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Aca

Biomedical

Biological

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in bilothio Noas, 175 Sea

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Laboratory

motor: Dr.

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Acknowledgement

This thesis is the result of hard work from different people. My part was only a

piece of the puzzle. I appreciate that the Ruđer Bošković Institute allowed me to do

my internship there. Because of this I met a lot of new people who were very

welcoming. I am very grateful for the work that the persons did from my university

college HoGent, i.e. the teachers from the department Biomedical Laboratory

technology for making the internship happen through a lot of effort and

communication and the administrative staff who helped me with the paper work

which was very welcome since I’m not very good at that.

I want to award some persons in particular and since the Nobel prizes aren’t

awarded yet, I want to make some suggestions.

I nominate my promotor Dr.Sc. Marijana Erk for the Nobel prize in Chemistry. I am

very glad that she accepted me to do my internship in her laboratory LBUM. I

learned a lot from her during those 4 months. I want to thank her for the effort she

put in helping me with my work and giving feedback on my thesis.

I would like to give the Nobel prize in Literature to my co-promotor Dr. Brita

Muyssen. I am very grateful for the patience that she had with me for answering all

my questions. I want to thank her for the time she spent in reading my thesis and

guide me with the writing part of my thesis.

The Nobel Peace Prize should go to the colleagues from the Laboratory for

Biological Effects of Metals (LBUM): Dr. Zrinka Dragun, Dr. Vlatka Filipović

Marijić, Dr. Dušica Ivanković and Nesrete Krasnići. They and my promotor were

very supportive and helped me where they could. They showered me with

kindness, I felt at home and also a part of the team. I am happy as a clam that they

learned me some Croatian, hvala najljepša.

The nomination for Nobel prize for Physics goes to the colleagues of IRB. This is for

a different kind of physics, i.e. football. The games of football were a perfect outlet

after a work day. Thanks, guys.

Th

Ser

un

Th

my

Spe

Za

pro

Me

and

rep

I a

sho

he Nobel P

rgeant for

nforgettable

he Sveriges

y family fo

ecial grati

dar for SC

ofessor at

elita Pehar

d Dr.Sc.

productive

also want

owing me

Prize in Ph

r the heal

e.

Riksbank

or supportin

tude goes

CUBA divi

University

rda Uljević

Sanja Pu

e status of s

to thank

the ropes o

ysiology o

lthy and

Prize in Ec

ng me in m

to Dr.Sc.

ing and co

y of Zadar

ć from the

ljas from

shell fish.

Dr. Sc. Sa

of field wo

or Medicin

less healt

conomic Sc

my choice t

Ivan Žup

ollecting sh

r for logisti

Institute f

the Univ

anja Gottst

ork.

ne goes to

thy diners

ciences in

to do my in

pan, assista

hell fish, to

ics and dis

for Oceano

versity of

tein and D

Steffi Lem

s. You m

Memory o

nternship i

ant profess

o Dr.Sc. Jel

ssection of

ography an

f Split for

Dr. Sc. Mir

mmens and

made the e

of Alfred N

in Zagreb.

sor at Uni

lena Čulin

f shell fish

nd Fisheri

r determi

irela Sertić

d Bronwen

experience

Nobel is for

iversity of

n, assistant

h, to Dr.Sc.

ies in Split

ination of

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Table of contents

1  Introduction ...................................................................................................... 9 1.1  Noah’s ark shell ................................................................................................ 9 1.1.1  Taxonomy .......................................................................................................... 9 1.1.2  Morphology ...................................................................................................... 9 1.1.3  Reproduction .................................................................................................. 10 1.1.4  Habitat ............................................................................................................. 11 1.1.5  Consumption .................................................................................................. 12 1.2  Pollution .......................................................................................................... 12 1.2.1  Bivalves as indicator organisms in environmental studies ...................... 13 1.3  Sampling sites ................................................................................................. 15 1.3.1  Telaščica ........................................................................................................... 16 1.3.2  Pašman kanal/channel .................................................................................. 16 1.4  Biomarkers ...................................................................................................... 17 1.4.1  Metallothionein .............................................................................................. 20 1.4.2  Proteins, carbohydrates and lipids .............................................................. 23 1.5  Biotic and abiotic factors that affect biomarker variability ...................... 24 1.6  Aim of this study ............................................................................................ 26 

2  Materials and methods ................................................................................. 27 2.1  Homogenization ............................................................................................. 27 2.1.1  Materials .......................................................................................................... 27 2.1.2  Protocol ............................................................................................................ 28 2.2  Quantification of metallothioneins and cytosolic proteins ...................... 30 2.2.1  Materials .......................................................................................................... 30 2.2.2  Protocol ............................................................................................................ 31 2.3  Quantification of total tissue proteins ......................................................... 35 2.3.1  Materials .......................................................................................................... 35 2.3.2  Protocol ............................................................................................................ 36 2.4  Quantification of total tissue carbohydrates .............................................. 37 2.4.1  Materials .......................................................................................................... 37 2.4.2  Protocol ............................................................................................................ 38 2.5  Quantification of total tissue lipids ............................................................. 40 2.5.1  Materials .......................................................................................................... 40 2.5.2  Protocol ............................................................................................................ 40 2.6  Statistics ........................................................................................................... 42 

3  Results ............................................................................................................. 43 3.1  Metallothionein .............................................................................................. 43 3.2  Proteins ............................................................................................................ 46 3.3  Carbohydrates ................................................................................................ 49 3.4  Lipids ............................................................................................................... 52 3.5  Correlations between measured parameters ............................................. 55 3.6  Seasonal and spatial variability of measured parameters ........................ 60 3.6.1  Metallothionein .............................................................................................. 60 3.6.2  Proteins ............................................................................................................ 62 3.6.3  Carbohydrates ................................................................................................ 63 3.6.4  Lipids ............................................................................................................... 65 

4  Discussion ...................................................................................................... 67 

5  Conclusion ...................................................................................................... 74 

Bibliography ..................................................................................................................... 75 

Abbreviations

MT Metallothionein

PK 1 Pašman kanal 1

PK 2 Pašman kanal 2

TE Telaščica

d.w. dry weight

w.w. wet weight

MGI Mean gonadal index

CI Condition index

ISQG Interim Marine Sediment Quality Guidelines

PEL Probable Effect Level

MCL Maximum contaminant level

n.v. no value

Glossary Periostracum = an organic, outside layer that is found on shells of a mollusk.

Protandric = having male sexual organs in young stadium of life, and female in the later stadia.

Abstract English Noah’s Ark shell (Arca noae) is a bivalve that is common in the Adriatic Sea, it is

exploited for culinary purposes from natural populations. The aim of this study is to

determine the concentrations of total lipids, proteins, carbohydrates and

metallothionein to differentiate between the sampling sites and their respectively

different environmental pressures and potential metal exposure. Three sampling

sites were selected: one from nature park Telaščica and two from the Pašman kanal-

the main harvesting area in the central part of Eastern Adriatic Sea in Croatia. From

each site, 36 individuals were sampled monthly in the period from March 2013 to

February 2014 . The digestive gland was used for measuring total lipids, total

proteins, total carbohydrates and metallothionein concentration, all by

spectrophotometric methods. Correlations between parameters were calculated and

two-way ANOVA was used to investigate the seasonal and spational variability

between the different sampling stations. Correlations were found for metallothionein

concentrations and reproductive cycle as well as for temperature and total lipids.

This study provided first data on metallotioneins in Arca noae. Levels of

metallothionein showed the impact that nautical tourism has on nature parks. In

summer the level of metallothionein in A. noae from Telaščica is higher than that for

the sampling sites where anthropogenic stress is expected. From the results we can

conclude that metallothionein is a possible biomarker for A. noae. More research with

organisms from sampling sites of which pollution levels are more elevated is

necessary to confirm this conclusion.

Keywords: Arca Noae, biochemical composition, seasonal variation, metallothionein, digestive gland

Abstract Nederlands Arca noae is een tweekleppige schelp die voorkomt in de Adriatische Zee. Schelpen

uit de natuurlijke populatie worden gevangen genomen voor menselijke consumptie.

Het doel van de studie is het achterhalen of de concentratie van alle lipiden,

proteïnes, carbohydraten en metallothioneïne beïnvloed wordt door de verschillende

staalafnameplaatsen en hun eigen verschillen in ecologische druk en eventuele

metaal vervuiling. Er zijn drie staalafnameplaatsen waarvan één in het natuurpark

Telaščica en twee uit het Pašman kanal wat het grootste oogstgebied is, in het

centrale gedeelte van de Adriatische Zee van Kroatië. Voor elke plaats zijn er

maandelijks 36 individuen genomen over een periode van maart 2013 tot februari

2014. Voor de metingen van alle lipiden, alle proteïnes, alle carbohydraten en

metallothioneïne werd de spijsverteringsklier gebruikt en gemeten door

spectrofotometrische bepalingen. De correlaties tussen de parameters werden

onderzocht en two way ANOVA berekende de seizoens- en plaatsschommelingen

voor de verschillende sites. Er zijn correlaties gevonden tussen metallothioneïne

concentratie en de voortplantingscyclus alsook voor de temperatuur en de lipiden.

Het is voor het eerst dat er concentraties van metallothioneïne zijn gemeten bij de

Arca noae. De metallothioneïnewaarde in de zomer voor Telaščica ligt hoger dan de

plaatsen waar er antropogenische invloeden werden verwacht. Dit toont de invloed

van boottoerisme op natuurparken. Metallothioneïne zou een mogelijke biomarker

kunnen zijn voor A. noae maar verder onderzoek is aangewezen, aangezien de

concentraties van de verontreinigde stoffen niet hoog lagen.

Keywords: Arca Noae, biochemische samenstelling, seizoensvariatie,

metallothioneïne, spijsverteringsklier

BIO

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CONTENTS

Education

to the phy

orphia and

family Ar

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at a numbe

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COMPOSITIO

al., 2014).

old populat

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METALLOT

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2 below.

CONTENTS

Education

o of tissue

spawning

d mean go

mber of in

stage, and

ed (Gosling

ng numeri

g: inactive

pent (1). (P

pawning p

mpled shell

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from each

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s used for

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al., 2006)

in A. noae

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n rocks or

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COMPOSITIO

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OCHEMICAL

geschool Gen

ese produc

mpound. A

pan in 195

vironment

sulted in

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the richest

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valves ha

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METALLOT

culture. M

bout this p

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sediments

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by a very h

cosystems o

Adriatic Sea

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utants in th

THIONEIN C

Methylated

pollutant is

leased me

then me

to fish. 10

high biodiv

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a are the r

sm and la

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l pollutants

e, due to

nal consum

anisms inadvantage

ween the c

quently, i

vironment

sentinel

ct either na

that mak

e substratemedia

he organism

CONTENTS

Education

mercury i

s the accide

tallic merc

thylated t

00 people

versity and

Main sourc

residences

and-use pr

ustaceans

s in high co

metal toxi

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m

IN ARCA N

BLT - specia

is an organ

ent in Mini

cury in th

the mercu

died of e

d they incl

ces of anth

in the coa

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and fish) t

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er et al., 20

nmental ntaminant a

ions of con

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ring tasks (

ator organ

anthropoge

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NOAE 13

alization FBT

nometallic

imata bay,

he aquatic

ury which

eating the

lude some

hropogenic

astal zone,

griculture,

that live in

ons. Metal

he aquatic

001).

studiesanalysis of

ntaminants

uded that

(Phillips &

nisms for

enic stress.

ioindicator

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c

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r

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Currently

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organisms

substance

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parent an

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tissue. Dig

as import

therefore

exposure

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general co

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performed

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ds (PFCs)

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ations of th

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is has bee

by means

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ondition of

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ing of con

d within the

e marine e

described

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mme of che

JAMP, an

nd snails)

sentatives o

etals and o

henyls, DD

ed PAHs, b

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of MTs as

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aminants,

ation (Livi

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ON AND ME

taminants

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as well as

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types of c

main meta

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ngstone &

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tive gland

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in marine

ork of OSP

nt of this a

nt Assessm

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r priority a

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biota are s

mpounds i

tabolites, H

d flame re

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unds (TBT

SPAR JAM

contaminan

abolic and

ic contami

s the targe

biomarker

/MAP, 19

the bioi

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he breakdo

onse, resul

o physiolo

& Pipe, 19

ell-type ra

ant than d

pithelium w

(Cajaravil

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e biota in

AR as the

area. The o

ment and M

egy, provi

action, and

nants are

nd seabird

suitable for

including c

HCH isom

etardants s

odecane (

T and its

MP, 2012).

nts can dif

storage ti

inants (Ge

et tissue fo

r (e.g. with

999). With

indicator

land (OSPA

own of th

lting not o

ogical extre

992). It is

atios in th

igestive ce

was exami

le et al., 19

ONTENTS IN

the North-

regional co

objectives o

Monitoring

ding the

d hazardou

performed

d eggs. The

r monitorin

chlorinated

ers, HCB a

uch as pol

HBCD), p

breakdow

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ssue for m

effard et a

or the sur

in the fram

hin the O

organism

AR JAMP,

he digestiv

only from e

emes such

known th

he digestiv

ells), and

ined as a m

990).

N ARCA NOA

h-east Atlan

onvention

of monitor

g Program

basis for

us substan

d in shellf

ese groups

ng hazardo

d compoun

and dieldr

lybromina

perfluorina

wn produc

erent types

metals, as w

al., 2001) a

rvey of me

mework of

OSPAR Jo

m is Myt

2007).

ve epitheli

exposure t

h as increa

hat pollut

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marker of

AE

ntic

for

ring

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nces

fish

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hey give ti

ater column

3 Sahe study a

mpling site

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L COMPOSI

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, for det

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ITION AND

s Vesalius

tecting na

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ution becau

pollution

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sites cated in th

lected, as s

central Adrthe Pašman

METALLOT

atural env

energy allo

ntle (Erk et

es that are b

use their b

sources o

mation on

goli, 1998)

he central

shown in fi

riatic Sea in n channel ha

THIONEIN C

vironment

ocation (CE

al., 2012).

basically se

body conta

or locally p

n the bioav

.

l Adriatic

igure 3.

Croatia witharvesting are

CONTENTS

Education

tal stresso

EA) metho

edentary a

aminant bu

polluted w

vailability

Sea in C

h indicated sea and TE is

IN ARCA N

BLT - specia

ors in th

odology the

are suitable

urdens nor

water and

of chemic

Croatia, wh

sampling sits a sampling

NOAE 15

alization FBT

he mussel

e digestive

e indicator

rmally can

sediment.

cals in the

here three

tes. PC1 andg site in the

T

l

e

r

n

.

e

e

d e

16 BIOCH

1.3.1 The samp

m) located

impact. H

vessels in

daily visit

because o

(Favro &

indeed be

Nickel an

compared

µg/g dry

weight. T

other unp

1.3.2 The Pašm

in the cen

This area

Adriatic.

and orga

different

municipal

industrial

transport

Two samp

northwest

Sampling

while site

potential

waters) is

HEMICAL C

Telaščicpling site w

d on the is

However th

n the bay. D

ts and excu

of the fuel

Gržetić, 2

e used as a

nd chrom

d to the un

weight w

The levels o

polluted are

Pašmanman channe

ntral Adria

represents

However,

anisms livi

sources. P

l waste fro

l waste of

of chemica

pling sites

t–southeas

site PK 1

e PK 2 (at

influence

to be expe

COMPOSITIO

ca was selecte

sland Dugi

he nature p

During the

ursions. Th

from the

2008). So w

reference

mium conc

npolluted A

while the u

of copper,

eas (Mihel

n kanal/chel includes

atic, (Figure

s the main

this sea r

ing there

Potential c

om the tow

f the city

als) in the n

were sele

st directio

(at a dept

a depth o

of anthr

ected on bo

ON AND ME

d in the na

i otok, and

park is a p

e touristic

his leads to

boats and

we have to

site.

centrations

Adriatic se

unpolluted

lead and

čić et al., 2

hannel one of the

e 3). It will

n harvestin

region is u

can be ex

contaminat

wn Biograd

y of Zadar

northwest

cted in Pa

on between

h of 12-15

of 5-7 m)

ropogenic

oth PK 1 an

ETALLOTH

ature park

d represen

popular pla

season (Ju

o excessive

d of the tra

o investiga

s in sedi

ediments.

area give

zinc are l

2010).

e most imp

l further o

ng area for

under cons

xposed to

tion in the

d (hospital

r (interna

.

šman kana

n the coa

m) is loca

is located

impact (m

nd PK 2 sa

IONEIN CO

Telašćica

nted the sit

ace for tou

une, July, A

crowds an

ash that th

ate in this

iments of

For nickel

s values lo

ower at TE

portant nat

n be referr

r Noah’s A

siderable a

o contamin

e Pašman

and marin

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al (PK), wh

astline an

ated in the

close to t

municipal

ampling sit

ONTENTS IN

(TE; at a d

e of low a

urists. Ther

August) th

nd pollutio

he tourists

s study if

Telašćica

l there are

ower than

E than the

tural habit

red to as P

Ark shells i

anthropoge

nants orig

kanal is

na) in the s

rbor - pas

hich is spr

d the isla

proximity

he island

and indu

tes.

N ARCA NOA

depth of 10

anthropoge

re are a lo

here are ma

on of the ar

leave beh

Telašćica

a are hig

e values of

n 52 µg/g

e values fr

tats of A. n

Pašman kan

in the cen

enic press

ginating fr

the result

southeast a

ssengers a

reading in

and Pašm

y of the co

Pašman. T

ustrial wa

AE

0-14

enic

t of

any

rea,

hind

can

gher

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dry

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noae

nal.

tral

ure

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t of

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and

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man.

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aste

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kan

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wa

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OCHEMICAL

geschool Gen

here are no

nal.

4 Biomarkers

posure to

vironment

omarkers w

be very us

arning indi

ological re

oindicators

omarkers (

anges at th

gure 4. Concess/ pollutio

the first le

tegrity is a

netics cau

L COMPOSI

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data avail

iomarkerare bioch

or effects

tal scientis

were alread

seful as m

icators of s

sponses at

s. Changes

(Walker et

he level of c

eptual diagrn (graphic: M

evel expose

affected, e.

used by

ITION AND

s Vesalius

able of me

rs emical, ph

s of xenob

sts around

dy known

easures of

specific dis

t the level

s at individ

t al., 2001

cells and ti

ram of diffeMichael Ahr

ed to envir

.g sister ch

chemical

METALLOT

easured con

hysiologica

biotic chem

d 1981 (W

n in human

f human ex

seases or sy

of popula

dual level

1). The firs

issues (Fig

erent scales rens).

ronmental

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ls that c

THIONEIN C

ncentration

al, or his

micals. Bio

Walker et

n toxicolog

xposure to

yndromes

ation and

or below,

st level of

gure 4).

of ecosystem

stress, i.e.

exchange i

cause chr

CONTENTS

Education

ns of any p

tological i

omarkers

al., 2001)

gy, in whic

o specific c

(Hill, 1965

communit

i.e. cell an

f environm

m health ca

cell and ti

is a bioma

romosoma

IN ARCA N

BLT - specia

pollutants i

indicators

were first

). Before

ch they ha

chemicals o

5; Forbes et

ty are con

nd tissue,

mental stre

aused by env

issues, the

arker for c

al aberrat

NOAE 17

alization FBT

in Pašman

of either

t used by

that time

ve proven

or as early

t al., 2006).

sidered as

are called

ess causes

vironmental

structural

changes in

tions e.g.

T

n

r

y

e

n

y

s

d

s

l

l

n

.

18 BIOCH

dichlorop

organism,

the organ

heavy me

Nowaday

effects of e

In order

criteria sh

- If

ind

am

ace

pes

- Rel

bio

and

- Bio

am

- Ch

rea

inh

wh

- It s

as a

- Oth

It i

not

Knowing

conclusion

HEMICAL C

phenol (Wa

, it can lea

nism occur

etals (Walk

ys, biomark

environme

to evalua

hould be ch

a biomark

dicator, it

minolaevuli

etylcholine

sticides an

lative sen

omarker co

d to other c

ological sp

mong differ

hemical spe

act differen

hibit ALAD

hich is a pr

should be c

a reaction

her criteria

s importan

t only in th

the limita

ns (Hugge

COMPOSITIO

alker et al

ad to an in

r, e.g. avoi

ker et al., 20

kers are oft

ental pollu

ate candid

hecked.

ker respon

is more u

inic acid d

esterase ge

d a comple

sitivity is

ompared t

candidate

pecificity m

rent species

ecificity is

ntly to ch

D which ca

otective m

checked if

to exposur

a are appli

nt to know

he laborato

ations of a

ett, 1989).

ON AND ME

., 2001). Th

ncrease of d

idance beh

001).

ten used as

utants.

date bioma

ds to a va

useful in

ehydratase

ets inhibite

ex mixture

a criterio

o endpoin

biomarker

must be k

s (Huggett

s also diffe

hemicals (H

an be mor

mechanism

the bioma

re of pollut

icability to

w if the biom

ory.

a method

ETALLOTH

he second

diseases o

haviour ha

s monitors

arkers’ str

ariety of di

monitorin

e (ALAD)

ed by orga

e of polluta

on which

nts such as

rs (Hugget

kept in m

t, 1989).

erent for ev

Huggett, 1

rtal while l

(Walker et

arker is a tr

tants (Hug

field cond

marker is u

should be

IONEIN CO

level of t

r death. C

as been us

s for quanti

rengths an

ifferent ch

ng (Hugge

is only inh

anophosph

ants (Walk

looks at

s lethality

tt, 1989).

mind since

very bioma

1989), e.g.

lead also in

t al., 2001).

ransient or

ggett, 1989)

ditions and

useful for f

e consider

ONTENTS IN

the respon

hanges in

sed as a b

ifying expo

nd weakn

hemicals, th

ett, 1989).

hibited by

horous and

er et al., 20

the sensi

or growth

biomarke

arker, som

pollution

nduces me

r permanen

).

d validation

field measu

red before

N ARCA NOA

nse affects

behaviour

biomarker

osure and

nesses seve

hus a gene

The enzy

lead wher

d carbam

001).

itivity of

h impairm

ers can dif

me biomark

of lead w

etallothion

nt occurre

n in the fie

urements a

e making a

AE

the

r of

for

the

eral

eral

yme

reas

mate

the

ment

ffer

kers

will

nein

nce

eld.

and

any

BIO

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If b

not

the

cap

situ

any

inc

occ

sea

che

In

bio

tetr

Th

one

cha

Bef

est

OCHEMICAL

geschool Gen

biomarker

tice small

e health of

pacity to w

uation wit

y conditio

crease its e

cur in the e

asonal fluc

emical poll

case of ch

ochemical

rachlorodi

his biochem

e of the wh

anges are v

fore you c

tablished:

- The as

- Baselin

order

induce

- The ba

of unc

- All the

- It shou

to phy

- And

health

L COMPOSI

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rs are mea

changes a

the organi

withstand

hout havin

on or situa

energy exp

environme

ctuation in

lution) (Hu

hemical po

receptor

ibenzodiox

mical respo

hole organ

visible in th

can use a

ssay to qua

ne data fo

to be able

ed stress.

asic physio

controlled v

e factors th

uld be esta

ysiological

the increa

h of the org

ITION AND

s Vesalius

asured in a

and therefo

ism. The h

stress. If

ng negativ

ation that

penditure,

ent can be

n the seawa

uggett, 198

ollution th

in the o

xin on the

onse is exp

nism. So yo

he organis

protein as

antify the b

or the conc

to disting

ology of th

variation c

hat affect th

ablished wh

acclimatio

ased levels

ganism (Hu

METALLOT

a dose- or

ore biomar

health of an

an organ

ve effects, t

t causes a

e.g.. a dr

natural (e.

ater and a

89).

here is an

organism

e aryl hydr

pected to b

ou can dete

sm (Hugge

s a biomar

biomarker

ncentration

guish betwe

he test org

can be min

he biomark

hether cha

on or to gen

s of the bi

uggett, 198

THIONEIN C

time-depe

rkers can

n organism

nism cann

the organis

system t

op in wat

.g. salinity

air tempera

interaction

(Huggett,

rocarbon r

be the mo

ect pollutio

ett, 1989).

rker the fo

should be

of the bio

een natura

ganism sho

nimized.

ker should

anges in bio

netic adap

iomarker s

89).

CONTENTS

Education

endent ma

allow for

m can be de

ot endure

sm is not h

o mobilize

er tempera

fluctuatio

ature) and

n between

1989), e

receptor (W

ost immedi

on with bio

ollowing p

sensitive,

omarker s

al variabilit

ould be kn

d be known

omarker co

tion.

should be

IN ARCA N

BLT - specia

anner, it is

rapid asse

efined as th

e a limited

healthy. A

ze its reso

ature. Stre

on inside th

d anthropo

n the toxic

.g. the b

Walker et

iate, and f

omarkers b

properties

reliable an

should be

ty and con

nown so th

n.

oncentratio

correlated

NOAE 19

alization FBT

s easier to

essment of

he residual

d stressful

stressor is

urces and

essors that

he estuary,

genic (e.g.

cant and a

inding of

al., 2001).

faster than

before any

should be

nd easy.

known in

ntaminant-

hat sources

on are due

d with the

T

o

f

l

l

s

d

t

,

.

a

f

.

n

y

e

n

-

s

e

e

20 BIOCH

1.4.1 Metalloth

cysteine-r

serve as li

variety of

bismuth, a

essential

contain ca

MTs from

three met

and inver

MTs are

metabolis

donation

is consiste

nucleic a

growing,

stressors s

increase o

until now

Figure 5. Mbeads are m

HEMICAL C

Metallohioneins (M

rich, metal

igands for

f metal io

and silver.

metals zin

admium, c

m marine c

tals. There

rtebrates co

thought to

sm, such a

of metals

ent with t

acid metab

injured or

such as co

of MT synt

w only know

Metallothionemetal atoms (

COMPOSITIO

othioneinMT) consti

l binding

metal che

ons, such

. Native M

nc and co

opper and

crustacean

is a differ

ompared to

o be invol

as metal

to metallo

he hypoth

bolism, pr

r regenera

old, heat, e

thesis seem

wn for mam

ein (MT) stru(e.g., Zn), an

ON AND ME

n itute a fam

proteins a

lation. The

as cadmiu

MTs often co

pper. Afte

zinc (Hug

ns consist o

rence betw

o the typic

lved in in

detoxificat

proteins. E

hesis that M

rotein syn

ting tissue

exercise, an

ms to be me

mmals, ins

ucture. Modnd small yell

ETALLOTH

mily of lo

and oligop

e synthesis

um, coppe

ontain mix

er exposur

ggett, 1989)

of two 9-

ween MTs f

al mamma

nterrelated

tion, regu

Evidence p

MTs supp

nthesis, an

es. MT als

nd injection

ediated by

sects and c

el of two binlow beads ar

IONEIN CO

ow molecu

peptides.

s of MT can

er, zinc, m

xed-metal c

re to cadm

).

cysteine d

from non-m

alian MT.

processes

ulation of

presented b

ly zinc, an

nd other m

o plays a

n of chem

y glucocort

rustaceans

nding sites ore sulfur atom

ONTENTS IN

ular weigh

The cyste

n be induc

mercury, c

clusters con

mium the

domains th

mammalia

associated

zinc and

by several

nd possibly

metabolic

role in the

icals. The

ticoid horm

s (Huggett,

of metallothims (Branisla

N ARCA NOA

ht (6-7 kD

einyl resid

ced by a w

cobalt, nick

ntaining b

protein m

hat each b

an vertebra

d with me

copper, a

investigat

ly copper,

processes

e response

stress rela

mones. Thi

, 1989).

ionein. Red av et al., 2013

AE

Da),

ues

wide

kel,

both

may

ind

ates

etal

and

tors

for

in

e to

ated

is is

big 3).

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Ho

MT

im

Th

ind

exp

on

cad

bio

In

dif

me

Th

me

Ac

me

we

me

spe

tak

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dig

tha

OCHEMICAL

geschool Gen

T has also

mportant in

hey discov

ducible MT

pression m

the metal

dmium re

odegradati

table I bas

fferent spec

ethod of de

he method

ethod whic

ccording to

ethods for

ere at least

easuremen

ecific reag

ken if the re

s can be inf

gestive gla

an in the w

L COMPOSI

nt – Campus

been sho

n normal c

vered in b

Ts are dif

may be met

bound. W

emains bo

on of MT.

al metallot

cies and di

eterminatio

used for

ch is differ

o the stud

quantifica

t one orde

nts depend

gents and

esults of d

ferred from

and than in

whole organ

ITION AND

s Vesalius

wn to be

cellular me

blue crabs

fferent pro

tal specific

When MT b

ound to

thionein co

ifferent tiss

on.

metallothi

rent than t

dy of Ivank

ation of m

r of magn

ding on th

reference

ifferent stu

m Table I,

n the whol

nism exam

METALLOT

an effectiv

etabolism

(Callinecte

oteins. Suc

. The rates

biodegrade

MT in t

oncentratio

sues with d

ionein qua

the one use

ković et a

metallothion

nitude lowe

he particu

standards

udies are c

the values

le organism

mple.g. in M

THIONEIN C

ve free-rad

and in m

es sapidus)

ch observ

s of biodeg

es copper a

he steady

ons are exp

differentia

antification

ed in this s

al. (2003) w

neins spec

er in comp

ular samp

applied.

ompared.

s of MT co

m, while th

M. edulis.

CONTENTS

Education

dical scave

odifying r

) that cad

ations sug

gradation o

and zinc w

y state of

pressed in

al pulse pol

n in Table

study, i.e.

who comp

trophotom

parison wi

le treatme

Therefore,

oncentratio

he values i

IN ARCA N

BLT - specia

enger, whi

radiation s

dmium- an

ggest that

of MT are d

will be lost.

f biosynth

d.w. or w.

larography

I is a vol

spectroph

pared two

metric mea

ith the vol

ent as we

, caution

ons are hig

in the gills

NOAE 21

alization FBT

ich can be

sensitivity.

nd copper

MT gene

dependent

. However

hesis and

w. for

y as the

ltammetric

hotometric.

SH-based

surements

ltammetric

ell as two

should be

gher in the

are lower

T

e

.

r

e

t

r

d

c

.

d

s

c

o

e

e

r

22 BIOCH

Table I. Babivalves fro

In the stu

gland for

from 520–

and autum

these resu

interspeci

HEMICAL C

sal metallotom sites con

udy perform

r M. gallop

–870, 360–7

mn (0.52-0

ults could

ies differen

COMPOSITIO

thionein connsidered to b

med by (Iv

provincialis

760, 225–6

0.87, 0.36-0

be comp

nces would

ON AND ME

ncentrations e uncontami

vanković e

determin

15 and 180

0.76, 0.225-

ared to th

d be expect

ETALLOTH

determinedinated (Ami

et al., 2005

ned by spe

0–860 µg/

-0.615 and

he data ob

ted - as lon

IONEIN CO

d by Differeniard et al., 20

5) MT conc

ectrophoto

g d.w. in w

0.18-0.86

btained in

ng as you c

ONTENTS IN

ntial Pulse P006).

centrations

ometric me

winter, spr

mg/g d.w

this thesi

convert w.w

N ARCA NOA

Polarograph

s of digest

ethod rang

ring, summ

w.). Therefo

is – althou

w. to d.w..

AE

y in

tive

ged

mer

ore,

ugh

BIO

Ho

1.4Ch

cau

bec

ma

Wh

req

ene

str

is n

(H

Gly

tox

fro

gla

ene

the

For

sto

198

wh

are

al.,

Lip

inv

sou

sea

of l

dar

by

as

OCHEMICAL

geschool Gen

4.2 Prhanges in

used by ph

cause of l

anifested u

hen the e

quirements

ergy reserv

ess or repr

not the ma

uggett, 198

ycogen is

xicant-indu

om the gon

abrata). Gen

ergy dema

erefore obs

r vertebrat

orage organ

89). Glyco

hen lipids a

e released.

, 2014).

pids are

vertebrates

urce. Altho

asons, spec

lipids is va

rk muscle

toxicant-in

well follow

L COMPOSI

nt – Campus

roteins, cthe concen

hysical and

long-term,

under acute

energy int

s of the or

ves are mo

roduction.

ain role be

89).

a storage

uced stress

nadal regio

nerally, th

and. Glyco

served alte

tes the pri

ns are the

gen reserv

are not ava

This proc

the most

s, fish and

ough it can

cies or stad

ariable in a

than in th

nduced str

wed by an

ITION AND

s Vesalius

carbohyntration o

d chemical

, sublethal

e exposure

take excee

ganism th

obilized w

Proteins c

ecause thei

form of g

s. Ansaldo

on can be u

ere will be

ogen storag

erations in

mary stora

hepatopan

ves are gen

ailable, sin

ess is follo

importan

birds. For

n tend tow

dia in life c

an organism

he white m

ress but a

increase in

METALLOT

ydrates aof stored e

stressors.

l exposure

e condition

eds the m

he energy w

when there

can also be

ir role is t

glucose. D

o et al. (20

used as a g

e a depleti

ge and mo

n glycogen

age locatio

ncreas (dig

nerally us

nce the rese

owed by th

nt, readily

r most of th

wards a se

cycle or re

m. For inst

muscle of a

decrease i

n specific c

THIONEIN C

nd lipidsenergy res

Changes in

e to a str

ns (Hugget

maintenanc

will be sto

is an incre

e used as an

that of a s

Depletion o

06) proved

general bio

ion of glyc

obilization

concentra

on is the li

gestive gla

sed during

erves of lip

he gametog

y available

he species

econdary e

productive

tance there

fish. A de

in total lip

component

CONTENTS

Education

s erves in in

n stored en

ressor and

t, 1989).

ce, growth

red as gly

eased ener

n energy s

structural u

of glycoge

d in their

marker for

cogen beca

is restricte

ations are o

iver. For m

and) and th

g the game

pids deplet

genetic dev

e energy

lipids are

energy sou

e cycle. Th

e are more

ecrease in l

id concent

ts of the lip

IN ARCA N

BLT - specia

invertebrat

nergy rese

d are nor

h and rep

ycogen or l

rgy deman

source, alth

unit of an

en can occ

study that

r snails (Bi

ause of an

ed to certa

often tissu

mussels th

he mantle

etogenetic

tes when t

evelopmen

source fo

e the prima

urce among

he tissue di

lipids pre

lipids can

trations m

pid constit

NOAE 23

alization FBT

tes can be

rves occur

mally not

productive

lipids. The

nd such as

hough that

n organism

cur due to

t glycogen

iomphalaria

increased

ain tissues,

ue specific.

he primary

(Huggett,

processes

he gonads

t (Radić et

or aquatic

ary energy

g different

istribution

sent in the

be caused

might occur

tuents, e.g.

T

e

r

t

e

e

s

t

m

o

n

a

d

,

.

y

,

s

s

t

c

y

t

n

e

d

r

.

24 BIOCH

an increas

term stres

occur dur

humans to

Structural

an energy

mobilize p

but depen

circumsta

used as an

the last so

(Huggett,

nutritiona

1.5 In genera

and seaso

such as th

decussate (

and chron

an increa

1989). Sim

Ruditapes

noae is ne

hydrate v

by Radić e

coinciding

reserves i

demand (

HEMICAL C

se of trigly

ss, althoug

ring acute

o detect pr

l proteins a

y source. H

proteins as

nding on

ances, prot

n individu

ource of en

1989). To

al stress, ho

Biotic aal the glyco

on (Hugge

he mussel M

(Radić et a

nic exposu

ased energ

milar result

decussatus

egatively a

value was d

et al. (2014

g with high

is strongly

Radić et al

COMPOSITIO

ycerids. Li

gh it is po

e stress (H

rostate can

are abunda

However

s an energ

the seaso

teins can b

ual biomark

nergy used

otal protei

owever for

and abiotogen conte

ett, 1989).

M. gallopro

al., 2014). T

ure to meta

gy demand

ts have bee

(Hamza-C

affected by

detected in

4), a second

h phytopla

y influence

l., 2014).

ON AND ME

ipids are m

ossible that

Huggett, 1

ncer (Zhou

ant in an o

under con

y source. N

n, reprodu

be a quite

ker, becau

d and there

in concent

r chemical

tic factorent can be

This is de

ovincialis an

The glycoge

als. A decr

d combine

en found f

Chaffai et a

y seawater

n December

d carbohyd

ankton abu

ed by food

ETALLOTH

most usefu

t changes

1989). Lipi

et al., 2012

organism. T

nditions of

Normally l

uctive stat

important

use of the fa

efore will n

tration in

pollutants

rs that a influenced

emonstrate

nd the clam

en content

rease in gly

ed with ch

for snails (A

al., 2003).

r temperat

r (Radić et

drate peak

undance. T

d availabi

IONEIN CO

ul as suble

in specific

ids are us

2).

These prote

f severe st

lipids and

tus and th

t energy s

act that ge

not be a ve

the plasm

s it is not se

ffect biod by diet,

ed in vario

ms Venerup

t of fish tis

ycogen us

hemical-in

Ansaldo et

Carbohydr

ture, there

t al., 2014).

k in A. noae

The season

ility, in ad

ONTENTS IN

thal indica

c lipid com

sed as a b

eins are no

tress the o

glycogen

he length

ource. Pro

enerally pr

ery sensitiv

ma of a fis

ensitive (H

marker vreproduct

ous specie

pis philippi

ssue is affe

ually occu

duced stre

t al., 2006)

rate concen

fore the h

In the stud

e was obser

al variation

ddition to

N ARCA NOA

ators of lo

mponents

biomarker

ot intended

organism

are used f

of the str

oteins are

roteins will

ive biomar

sh can det

Heath, 1995

variabilittive condit

es of bival

inarum and

ected by ac

urs because

ess (Hugg

and the sh

ntration in

highest car

dy perform

rved in Ap

n in glycog

reproduct

AE

ng-

can

r in

d as

can

first

ress

not

l be

rker

tect

5).

ty tion

lves

d V.

cute

e of

gett,

hell

n A.

rbo-

med

pril,

gen

tive

BIO

Ho

In

rea

in t

Pro

pri

aft

biv

and

Lip

gro

gen

dec

car

gly

201

Th

tem

in

sig

rec

gon

Au

For

mu

sea

(Iv

For

gro

me

exp

wit

OCHEMICAL

geschool Gen

a study p

ached the m

the concen

otein is a m

ior to spaw

er spawni

valves. In m

d constitut

pid conten

owth, repr

neral, lipid

creases. In

rbohydrate

ycogen to

14).

here is no

mperature

mean go

gnificant c

corded in t

nad index

ugust) (Peh

r the mus

ussel repro

ason of sa

vanković et

r oysters i

owth and t

easuremen

posure to

thout the i

L COMPOSI

nt – Campus

performed

maximum

ntration of

major organ

wning (Jun

ing (from

marine biv

te a major c

nt of variou

roductive

d content i

n A. noae, li

e content.

lipids bios

o proof o

for Arca na

nad index

orrelation

the period

x during t

harda et al.

ssel M. ga

oductive st

ampling, c

t al., 2005).

it is the rep

the molt c

nt of MT

metals (H

influence o

ITION AND

s Vesalius

by Radić

value in J

proteins an

nic compon

e) may sup

July to

valves, lipi

componen

us species

condition

increases b

ipid variati

This relati

synthesize

of a signi

aoe in Mali

x is follow

between

from July

the period

., 2006).

lloprovincia

tate, age an

contribute

productive

ycle that c

in organi

Huggett, 19

of the repro

METALLOT

et al. (20

June, just b

nd lipids.

nent of biv

pport this h

September

ids play a

nt of reprod

of bivalve

n, salinity

before mas

ions from

ionship is

ed during

ificant cor

i Ston Bay

wed by a

these two

to Decemb

d when tem

alis differe

nd sex, sea

to variat

e cycle/sea

can affect M

isms does

989). For

oductive cy

THIONEIN C

14), protei

before spaw

valve oocyt

hypothesis

r) was sim

crucial rol

ductive ma

es (Radić e

and tem

ss spawnin

February t

usually a

the forma

rrelation b

. However

an increase

o variable

ber when s

mperature

ent biotic

a temperatu

tions in th

ason of th

MT concen

not nece

detecting

ycle on the

CONTENTS

Education

in and lip

wning, foll

tes. Theref

s. The decr

milar to m

le in matur

aterial (Rad

et al., 2014)

perature

ng occurs,

to June are

ttributed t

ation of ga

between g

r, most of t

e in temp

s is proba

spawning c

e was still

and abiot

ure and sa

he MT ce

e year and

ntration. It

essarily re

exposure

e level of M

IN ARCA N

BLT - specia

pid levels

lowed by

fore, protei

rease in pr

many othe

ring gonad

dić et al., 2

4) is affecte

(Huggett,

and then

e inversely

to the con

ametes (Ra

gonadal in

the times a

perature. L

ably due

caused a d

increasin

tic factors

alinity, as w

ellular con

d for blue

t is thus ev

eflect the

in the env

MT, a meth

NOAE 25

alization FBT

in A. noae

a decrease

in maxima

otein level

er marine

dal tissues

2014).

ed by diet,

1989). In

markedly

y related to

version of

adić et al.,

ndex and

an increase

Lack of a

to values

decrease in

ng (July to

including

well as the

ncentration

crabs it is

vident that

degree of

vironment

hod is used

T

e

e

a

l

e

s

,

n

y

o

f

,

d

e

a

s

n

o

g

e

n

s

t

f

t

d

26 BIOCH

where the

The conce

between

(Pellerin &

Therefore

differentia

example

example

caused by

of biomar

induced b

1.6 This thesi

much larg

determina

seawater

metals in

was not th

The aim o

and carbo

differentia

environm

to provid

on the ob

bioindicat

environm

the levels

Peharda U

the gonad

HEMICAL C

ey transpo

entrations

the two s

& Amiard,

e, when u

ate betwee

gonadal

temperatu

y the anthr

rker respo

by anthrop

Aim of is focused

ger resear

ation of m

and the s

the soft ti

he main aim

of this stud

ohydrates i

ate betwe

mental pres

e informat

btained res

tor species

mental stres

s of metall

Uljević and

ds.

COMPOSITIO

ort species

are meas

sites to co

2009)

sing biom

en the phy

developm

ure and sa

opogenic s

onse may i

pogenic sou

this stud on analys

rch that w

metal conce

sediments.

issue. Accu

m of that r

dy was to

in the tissu

een the

sures. The

tion about

ults of this

s and dige

ss that coul

lothionein

d Dr. Sc. Sa

ON AND ME

from an u

ured at bo

onclude if

markers in

ysiological

ent and

alinity) an

stressor stu

interfere w

urces.

dy ses of the

was done o

entrations

. The unp

umulation

research.

determine

ue of diges

sampling

e levels of

t potential

s study we

estive glan

ld be cause

are measu

anja Puljas

ETALLOTH

unpolluted

oth sites.

there is a

environm

l changes

food abun

nd the co

udied. In o

with the es

digestive g

on this spe

on the wh

published

was only

e the conce

stive gland

sites an

metallothi

metal exp

e will eval

nd as the t

ed by meta

ured for th

s were in ch

IONEIN CO

area to th

Then they

any expos

mental stud

caused by

ndance),

ntribution

other word

stimation o

gland of A

ecies. The

hole soft ti

results sh

measured

entrations

d of Noah’

nd their

ionein will

posure in t

luate the p

target orga

al pollution

he species

harge of st

ONTENTS IN

he area of i

y compare

ure in sit

dies it is

y biotic in

abiotic inf

to bioma

s, the natu

of the leve

A. noae as

other par

issue, as w

howed accu

d in March

of total pro

s Ark shel

respective

l be determ

the environ

potential o

an in the a

n. It is the f

A. noae. D

tudying the

N ARCA NOA

investigati

e the chan

te of inter

important

nfluences (

nfluences (

arker chan

ural variatio

el of chan

one part o

rt focused

well as in

umulation

h 2013 sinc

roteins, lipi

lls in order

ely differ

mined aim

nment. Ba

of A. noae a

assessment

first time t

Dr. Sc. Me

e histology

AE

ion.

nges

rest.

t to

(for

(for

nge

ons

nges

of a

on

the

n of

ce it

ids,

r to

rent

ming

sed

as a

t of

that

elita

y of

BIO

Ho

2 Th

Ma

tra

the

usi

sam

con

Ou

site

so

nec

pro

to

bio

sam

80°

2.1

2.1In

Tab

Pro

β-

Leu

PM

Tri

Tri

Ab

OCHEMICAL

geschool Gen

Mhe Noah’s

arch 2013 u

ansported i

ey were im

ing stainle

mples wer

ntained dig

ur samples

e there are

for one s

cessary wh

oteins and

be homo

ochemical c

mples for a

°C for later

1 Ho

1.1 MTable II th

ble II. Produ

oduct/soluti

mercaptoe

upeptine

MSF (pheny

is- HCl

is- base

bsolute eth

L COMPOSI

nt – Campus

aterials Ark shell

until Febru

in dark an

mmediately

ess steel e

re collected

gestive gla

consist of

e four diffe

ampling s

hen measu

the level o

ogenized.

compositio

analysis of

r use.

omogen

Materials he products

cts used for h

ion

ethanol

ylmethylsu

anol

ITION AND

s Vesalius

and metls from na

uary 2014 b

nd cool co

y washed w

equipment

d for each

ands dissec

tissue of t

erent pools

site there a

uring bioch

of metallot

There is

on and MT

f biochemi

ization

s needed fo

homogenizat

ulfonyl fluo

METALLOT

hods atural pop

by SCUBA

ontainers t

with seawa

t to extrac

h sampling

cted from 9

the digestiv

s; A, B, C

are 36 ind

hemical com

thionein (M

a differen

T measurem

ical compo

or homoge

tion.

B

S

S

oride) S

S

S

K

THIONEIN C

pulations w

A diving d

o the labo

ater and di

ct the dige

g site and

9 individua

ve gland o

and D. On

dividuals. P

mpositions

MT). As a f

nce in the

ments. Afte

osition are

enization o

Brand/firm

Sigma

Sigma

Sigma

Sigma

Sigma

Kemika

CONTENTS

Education

were sam

one by Dr

oratory wit

issected by

estive glan

time. Each

als.

of Arca noae

ne pool con

Preparatio

s such as li

first step, a

e homoge

er the hom

stored in

of the samp

IN ARCA N

BLT - specia

mpled mon

r.Sc. Ivan Ž

thin 6 hou

y Dr.Sc. Jel

nd. Four

h composi

e. For each

ntains 9 in

on of the s

ipids, carbo

all the sam

enization b

mogenizatio

several al

ples are list

NOAE 27

alization FBT

nthly from

Župan and

urs, where

lena Čulin

composite

ite sample

h sampling

ndividuals,

samples is

ohydrates,

mples need

buffer for

on process

iquots at -

ted.

T

m

d

e

n

e

e

g

,

s

,

d

r

s

-

28 BIOCH

PreparatBiochemic

100

MT homo

20

+ P

hom

+ l

3µl

+ β

add

2.1.2 ‐ Tra

‐ Sw

‐ Tur

‐ Lab

ice

‐ We

‐ Tak

‐ We

‐ We

sto

Procedure

We use t

Dondero,

Add 3 m

homogeni

HEMICAL C

tion of bucal compos

0 mM Tris-

ogenization

mM Tris-H

PMSF diss

mogenizat

leupeptin

l/ml homo

β- mercap

dition 1 µl/

Protocoansfer the t

witch on the

rn on the c

bel the sam

.

eigh the tis

ke 1 g of th

eigh about

eigh the re

ore them at

e

the metho

& Fabbri,

ml homog

ization tub

COMPOSITIO

uffers sition hom

-HCl pH =

n buffer (ad

HCl pH = 8

solved in

tion buffer

dissolved

ogenization

ptoethanol

/ml homo

ol tissues of t

e ice maker

centrifuge

mple tubes

ssue and cu

he tissue fo

0.4 g for b

emaining t

t -20 °C. Th

for

od accord

1997)

enization

be.

ON AND ME

mogenizatio

8.6 (at 5 °C

dd fresh ad

8.6 (at 5 °C

ethanol (s

.

in 20 mM

n buffer.

dissolved

genization

the digestiv

r.

to cool the

and put th

ut with scis

or MT mea

biochemica

issue and

hese will be

samples

ing to Vi

buffer pe

ETALLOTH

on buffer:

C)

dditives ev

C)

stock solut

M Tris-HCl

d in MQ-

n buffer.

ve gland fr

e rotor (4-9

he tubes an

ssors until

asurement.

al composit

put in a cr

e used for

s

iarengo et

er 1 g of

IONEIN CO

very day):

tion 58 mg

(stock sol

H2O(stock

rom -80 °C

°C).

nd the hom

you get a

tion measu

ryogenic v

lyophiliza

to

t al (1997)

tissue an

ONTENTS IN

g/ml) - ad

lution 1 m

k solution

C to -20 °C.

mogenizati

pulp.

urement.

vial of 1.5 o

tion .

measure

). (Viareng

nd transfe

N ARCA NOA

dd 1.5 µl/

mg/ml) – a

n 100 µl/m

ion buffers

or 1.8 ml a

M

go, Ponza

er to a gl

AE

/ml

add

ml)-

s on

and

MT:

ano,

lass

BIO

Ho

Pro

OCHEMICAL

geschool Gen

‐ Homo

‐ Rinse

sampl

‐ Transf

centrif

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balanc

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pipetti

fat, cen

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Make

defros

(SN) f

3 subs

furthe

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‐ Add 1

homog

ml buf

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PTFE

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sampl

L COMPOSI

nt – Campus

ogenize on

the pestl

les.

fer the hom

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ifuge for 12

ate the su

ing fat (on

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x the supe

aliquots fo

sted and f

or determi

samples of

er be referr

the aliquot

or samples

10 volume

genization

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ogenize the

pestle.

the pestl

les.

ITION AND

s Vesalius

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le and ot

mogenates

er with pa

ing the tu

trifuge rot

wed.

20 min at 5

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or an additi

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frozen. Ad

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30 µl for c

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to measur

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n tube, here

e samples

le and ot

METALLOT

a motor-dr

ther equip

into the ce

arafilm.

ubes in the

tor. A diffe

50000 ×g (t

t using a

pellet (at t

ional 15 m

nd transfer

lyses neede

dditionally

cytosolic p

cytosolic pr

50.

les at -80 °C

re biochem

ogenization

e the ratio

s on ice us

ther equip

THIONEIN C

riven homo

pment use

entrifugati

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the bottom

min.

r it to labe

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proteins. W

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C.

mical comp

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for digest

sing a mo

pment use

CONTENTS

Education

ogenizer w

ed with D

ion tubes a

heck their

s than 0.5

ivalent to 1

pipette tak

m); if there

lled Eppen

es should

several p

We collected

hese supern

position:

er volume

tive gland

otor-driven

ed with D

IN ARCA N

BLT - specia

with PTFE p

De-H2O in

and keep o

weight in

g between

1 h at 10000

king care

is a large

ndorf tube

never be r

parts of su

d 2x250 µl f

rnatant sam

e of tissue

is 0.4 g tis

n homogen

De-H2O in

NOAE 29

alization FBT

pestle.

n between

on ice until

n order to

n opposite

00 ×g).

to avoid

fraction of

es (on ice);

repeatedly

upernatant

for MT and

mples will

to a glass

ssue and 4

nizer with

n between

T

n

l

o

e

d

f

;

y

t

d

l

s

4

h

n

30 BIOCH

‐ Tra

cov

sam

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tub

for

det

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‐ Sto

2.2

2.2.1 The matecytosolic pTable III. P

Product/so

Chloroph

Ethanol, 9

NaCl

EDTA

HCl

DTNB acid)

GSH (glut

KH2PO4

NaOH

BSA (bovi

H3PO4

G- Cooma

HEMICAL C

ansfer hom

ver with p

mples.

rtex befor

bes, use the

r determina

termination

ansfer the r

ore the aliq

Quantif

Materiaerials that proteins arroducts used

olution

horm

95%

(5,5-dithi

tathion)

ine serum

assie brillia

COMPOSITIO

mogenates

arafilm an

re you ma

e blue pipet

ation of lip