Lysozyme-agarose interaction

Vol. 74, No. 4, 1977

BIOCHEMICAL

AND BiOPHYSICAL

RESEARCH COMMUNICATIONS

LYSOZYME-AGAROSE INTERACTION J.M.Ferndndez-Sousa Department

of Biochemistry.

Complutensis Received

November

and R-Rodriguez Faculty

University.

Madrid.

of Sciences Spain

16,1976

Summary. It has been observed that the elution volumes of hen eggwhite lysozyme on agarose columns exceeded by far the total volumes of the corresponding columns. Similar results have been obtained also on polyacrylamide-agarose gel columns. This anomalous behaviour of lysozyme on agarose columns is interpreted in terms of an interaction between the enzyme and agarose which resembles in some way the physiological substrates of lysozyme. It has been already reported pancreatic a-amylase elutes later volumes on gel filtration The possible retention was ruled the ionic ed to

that than

chromatography of these proteins

lysozyme and before the total

in dextran columns (l-4). by an ion exchange mechanism

out as the elution volumes were found to be independent of strength. Thus, this anomalous behaviour has been attribut-

an interaction While modified

of lysozyme and a-amylase with dextran gels (3). dextrans used as sieves are three-dimensional agarose is a linear chain polymer of -Dchains,

networks of glucan galactose6(1+4)-3,6-anhydro-L-galactoseu(l+3)-. the structure of bacterial cell walls chains built muramic acid similar tention

hen egg-white expected but

up of alternating residues linked

is

On the other hand, composed of polysaccharide

N-acetylglucosamine and N-acetyl throughout B(1+4) linkages, and thus

to agarose. Accordingly, if the reason of hen egg lysozyme on dextran gels is

for the

the abnormal interaction

re -

between the enzyme and the polysaccharide structure, a stronger interaction would be expected with agarose and consequently greater elution volumes of lysozyme on agarose filtration chromatography than

on dextran gel columns. These considerations led us to investigate lysozyme with agarose through the comparative enzyme in gel

filtration

chromatography

MATERIALS AND METHODS Hen egg-white lysozyme, cell walls from Michococcun

Copyright 0 1977 by Academic All rights of reproduction in any

Press, Inc. form reserved.

horse

heart

.fLqbodeikXicub

1426

the interaction behaviour of the

on different

of

media.

cytochrome c, bacterial and 2-mercaptoethanol

were

ISSN

0006-291X

BIOCHEMICAL

Vol. 74, No. 4, 1977

obtained

from Sigma Chemical

and Blue Dextran Biogel A, 0.5m,

(St.Louis,USA).

Sephadex G-75,fine,

were purchased from Pharmacia (10% agarose) was from Bio-Rad

Co.

(Upsala, Sweden). Lab. (Richmond,USA).

Ultrogel AcA 54 (5% acrylamide (Sweden). All other chemicals 6.3,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and 4% agarose) was from LKB Products were reagent grade.

Sephadex was suspended in 0.05 M potassium phosphate and allowed to swell for 2-3 days at room temperature.

swollen Ultrogel

buffer, The

pH

Sephadex suspension as well as the comercial suspensions of AcA 54 and Biogel A were degassed before poured into the

(lx50cm) columns, for chromatography. were calculated

and then equilibrated Void (V,) and total

with

Blue

Dextran

with (Vt)

the same solutions used volumes of the columns

and ammonium hydroxide

respectively.

A solution of 2.2 mg of hen egg-white lysozyme and 2.2 mg of horse heart cytochrome c in 250 ul was applied to the Sephadex G-75, Biogel A and Ultrogel AcA 54 columns, previously equilibrated, and eluted with a) 0.05 M potassium phosphate buffer, pH 6.3,(I'/2=0.07); b) 0.05 M phosphate buffer, pH 6.3, containing 0.28 M KCl(T/2=0.35); c) 0.05 M phosphate d) 0.05 M phosphate phosphate ethanol.

buffer,

buffer, buffer, pH 6.3,

pH 6.3, pH 6.3,

containing containing

containing

1.0 M KCl(I'/2=1.07) 6 M urea and e)0.05

6 M urea

Lysozyme was determined either absorbance at 280 nm or by its lytic

and 25 mM mercapto

; M -

spectrophotometrically by its activity towards Michococcun

cell walls at pH 6.3 and 0.1 ionic strength. Cyto chrome c was determined by its absorbance at either 280 nm or 410 nm

Cynodeihticud

or 550 nm, according

to the

experiment.

Discrimination between cytochrome c and lysozyme was occasionally done on polyacrylamide gel electrophoresis performed according to Panyim and Chalkley (5), using

Amido Black

dye for

gel

staining.

RESULTS AND DISCUSSION Fig.la heart

shows a typical

cytochrome

elution

pattern

c and hen egg-white

lysozyme

of a mixture

of horse

from a Sephadex

G-75

column: this elution profile shows clearly that both proteins overlap partially in spite of the differences in their molecular weight. It is interesting to note, however, that hen lysozyme (mw.14307) elutes slightly later than horse cytochrome c (mw.12398). Concerning the mechanisms underlying this fact, it has previously reported that basic proteins such as horse heart cytochrome c (p1 10.8) (6) and hen lysozyme

(p1 11-O-11.3)

(7) eluted

later

1427

than

expected

from dextran

Vol. 74, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1.6-

SEPHADEX

G-75

ULTROGEL

AcA

54

-5

BIOGEL

A 0.5m -10

-5

25

30

35

40

45

50

55

60

65

rnl

Figure 1. Elution profiles of cytochrome c and lysozyme from Sephadex G-75 (a), Ultrogel AcA 54 (b) and Biogel A (c). Total recorded proteins are given by the continuous line. Cytochrome c was evaluated by absorption at 550 nm ($@:l') and lysozyme was determined by its lytic activity (m).

gels due to weak ion-exchange properties of the gels (1). This result has been later attributed to a specific interaction of lysozyme with the dextran gel matrix rather than to the more basic character of the molecule of lysozyme (3,4). Elution profiles of the mixture of horse cytochrome c and hen lysozyme from columns of Ultrogel AcA 54 (polyacrylamide/agarose) and Biogel A 0.5m (agarose) (figs.lb and lc, respectively)clearly show the high resolution of both proteins. Cytochrome c elutes from these columns at the expected volume, but hen lysozyme elutes with a volume

1428

Vol. 74, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

C”3

co kn

CH3

$0

Cl+ll

...+iCVH ,,“,:.+o+J CH co no-

_ A0

un-

CELL

b43

. . ..&.e.

=

/-

WALL

NCI

xao..

co

POLYSACCHARIDE

=“3

“07---.JJo~o... qon

NW

Nbl

CO &I3

CO dH3

CHITIN

AGAROSE

Figure 2. Fragments of the structure of bacterial saccharide, chitin and agarose matrix.

cell

wall

poly

-

greater than the total volume of the corresponding agarose columns. Moreover, lysozyme elutes from Biogel A (10% agarose) columns with a greater volume than from the Ultrogel AcA 54 (5% polyacrylamide and 4% agarose) columns. Ionic strength (0.07) and pH (6.3) of the buffer used in these gel filtration chromatography experiments are those at which could

lysozyme exhibits maximal be explained on the basis

activity (8). of a specific

Thus, these interaction

results of lyso

-

zyme with the agarose gel matrix, possibly due to the similarity of the structure among agarose and bacterial cell wall polysaccharides and chitin. In fig.2 a fragment of these structures is given. If the mixture of horse cytochrome c and hen lysozyme is chromate graphed on agarose columns equilibrated with 0.05 M potassium phos phate buffer, pH 6.3, containing 6 M urea, lysozyme is eluted slightly later than cytochrome c and before the total volume of the corresponding columns (Table 1). This fact could be explained through a weak interaction of lysozyme in 6 M urea with the agarose matrices that account

for

an elution

volume

higher

than

that

is in coincidence with a remanent enzyme activity tributed to the remaining unfold of the protein 9-10 M urea

(9,lO). 1429

of cytochrome

c.

It

of about 3-5% atin the presence of

Vol. 74, No. 4, 1977

Elution

Elution Solvent

BIOCHEMICAL

volumes lysozyme

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1 of horse cytochrome c and hen egg-white on gel chromatography columns Sephadex G-75 Ultrogel AcA 54 Biogel A, 0.5 m Vt=34.6 ml Vt=37.5 ml Vt=3B.0 ml

Protein

a

Cytochrome Lysozyme

c

b

Cytochrome

c

Lysozyme

28.2 ml 30.9

36.8 ml

37.5

46.4

57.2

27.6 29.7

35.7 41.4

35.5 41.7

C

Cytochrome Lysozyme

c

27.5 29.5

35.5 41.2

35.5 41.4

d

Cytochrome Lysozyme

c

27.8 28.8

34.6 36.6

31.5 34.7

Cytochrome

c

27.6

34.5

32.1

28.2

35.4

33.6

e

Lysozyme

Similarly,

it

has been reported

that

the activity

ml

of hen lysozyme

at 0.35 ionic strength decreased to S-10% of the maximal activity at 0.07 ionic strenth, pH 6.25. Thus, Davies et al. (8)suggest that the electrostatic interaction of lysozyme with the bacterial cell wall polysaccharide used as substrate decreased as the ionic strength of the medium raised. Effectively, the elution patterns of the mixture cytochrome could also lysozyme table 1 variation therefore, strengths. columns retention matrices If carried

c-lysozyme from agarose columne (see fig.1 and table 1) be interpreted in terms of the remanent activity of hen

at 0.35 ionic strength. However, it can also be observed in that by increasing the ionic strength up to 1.1, a very low of the elution volume is registered and it can be concluded, that lysozyme exhibits some activity even at high ionic On the other hand, this retention of lysozyme on agarose at high ionic strengths also rule out the possibility that the is due to ion exchange properties of the polysaccharide of the gels. the gel chromatography of the mixture cytochrome c-lysozyme is out in the presence of 6 M urea containing 25 mM 2-mercapto -

ethanol , a different elution profile presence of 25 mM 2-mercaptoethanol,

1430

is obtained (Table 1). In the disulfide bridges of the enzyme

BIOCHEMICAL

Vol. 74, No. 4, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are reduced (7) and lysozyme is totally devoid of lytic activity. Thus, chromatography on agarose columns with 2-mercaptoethanol shows that lysozyme regains its overlap with cytochrome c. All these facts suggest that the anomalous behaviour of native lysozyme interaction saccharide structure

on agarose

gels

can be explained

on the basis

of a specific

between the active center of the enzyme and the poly matrix due to a certain resemblance of the agarose with those of the bacterial cell wall polysaccharides and

chitin. Concerning

the

functional

groups

involved

in these

interactions,

acetamide and 8(1+4) linkage are the main groups offering significant challenges. Both groups are necessary for the lytic activity of lysonevertheless, amide groups have no equivalent in the agarose zyme (7); structure

whereas

8(1+4)linkages

are common to the

lysozyme

and to the agarose matrix simmilarities in structure

(fig.2). This fact, and conformation,

that with

with lysozyme due to their cell wall polysaccharides.

agarose gels interact chitin and bacterial Chromatography

on agarose

selective method to purify ing any contamination with the gel matrix; molecules that

substrates

jointly with other allows one to consider resemblance

columns has been described

to be a

lysozymes due to these facts, thus other proteins that do not interact

avoid with

-

simultaneously, lysozymes are obtained free of small should elute at approximately the Vt of the column(l1).

REFERENCES 1. Miranda,F., 2. Gelotte,B. ion in gel 3. Whitaker,J.R., 4. Kramer,K.J.,

H.Rochat

and S.Lissitsky,

Quoted by P.Flodin, filtration", Upsala Anal.Chem., Ph.D.Thesis,

J.Chromatog.,

1,

142 (1962)

in "Dextran gels and their (Sweden), Pharmacia, 1962.

35, 1950 (1963) University of Arizona

applicat-

(1971)

5. Panyin,S. and R.Chalkley, Arch.Biochem.Biophys., 130, 337 (1969) 6. Lemberg,P. and Barret,J., In "Cytochromes" pp.122-216, Academic Press (New York), 1973. 7. Imoto,T., L.N.Johnson, A.C.T.North, D.C.Phillips and J.A.Rupley, In "The Enzymes", ~01.7, pp.665-868 (ed.P.D.Boyer), Academic Press, New York, 1972 A.Neuberger and B.M.Wilson, Biochim.Biophys.Acta, 178, 8. Davies,R.C., 293-305 (1969) 9. Hamaguchi,K. and A.Kurono, J.Biochem.(Tokyo) 54, 111 (1963) Biochim.Biophys.Acta, 178, 293 (1969) lO.Steiner,R.F., ll.Rodriguez,R. Ph.D.Thesis. Complutensis University, Madrid (1976)

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