Analysis of aquatic humic material and high molecular weight components of bleached kraft mill effluent (BKME) by gradient gel electrophoresis

Chemosphere,Vol.24, No.12, pp 1745-1753, 1992 Printed in Great Britain

ANALYSIS

OF

COMPONENTS

AQUATIC

0045-6535/92 $5.00 + 0.00 Pergamon Press Ltd.

HUMIC

OF B L E A C H E D

MATERIAL

AND

HIGH MOLECULAR

KRAFT MILL EFFLUENT

WEIGHT

(BKIKE) BY G R A D I E N T

GEL E L E C T R O P H O H E S I S

R.M. Baxter* & John Malyszl National Water R e s e a r c h Institute P.O. Box 5050, Burlington, Ontario, Canada L7R 4A6 (1McMaster U n i v e r s i t y Co-op student, 1991)

ABSTRACT

Three preparations of humic material (a commercial humic acid and material isolated from soil and from water) were a n a l y s e d by electrophoresis on p o l y a c r y l a m i d e gradient gel slabs. All gave similar p a t t e r n s showing four bands of material of m o l e c u l a r weights a p p a r e n t l y r a n g i n g from a few h u n d r e d to about 20,000 as e s t i m a t e d by c o m p a r i n g their mobilities with those of p r o t e i n markers. The high molecular weight material from b l e a c h e d kraft mill effluent (BKME) showed similar p a t t e r n s w i t h the a d d i t i o n of completely unresolved material of m o l e c u l a r weight up to about I00,000. E l e c t r o p h o r e s i s on p o l y a c r y l a m i d e slabs may prove v a l u a b l e for the study of humic substances and other i l l - d e f i n e d p o l y m e r i c materials.

INTRODUCTION

Humic material the

is the

decomposition

responsible

is soluble

solution

is

to consist

in a l k a l i n e

acidified;

after acidification; conditions.

The

In

the

organic

material p r o d u c e d by substances,

and

manufacture fibres

with an alkaline

of three

components:

fulvic

acid, w h i c h remains

humin,

which

is

of soil

of

wood

are r e l e a s e d solution

pulp

by

are

material

subsequently

m o l e c u l a r weight

when the in s o l u t i o n

insoluble

under all and

(Malcolm 1990).

the

kraft

process the

from wood by d e g r a d i n g the lignin

of sodium

sulphide.

The d a r k - c o l o u r e d

liquid c o n t a i n i n g most of the d e g r a d a t i o n p r o d u c t s

some

is

It is

is m o s t l y humic acid,

is largely fulvic acid

is then s e p a r a t e d from the w o o d fibres, However

which

humic acid,

s o l u t i o n but p r e c i p i t a t e s

humlc material

that of natural waters

cellulose

natural

for the colour of soil and some natural waters.

usually considered which

d a r k - c o l o u r e d acidic of

(black liquor)

concentrated

and burned.

remains w i t h the w o o d fibres,

and if these

bleached

with

chlorine

c h l o r i n a t e d material 1745

dark-coloured

high

is p r o d u c e d and d i s c h a r g e d

1746

into the effluent may

survive

stream

subsequent

into the environment, appears kraft

to

1991;

effluents

Wolfe

&

humic

Jones,

field.

This

of the

biological 1930

is

the

century,

use

have been supporting

the

When

medium

of

for

as

sample

appear

as

more

electrophoresis),

or

bands.

may be b a s e d

Separation

components

as a

(isoelectric

These p r o c e d u r e s and nucleic

have proved

acids

of soil humic material by

Stepanov

and

Curvetto

et al.

Kasparov

et

(1981), al.

Ceccanti

(1990)

material

and

has

components. (Kl~cking

et al. others

usually The

1973)

or

focusing

et al

1975;

1974, 1989;

reproducible molecules not have

but

many more

their

are thought

have

increased

Lu, et al

to bear points

been

components

made

Ceccanti

is

only n e g a t i v e 1976;

apply

(1973),

(1978,

1979), et al.

De Nobili

these

studies

the six

presence al

of SDS

1978).

et

patterns unclear

et

to about

are o b t a i n e d

The

to

al.

the

1983;

(Thornton

of proteins

De Gonzalez

In

1990).

significance

weights.

first d e s c r i b e d

from two

et

of

of the

electrophoresis

(1988),

in

of

(disk

as a series

study

et

into

The

zones

points

Disc

(1983),

(Castagnola

Zhang &

isoelectric

Few attempts

is

appear

gel was

1989).

proved methods

later by K l o c k i n g

Nobili

resolved

urea

De Nobili

De

(Duxbury

been

number

isoelectric

Nobili

(1986),

Lu

for

the components

for the

and

into

in a solid

defined

1991).

Castagnola

Zhang &

studies

or on their m o l e c u l a r

(1969)

1975),

(1981),

came useful

however

isoelectric

in p o l y a c r y l a m i d e

(1974,

al.

they

the

of

about

polyacrylamide.

sharply

& Bocek

Pakhanov

Tiselius

prove

so that

invaluable

(Kleparnik

the middle

first

also

or

slab where

focusing)

by

in

electric

separation

out e l e c t r o p h o r e s i s

less

on

to resemble

since the

recently

in a tube

or

least for

but early

More

agarose

m e d i u m may be cast

it

et al

particles

technique

humus,

carrying

such

at

would

1982).

seems

of an applied

recognized

this it

components

(Stevenson

developed

supporting

first

(Pillai

charged

studied

1989).

the

disappointing

of

influence

usefulness

it was h o p e d that

separating

material

has been

was

because

of b l e a c h e d

1988).

but

its

for concern

organisms

This et al.

the

of this material and be d i s c h a r g e d

of the t o x i c i t y

migration

macromolecules

(Vesterberg

cause

to certain

under

phenomenon

last

general

it gives

1991).

Some

treatment

for much

(Virkki

or s u s p e n s i o n

plant.

water

(BKME)

material

Electrophoresis solution

where

be r e s p o n s i b l e

mill

natural

from the waste

al

With

(Curvetto 1986;

obtained because

De are

humic

charges

and so should

Duxbury

1989).

modern

electrophoretic

1747

methods to Gjerdahl humic

aquatic humic

1973)

carried

materials

reproducible

in

materials. out

aqueous

solution

(1975; G j e s s i n g &

focusing and

of aquatic

obtained

several

fractions.

In this paper we d e s c r i b e attempts material

Gjessing

isoelectrical

and

to fractionate

aquatic humlc

h i g h m o l e c u l a r weight components of BKNE on precaat

polyacryiamide

gradient gel slabs d e s i g n e d

for

the s e p a r a t i o n of

p r o t e i n s a c c o r d i n g to their m o l e c u l a r weights. H A T E R I A L S AND HETHODS Lake water

was o b t a i n e d

north of Montreal carbon

(Baxter

from Lac Cloche,

containing

& Carey

about

1962).

10

a small s o f t w a t e r mglL

lake

d i s s o l v e d organic

75L of water was c o n c e n t r a t e d to

20L in a n a n o f i l t r a t i o n apparatus u s i n g a m e m b r a n e w i t h a nominal molecular

weight

cut-off

further c o n c e n t r a t e d

by

yield

of

400

Daltons.

ultrafiltration

apparatus

to

300

ml

of

dark

components

of MW > i000. This was stored

The

in

a

filtrate

was

tangential

flow

concentrate at 4C

containing

and c o n c e n t r a t e d

tenfold by e v a p o r a t i o n under r e d u c e d p r e s s u r e at room t e m p e r a t u r e in a r o t a r y e v a p o r a t o r before use.

A soil extract was p r e p a r e d by shaking 10g garden soil 0.5 M

NaOH and

centrifuging.

w i t h 50ml

A p o r t i o n of the r e s u l t i n g extract

was s e p a r a t e d into humic and fulvic fractions by a d j u s t i n g the pH to

i

with

precipitate

conc.

centrifuging,

and

redissolving

the

of humic acid in NaOH.

A solution o f shaking

HCI,

200mg

commercial humic of

the

acid

material

(Aldrich)

for

was

p r e p a r e d by

three days in 100ml 0.01N

NaOH.

S a m p l e s o f BKME o r g a n i c

material

were

by

kindly

bleached bleaching

provided

soft-wood agent.

pulps, Effluent

further

concentrated

and 1000 - 10,000

(Burnison,

This

molecular

samples

mill

chlorine

Quebec, produces as

the

were initially c o n c e n t r a t e d

400 Daltons,

by tangential

y i e l d fractions of NW > 30,000,

in LaTuque,

Burnison.

using

by n a n o f i l t r a t i o n at a cut-off of was

from a mill

B.K.

I0,000 -

and the material

flow u l t r a f i l t r a t i o n to 30,000,

in preparation).

1000

- I0,000

1748

Rainbow(Tm)

protein

molecular

Amersham

Corporation.

molecular

weights

weight

These

ranging

are

markers

were

obtained

dye-labelled

from 2350

(insulin

proteins

a-chain)

from with

to 200,000

(myosin).

Electrophoresis

was

polyacrylamide

gels

(Amersham total

in

Corporation

gel

linker.

carried

Daltons.

the

specified

The

of

procedure (Amersham

gel was m o u n t e d

at the bottom.

ethyl

1,3-propanediol)

ammediol-glycine, (SDS).

The

glycine

buffer

obscured

the

experiments by cathode were

voltage

being

containing blue

most

were

buffer

analysed

with

the

or at constant

Prussian

blue

staining

chloride

and potassium

unless

was

ammediol

SDS,

of

dye

was

replaced

The electrodes

Similar

carried

ammediol-

our samples,

this b u f f e r

supply.

was

and glycerol,

the t r a c k i n g

component

was

noted.

sulphate

with

of glycerol.

power

experiment

Because

the

with the

buffer

dodecyl

mixed

in w h i c h

200,000 by

(2-amino-2-

cathode

were

the a d d i t i o n

is cross-

to

otherwise

sodium

dye.

with a

recommended

mercaptoethanol,

rapidly-running

2.5%

2000

in the apparatus

containing

to a B r i n k m a n n

whether

that

1988)

as a t r a c k i n g

gels,

23% of which is

precast

for the purpose

gradient

pH 9.5 and the

also p e r f o r m e d

buffer

connected

obtained

9.5,

designed

range

vertically

sulfate,

samples

-

Corporation

PhorCast(T~)

are

was

The anode

pH

with bromophenol

ii

used

Briefly, anode

These

separation

manufacturer the

on

apparatus

1988).

concentration

The

out

out

results

were

at constant

current.

was

carried

ferricyanide

out

by treatment

(Castagnola

et

with al.

ferric

1979).

1749

R E S U L T S AND D I S C U S S I O N

Typical

results

are presented

a

b

c

d

in F i g u r e

e

f

g

h

1.

i

j

k

Figure i. Electropherogram of humic preparations a n d BKME fractions. (a) Soil humic acid fraction. (b) BKNE > 30,000 MW. (c) BKME 10,000 - 30,000 MW. (d) BKME I000 - I0,000 NW. (e) Lake w a t e r concentrate. (f) Commercial humic acid. (g) R e - r u n of lake concentrate, band 3. (h) R e - r u n of BKME > 30,000 MW, band 3. (i) R e - r u n of BKNE >30,000 MW, b e t w e e n bands 1 and 2. (j) R e - r u n of BKNE > 30,000 NW, band I. (k). R e r u n of BKME >30,000 NW,

b a n d 2.

Results those the

with

preparations

reported

samples

small

tested

amount

but most smear

than

to form a diffuse

spots

of

0.80 i was and

lysozyme

lowest

and

(2),

(M.W.

14,300) 3 and 4

markers

used.

These

for

soil

highest diameter

humic

values

Rf 1.0

and spot

values

gel.

weight

A

well,

yellow-brown values (4)

of

(Figure

inhibitor

2 was close to

travelled

fractions

All

patterns.

trypsin

faster

are rather by

of 2.2 - 2.5 X 103

molecular of the

at

(3) and

in Rf b e t w e e n

Spots

with

in the s t a r t i n g

colour

0.96

3400).

calculated

of the pore

intense

0.88

intermediate

(N.W.

estimated who

more

(i),

the p r o t e i n

for the basis

electrophoretic

remained

21,500)

ai.(1981),

consistent

electrophoresis.

migrated

Spot

those

similar

were

disc

material

insulin b - c h a i n any of

gave very

material

using

of insoluble

with

(N.W.

of humic

literature

of the material

approximately 1,a,e,f).

in the

than lower

Kasparov

et

and 2 X 106

fractions

on the

1750

Rather

surprisingly

perhaps,

I0,000 and PTW I0,000 almost

-

BKME

showed

fractions

of MW I000-

e l e c t r o p h o r e t i c patterns

identical w i t h those of the humic preparations,

in the same positions as the materials bands

the

30,000

(Figure

but

contained

with bands

of the humic

completely

unresolved

material

of

(Figure l,b).

When the p o r t i o n of humic

gel

containing

material

or

sample well and subjected appeared

4 bands

comparable to those of proteins of m o l e c u l a r weight up

to about I00,000

either

3 and

l,c,d). The >30,000 fraction showed these same

also

mobilities

I, 2,

in

the

l,g,h,i,j,k).

Thus

weight fractions procedure.

same

polymers,

the

to a

second e l e c t r o p h o r e s i s as in the original

these

appear

(1978)

but..,

of

to

that

represent

contain

in a

the band

run (Figure

true m o l e c u l a r

and not artifacts of the

support "humic

bands of

cut out and placed

position

observations

al.

one

was

of the humic material,

These

C a s t a g n o l a et

any

BKNE

the

contention

of

acids are not statistical

constant

and

uniform

molecular

fractions". The

development

of

a

blue

colour

on

treatment

chloride followed by p o t a s s i u m ferricyanide indicates the (1979)

found

that only

their

test. W i t h

both of humic material with the

MacCarthy In and

humic

materials

This

acids

are

chemical

of

provides

SDS

processes

to them,

molecules

proportional

reliable proteins

different

and

water

solely

of

are

well

of the results

each

SDS-protein on their

procedure

for

(Weber & Osborne

from the

(Malcolm

&

and nucleic acids the physical

involved

conferring a to

evidence that

understood

is possible.

agent which

negative charge. The number of protein

molecule

complexes

through

m o l e c u l a r weights, determining 1969).

and an

SDS is an

denatures proteins

is

to the m o l e c u l a r weight of the protein.

the m o b i l i t y depends

bound

soil

proteins

interpretation

further

1987).

anionic detergent and d e n a t u r i n g and binds

intense blue colour

significantly

from

1986; Chiou et al.

unambiguous

gave a positive

those of the commercial humic acid, which

isolated

electrophoresis

MW fractions

and BKME d e v e l o p e d an

e x c e p t i o n of

commercial

low

blue test)

C a s t a g n o l a et al.

our material however all the fractions

remained yellowish-brown.

humic

(Prussian

presence of reducing substances.

P r u s s i a n blue

w i t h ferric

the

a

directly

Consequently sieving gel

and this provides molecular

a

weights of

1751

What

happens

with

substances

is

much

polycarboxylic alkaline other

anionic

may not

materials

clear.

which

are

would

molecules

of

not

such

be

as

be

d e p e n d e n t on

influenced and

comparisons

by

values with

approximate.

the

as

substances

expected

to

bind with

SDS as do a m p h o t e r i c p r o t e i n of the

various components

properties

their

such

molecular

mobilities

However whatever

mixtures

makes

possible

of

as charge/mass

weights d e r i v e d from

proteins

the physical

may

be

the

for humic

separation

preparations

only

and chemical basis

of

into a number of m o l e c u l a r fractions,

very similar

are

ionized in neutral or

for the e l e c t r o p h o r e t i c b e h a v i o u r of humic m o l e c u l e s may procedure

humic

their m o l e c u l a r weights but may

other

for

such

Humic

largely

C o n s e q u e n t l y the m o b i l i t i e s

be solely

ratios,

types

less

acids

s o l u t i o n and so

molecules.

also

other

be, the

these

complex

w h i c h appear to be

from soil and from water as

well as for commercial products.

W h a t e v e r constraints humic

substances

acting on the

high

fractions

MW

of

are

samples.

The

I000

-

for

weight

10,000

of

of

of

10,000

BKME.

The

- 30,000 y i e l d e d

identical with those of humic

same bands were o b t a i n e d from the highest m o l e c u l a r

lower mobility.

fractions of solely

d i s t r i b u t i o n of

material

and

apparently

the

fractions appear also to be

fraction too, but this also c o n t a i n e d a

material

BKME o b t a i n e d simple

m o l e c u l a r weights, units,

particular

molecular

electrophoretic patterns

weight

responsible

among

which

large q u a n t i t y of

These o b s e r v a t i o n s by tangential

covalently-bonded

suggest that the

flow

do not consist

molecules

but also contain a g g r e g a t e s

of

differing

of these covalent

may d i s s o c i a t e to a greater or lesser extent d u r i n g

subsequent m a n i p u l a t i o n s .

Electrophoresis

on p o l y a c r y l a m i d e

gel

requires

small

of

analysis

only

of a n u m b e r

apparatus we

used).

quantities of samples It

studies on humic substances and materials

from

of p a r t i c u l a r

fractions

for

chemical properties.

at a

should prove

c o m p a r i s o n of

slabs

time useful

similar different the

is

material,

study

(up

simple,

rapid,

and permits the to 16

w i t h the

in various types of

materials

such

as the

sources or the isolation of

their

physical

or

1752

ACKNOWLEDGEMENTS

We t h a n k Dr. B.K. B u r n i s o n and V. M a r t i n for assistance in the preparation of the lake w a te r concentrate as well as for providing the BKME samples, and Dr. S.A. Daniels for valuable help and advice. Emilie Finnerty-Baxter provided helpful encouragement.

REFERENCES A m e r s h a m Corporation. 1988. PhorCast(TM) Heights, Ill. U.S.A. 13 pp.

Handbook.

Arlington

Baxter, R.M. & J.H. Carey. 1982. R e a c t i o n s of single% oxygen in humic waters. F r e s h w a t e r Biol. 12, 285 - 292. Castagnola, M., R.G. De Las Heras, G.B. M a r i n i - B e t t ~ l o and C. Nigro. 1978. Effect of urea on e l e c t r o p h o r e t i c p a t t e r n of soil humic acids. J. Chromatogr. 147, 438 - 442. Castagnola, M., C. Nigro, G.B. M a r i n i - Bett~lo, A. Milana & R.G. De Las Heras. 1979. C h a r a c t e r i z a t i o n of soil humic acid by combined p o l y a c r y l a m i d e disc e l e c t r o p h o r e s i s and chromatic reactions. J. Chromatogr. 177, 130 - 134. Ceccanti, B., J.M. A l c a n i z - B a l d e l l o u , M. G i s p e r t - N e g r e l l & M. Gassiot-Matas. 1986. C h a r a c t e r i z a t i o n of organic m a t t e r from two different soils by p y r o l y s i s - g a s c h r o m a t o g r a p h y and isoelectric focusing. Soil Sci. 142, 83 - 90. Chiou, C.T., D.E. Kile, T.I. Brinton, R.L. Malcolm, J.A. L e e n h e e r & P. MacCarthy. 1987. A c o m p a r i s o n of water s o l u b i l i t y e n h a n c e m e n t s of organic solutes by aquatic humic m a t e r i a l s and commercial humic acids. Environ. Sci. Technol. 21, 1231-1234. Curvetto, N.R, N.A. B a l m a c e d a & G.A. Orioli. 1974. I s o t a c h o p h o r e s i s and isoelectric f o c u s i n g of soil humic substances in p o l y a c r y l a m i d e gel. d. Chromatogr. 93, 248 250. Curvetto, N.R, N.A. B a l m a c e d a & G.A. Orioli. 1975. E l e c t r o p h o r e t i c methods for f r a c t i o n a t i o n of humic and fulvic acids. T u r r i a l b a 25, 365 - 370. De Gonzalez, N.M., M. C a s t a g n o l a & D. Rossetti. 1981. Humic acid c h a r a c t e r i z a t i o n of C o l o m b i a n soil by disc e l e c t r o p h o r e s i s and infrared s p e c t r o s c o p y f o l l o w i n g gel filtration. J. Chromatogr. 209, 42i - 431. De Nobi-li, M. 1988. E 1 e c t r o p h o r e t i c evidence of the integrity of humic substances separated by means of e l e c t r o f o c u s i n g . J. Soil Sci. 39, 437 - 445. De Nobili, M., G. Bragato, J.M. A1caniz, A. Puigbo & L. Comellas. 1990. C h a r a c t e r i z a t i o n of e l e c t r o p h o r e t i c fractions of humic substances with different e l e c t r o f o c u s i n g behaviour. Soil Sci. 150, 763 - 770. Duxbury, J.M. 1989. Studies of the m o l e c u l a r size and charge of humic substances by e l e c t r o ph o r e s i s . In M.H.B. Hayes, P. MacCarthy, R,L. M a l c o l m & R.S. Swift, eds. Humic S u b s t a n c e s If. John W i l e y & Sons, Chichester. 764 pp. Pages 593 - 620. Gjessing, E.T. 1976. Physical and Chemical C h a r a c t e r i s t i c s of Aquatic Humus. Ann Arbor Science, Ann Arbor, Mich. 120 pp. Gjessing, E.T. & T. G j e r d a h l . 1973. E 1 e c t r o m o b i l i t y of aquatic humus: f r a c t i o n a t i o n by the use of the i s o e l e c t r i c focusing technique. In D. P o v o l e d o & H,L. Golterman, eds, Humlc Substances: their structure and f u n c t i o n in the biosphere. Centre for Agricultural Publishing & Documentation, Wageningen. Pages 43-50. Kasparov, S.V., F.A. T i k h o m i r o v & A.D. F1ess. 1981. Use of disk e l e c t r o p h o r e s i s to f r a c t i o n a t e humic acids. M o s c o w U n i v e r s i t y Soil Science B u l l e t i n 36, 21 - 28. (Translated from V e s t n i k M o s k o v s k o g o Universiteta. P o c h v o v e d e n i e 36, 23 - 30.) Kleparnik, K. & P. Bocek. 1991. T h e o r e t i c a l b a c k g r o u n d for clinical and biomedical a p p l i c a t i o n s of e l e c t r o m i g r a t i o n

1753

techniques.

J. Chromatogr.

569,

3 - 42.

K18cking, R. 1973. Ein System zur Polyacrylamidgelelektrophorese yon Huminsauren. J. Chromatogr. 78, 409 - 416. Malcolm, R.L. 1990. Variations between humic substances isolated from soils, stream waters, and groundwaters as revealed by lsC-NMR s p e c t r o s c o p y . In P. MacCarthy, C.E.Clapp, R.L. Malcolm & P.R. Bloom, eds. Humic Substances in Soil and Crop Sciences: Selected Readings. American Society of Agronomy Inc., Soil Science Society of America, Inc, Madison. 281 p p . Pages 13 - 35. Malcolm, R.L. & P.M. MacCarthy. 1966. L i m i t a t i o n s in the use of commercial humic acids in w a t e r and soil research. Environ. Sci. Technol. 20, 904 - 911. Pillai, M.C., G.N. Cherr & R.M. Higashi. 1991. A major c o n s t i t u e n t of b l e a c h e d kraft mill effluent (BKME) blocks sea u r c h i n f e r t i l i z a t i o n by i n h i b i t i n g the sperm a c r o s o m e reaction. A b s t r a c t s of the 12th Annual Meeting, S o c i e t y of E n v i r o n m e n t a l T o x i c o l o g y and Chemistry, Seattle, Nov.3-7, 1991. Page 31. Stepanov, V.V. & A.N. Pakhomov. 1969. E 1 e c t r o p h o r e s i s of humic substances in p o l y a c r y l a m i d e gel. Soviet Soil Science No.6, 742 -749. (Translated from P o c h v o v e d e n i e , 1959, N o . 1 2 : 4 6 56). Stevenson, F.J. 1982. Humus Chemistry. John W i l e y & Sons, New York. 443 pp. Thornton, J.I. 1975. I s o t a c h o p h o r e s i s and I s o e l e c t r i c f o c u s i n g of soil humic substances in p o l y a c r y l a m i d e gel. J. Chromatogr. 103, 402. V e s t e r b e r g , 0. 1989. H i s t o r y of e l e c t r o p h o r e t i c methods. J. Chromatogr. 480, 3 - 19. Virkki, L., J. Knuutinen, P. M a n n i l a & J. Paasivirta. 1988. NFIR studies of kraft pulp mill waste and natural humic substances. In G. A n g e l e t t i & A. Bjorseth, eds. Organic M i c r o p o l l u t a n t s in the Aquatic Environment. Proc. F i f t h E u r o p e a n Symposium. K l u w e r A c a d e m i c Publishers, Dordrecht. 512 pp. Pages 344 346. Weber, K. & M. Osborne. 1969. The r e l i a b i l i t y of m o l e c u l a r w e i g h t d e t e r m i n a t i o n s by dodecyl s u l f a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e e i s . J. Biol. Chem. 244, 4406 - 4412. Wolfe, M.F. & A.D. Jones. 1991. C h a r a c t e r i z a t i o n of a toxic h i g h m o l e c u l a r mass f r a c t i o n i s o l a t e d from b l e a c h e d kraft mill effluent. A b s t r a c t s of the 12th Annual Meeting, S o c i e t y of E n v i r o n m e n t a l T o x i c o l o g y and Chemistry, Seattle, Nov.3-7, 1991. Page 250. Zheng Dehe & Lu Shilin. 1983. S e p a r a t i o n and c h a r a c t e r i z a t i o n of humic and fulvic acids by gel e l e c t r o p h o r e s l s and isoelectric f o c u s i n g in p o l y a c r y l a m i d e gel. K e x u e T o n g b a o 28, 757 - 761. (Received in Germany 27 March 1992; accepted 25 May 1992)