station just 
opposite to the Biological Station is clayey 
and is always rich in organic matter. It is 
58 
often flushed with saline water except during 
the monsoon season. 
The organisms were cut open to remove 
all the sediments and sand grains and they 
were thoroughly washed with 50% seawater. 
The polychaetes were weighed separately. 
The weighed material was homogenised in 
3.0 ml of distilled water. 
Arylsulfatase activity was estimated spe-
ctrophotometrically in the red anionic form 
of 4-Nitrocatechol (4.NC), released during 
the enzyme hydrolysis (Shimony & Nigrelli, 
1972). The reaction mixture consisted of 
I ml of enzyme solution, 2 ml of 0.2 M 
acetate buffer at pH 6.2 and 1 ml of 0.002 M 
Nitrocatechol sulfate (NCS) as substrate 
and was incubated at room temperature 
(28 ± 2°C)for 12 h unless otherwise stated. At 
the end of the incubation period the reaction 
was stopped by the addition of 4 ml of 1 N 
NaOH. The liberated red anionic form of 
4·NC was measured following the modified 
procedure employed by Dhevendaran et a!. 
(1980) at a wave-length of 515 nm against a 
reagent blank. A standard curve was pre-
pared using 4.NC in the similar manner and 
the enzyme activity was expressed as 4. NC 
liberated/rug of organism. 
Results and Discussion 
Arylsulfatase activity was recorded in. all 
the four selected polychaetes. Of these, 
Heteromastus sp. displayed higher activity 
than that of others and hence this organism 
has been used for enzyme kinetic studies. 
It is understood from the results (Fig. 2) 
that the maximum activity was observed at 
pH 6.2 thereby confirming the earlier findings 
of Chandramohan et a!. (1974) in the marine 
sediments and the possibility for the contri-
bution towards the sedimentary enzyme 
pool and also supporting the previous reports 
in bacterium, Mycobacterium piscium and 
the mollusc, Achatina achatina (Whitehead 
et al., 1952; Agogbua et al., 1978). Conner 
et al. (1960) tested the arylsulfatase activity 
at pH 5.5 in Chaetopterus variopedatus but 
the optimum pH of this enzyme is still obs-
cure. Hence an attempt was made to study 
the pH optimum of this enzyme in Hetero-
mastus sp. and further study may throw more 
K. DHEVENDARAN 
100 
E 
::J 
E 
')( 
0 
E 
-0 
~ 
,.,50 
+-
:~ 
u 
<! 
oL---~4--~5~--~6~--7~--~s~--~9--,l~o 
pH 
Fig. 2. pH activity curve for arylsulfatase activity 
in Heteromastus sp. 
light on the possible function of this enzyme 
in marine polychaetes. Quite surprisingly 
when the pH was raised towards the alkaline 
side such as pH 8 and 9 the activity was 
very much reduced to 35~la and 19% of maxi-
mum activity respectively. This indicates 
that the arylsulfatase under natural condi-
tions must be at a very low level both quali-
tatively and quantitatively since the pH of 
the seawater and sediment ranged from 7.9 
to 8.3. Dhevendaran (1978) confirmed this 
by his study in the marine sediments. At 
pH 5.6, 88% of the maximum activity was 
observed which is very close to the pH opti-
mum in the limpet, Patella vulgata and in 
the fungus, Aspergillus oryzae (Dodgson., 
et a!., 1953; Robinson et a!., 1952) but 
Oshrain & Wiebe (1979) noticed this enzyme 
activity at the wider range of pH between 5 
and 9 in salt marshes. The earlier report 
in marine molluscs at the same pH optimum 
(pH 5.6) supports this (Dhevendaran et 
a/., 1980). At pH 7.1 about 54% of the 
maximum activity was recorded and this 
may be attributed to the microbial associa-
tion with polychaetes as suggested by Dodgson 
et al. (1953) in molluscs. Ammonia, chlo-
ride, citrate and acetate ions have a marked 
activation effect on the hydrolysis of· the 
substrate by bacterial arylsulfatase (Dodgson, 
1959; Harada & Spencer, 1964) and these 
ions may also have some influence on the 
arylsulfatase in polychaetes and it needs fur-
ther study and work on this line is in pro-
gress. However, the presence of activators 
FISHERY TECHNOLOGY 
ARYLSULFATASE ACTIVITY IN MARINE POLYCHAETES 59 
or inhibitors in the ·tissue of polychaetes, 
which are sensitive to pH may also cause 
some considerable changes in the enzyme 
activity curve as suggested by Shimony & 
Nigrelli (1972). 
Fig. 3 shows the influence of period of 
incubation on the enzyme activity. Maxi-
mum hydrolysis of nitrocatechol sulfate was 
10 
'E 
::J 
E 
x 
0 
.E 
..... 
0 
~ 0 
~ 
.... 
> 
-;:: 
0 
<! 
Fig. 3. 
16 20 24 
Time (hrs) 
Effect of incubation period on arylsulfatase 
activity in Heteromastus sp. 
obtained after 12 h of incubation, similar 
to the observation made by Dhevendaran 
et a!. (1980) in marine molluscs. Nearly 
77% of maximum activity could be recorded 
after 8 h of incubation. No change in the acti-
vity could be observed by extending the 
period of incubation. Dhevendaran (1978), 
however, observed maximum activity (92%) 
after 12 h of incubation in marine sediments. 
Tabatabai & Bremner (1970) reported that 
prolonging the period of incubation increased 
the activity in the soil. But a declined trend 
in activity with longer period of incubation 
was reported in Helix pvmatia (Dodgson & 
Powell, 1959). 
The optimum. temperature for arylsulfa-
tase activity in polychaetes was noticed at 
29° C (Fig. 4). It is obvious that it is an 
aquatic organism and showed maximum 
activity at this temperature, which is almost 
similar to the tern perature of the environment 
where the organism is inhabiting. This 
result fully supports the earlier observations 
Vol. 21, 1984 
100 
E 
::J 
E 
·x 
0 
E 
~ 
0 
~50 
;>, 
.... 
·:;: 
<= 
0 
<( 
Temperature ( oc l 
Fig. 4. Effect of temperature on arylsulfatase 
activity in Heteromastus sp. 
made by Dhevendaran (1978) and Dheven-
daran et a!. (1980) in marine sediments and 
marine gastropods respectively. For the first 
time we could obsetve that the 23% of the 
maximum activity was retained at 4°C. At 
40oC only 60~~~ of the activity was lost and 
further increase in tempe1atun;. to 65° C, 
there was a complete loss of enzyme 
activity. This may be either due to the 
proteolysis or to the inactivation of the 
enzyme by rise of temperatm e as suggested 
by Milanesi & Bird (1972). 
The stations from where polychaetes 
collected, were frequently influenced by the 
discharge of nearby agricultural field and 
there is every possibility for the influence of 
various herbicides and pesticides used in 
the agricultural field, over the polychaetes, 
which were discharged into the estuarine 
water. So an attempt has been made to 
study the effect of DDT on arylsulfatase 
activity in marine polychaetes (Table 1). 
The minimum concentration of (10 p.p.m.) 
DDT has got inhibiting effect on the arylsul-
fatase activity. It is understood that the 
inhibitors may be one of the factors for deter-
. mining the components of the arylsulfatase 
enzyme. Besides this, Kiss et al. (1975) 
suggested that the soil enzymes are very much 
influenced by differential compounds such 
as antibiotics, herbicides, pesticides etc. 
Similarly the inhibition of mercuric chloride, 
cyanide and phosphate on soil arlysulfatase, 
phosphate on the activity of arylsulfatase B 
in Balanus ebumeus and of cyanide, phosphate 
and sulfate on the marine sediments clearly 
60 
Table 1. Effect of DDT on m·ylsulfatase 
activity in H eteromastus sp. 
Concentration of 
DDT,p.p.m. 
10 
20 
50 
100 
Arylsulfatase 
activity, 
f-lgNCfmg 
1.177 
1.121 
0.188 
0.036 
indicates the possibility of occurrence of 
more than one type of arylsulfatase enzyme 
in the marine environment (Tabatabai & 
Bremner, 1970; Shimony & Nigrelli, 1972; 
Dhevendaran, 1978). Work on this line 
is in progress to identify the components of 
arylsulfatase enzyme. 
Table 2 shows the arylsulfatase activity 
in the marine polychaetes, inhabiting the 
Table 2. Arylsulfatase activity of d({ferent 
marine polychaetes 
Nature of Organisms Arylsulfatase 
sediments activity, 
f-lgNC/mg 
Sandy Monojissp. 0.322 
Sandy Lumbricon- 0.401 
eries sp. 
Clayey Po!ydora sp. 0.480 
ecologically varying niches. Po!ydora sp. 
exhibited considerably higher activity than 
other polychaetes (0.480 NC/mg). It is 
undoubtedly one of the potent sources foi· 
this enzyme. It is possible that the enzyme 
might help in the transfer of sulfate from 
arylsulfate to polysaccharides as suggested 
already (Suzuki et al., 1959; Dodgson & 
Powell, 1959). Dhevendaran (1978) found 
the above to be true in marine sediments. 
Since Polydora sp. feeds on sediments and 
detrital particles, the digestive system of 
this organism will certainly contain appre-
ciable amounts of sulfated polysaccharide 
material. Dodgson et a/. (1953) and Cor-
ner et a/. (1960) suggested that arylsulfatase 
activity was. greater in herbivorous species 
K. DHEVENDARAN 
possibily because of thick walled plant cells 
in the diet. Cornet & Jangoux: (1974) also 
reported that arylsulfatase in some echino-
derms and found higher. activity in. algivorous 
animals than in carnivores. All these o bser-
vations indicate the possibility of various 
roles for this enzyme, in. different forms (as 
indicated by the peaks of activity at various 
pH). Although it is not possible to differen-
tiate the arylsulfatases into A and B, as in 
mammals, the properties of this enzyme 
from polychaetes resemble the Type II aryl-
sulfatase (Dodgson & Spencer, 1957) of 
mammals. 
Thanks are due to Prof. R. Natarajan, Director, 
Marine Biological Station, Porto Novo for constant 
encouragement and critically going through the 
manuscript. The facilities provided by Anriamalai 
University, the financial assistance by UGC and 
Dr. B. Srikrishnadas Fisheries College, Tuticorin, 
Tamil Nadu for the identification of polychaetes are 
gratefully acknowledged. I am also thankful to 
Prof. N. R. Menon, Head of the Division of Marine 
Biology, Microbiology and Biochemistry, University 
of Cochin for his encouragement and· facilities 
provided. 
References 
Agogbua, S.I.O., Anosiki, E.O. & Ugochu-
kuru, E. N. (1978) Comp. Biochem. 
Physiol. 59B, 169 
Chandramohan, D., Dhevendaran, K. & 
Natarajan, R. (1974) Mar. Bioi. 27, 
89 
Corner, E. D.S., Leon, Y. A. & Bulbrook, 
R. D. (1960) J. mar, Bioi. Assoc. 
UK. 39, 51 
Comet, D. & Jangoux, M. (1974) Comp. 
Biochem. Physiol. 47, 45 
Dhevendaran, K. ( 1978) Studies in Aryl-
sulfatase Activity of Marine Sediments. 
Ph. D. Thesis, Annamalai University. 
p. 115 
Dhevendaran, K., Kannupandi, T. & Nata-
rajan, R. (1980) Mahasagar, 13, 173 
Dodgson, K. S. (1959) Enzymologia, 20, 
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FISHERY TECHNOLOGY 
ARYLSULFATASE ACTIVITY IN MARINE POLYCHAETES 61 
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Dodgson, K. S. & Powell. G. M. (1959) 
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Arylsulfatase activity in marine polychaetes

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Arylsulfatase activity in marine polychaetes

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