Adsorption and binding of peptides on homoionic montmorillonite and kaolinite

Copyright

cnl35-0717 84 s3.00 + 0.00 C 1984 Pergamon Press Lrd

ADSORPTION AND BINDING OF PEPTIDES ON HOMOIONIC MONTMORILLONITE AND KAOLINITE T. DASHMAN* and G. STOTZKY Laboratory of .Microbial Ecology, Department of Biology, New York University. New York, NY 10003, U.S.A. (.-tccepred20 Jr(/.v 1983)

Summary--The adsorption and binding (amount retained after extensive washing) of peptides (two. three or four amino acids with isoelectric points ranging from pH 3.31 to 6.81) on montmorillonite (M) and kaolinite (K) made homoionic to H, Na. Ca, Zn. Al or La were studied. The amount of peptide adsorbed or bound varied with the type of clay, the type of cation saturating the clay, and the constitutive amino acids of the peptides. Di-, tri- and tetraglycine were adsorbed to a greater extent on IV than on K. A larger amount of the basic peptide, r-alanyl-r-lysine, than of the acidic peptide. L-aspartyiglycine, was adsorbed. The heterocyclic peptides, L-histidylglycine and r-prolyl-L-phenylalanylglycyl-L-lysine, were preferentially adsorbed on &I homoionic to di- and trivalent cations. Only t.-aspartylglycine, t_-histidylglycine, L-alanyl-t,-lysine and r-prolyl-r-phenylalanylglycyi-t_-lysine were bound on M, and only digiycine, tetraglycine and L-prolyl-L-phenylalanylglycyl-L-lysine were bound on K.

INTRODUCTIOZI

The adsorption of low molecular weight (MW) peptides to clays appears to be governed by both their basicity and MW (Greenland et nl., 1962, 1965a, b;

Theng, 1974). Dashman and Stotzky (1982) found that the relative basicity and MW of amino acids were not as important in their adsorption and binding to clays as were other characteristics of the amino acids, such as additional thiol, guanido or carboxylic acid groups, and the physicochemical properties of the homoionic montmorillonite (M) or kaolinite (K) used. Harter and Stotzky (1971) showed that the adsorption of proteins with different isoelectric points (PI) in unbuffered suspensions was dependent on the physicochemical characteristics of the proteins and the homoionic M. We have investigated the influence of MW, basicity and functional groups of some peptides, ranging from two to four amino acid residues and in p1 from 3.31 to 6.81, on their adsorption and binding to M and K homoionic to mono-, di- or trivalent cations. SIATERIALS AND >lETHODS

Wyoming bentonite was obtained from Fisher Chemical Co. and kaolin from iMerck and Co. L-Aspartylglycine (aspgL/), L-histidylglycine (hisg/y). glycylglycine (dig/y), glycylglycylglycine (trig/y) and glycylglycylglycylglycine (tetrug/y) were obtained from Schwartz Mann Co., and L-alanyl+lysine (alunyllys) and L-prolyl-L-phenylalanylglycyl-L-lysine (prphgly) from Miles Laboratories. These compounds were of 907: or greater purity and were used

*Present address: Department of Biochemistry and Drug &letabolism, Hoffmann-La Roche Inc. Nutley, NJ 07 I 10, U.S.A.

as received. All other chemicals were of reagent grade. The homoionic M and K were prepared and the equilibrium adsorption and binding of the peptides on the clays in unbuffered systems were determined as described by Dashman and Stotzky (1982). Harter and Stotzky (1971) and Stotzky (1980) except that the concentration of clays used was 50 mg. RESULTS The di-, tri- and tetrugly peptides were adsorbed at equilibrium on M-H. Monomeric glycine also showed a similar preference for adsorption on M-H as compared to M homoionic to Na, Ca, Zn, Al or La (Dashman and Stotzky, 1982). The trend in the order of equilibrium adsorption of di- and rrigly on the homoionic M was monovalent > trivalent > divalent cations (Table 1). Dig/y was adsorbed to about the same extent (33%) on M-H and M-Na, to about the same extent on M-Ca and M-Zn (4--5x), and more than twice (12%) as much was adsorbed on M-AI than on M-Ca and M-Zn. Digly was not adsorbed on M-La. More trigly was adsorbed on M-H (23:/,) than on M-Na (17x), which was more than that adsorbed on M-Zn (11%). M-AI (14%) and M-La (12%). Trigfy was not adsorbed on M-Ca. Tetrugly was the least adsorbed glycine peptide used, and it was adsorbed to about the same extent (3-7x) on all homoionic M, except on M-Ca (< 1%) (Table 1). Neither the polygly peptides (Table 2) nor glycine (Dashman and Stotzky, 1982) were bound on any of the homoionic M studied. The equilibrium adsorption and binding of the polyglycine peptides (Tables 3 and 4, respectively) on homoionic K did not follow the same pattern as adsorption and binding on homoionic M. The order of adsorption of dig/y on the homoionic K was

T.

DASHMAN and

G.

STOTZE’I

Na > Ca > H > Al = La. and no dig& was adsorbed on K-Zn. Trig/~ was adsorbed to about the same extent on K-H (8”:) and K-Na (5”“). and it vvas not adsorbed on K homoionic to di- and trivalent cations. Teetrag/>,was adsorbed on all the homoionic K but only at low concentrations. Although none of the glycyl peptides was bound on M. digI>,vvas bound on K-Na and K-Ca. and retrug/!, was bound on K-H and K-Na. Trigl_vwas not bound on any K (Table 4). Acidic aspgly was adsorbed most on M-H. and although some adsorption to the other homoionic M occurred, aspgly was bound only on ,M-H (Table 2). Aspgly was adsorbed only on K homoionic to Ca or Al (Table 3). and the small amount adsorbed (< 1”;) was completely removed by three vvashings vvith distilled water (Table 4). The heterocyclic peptide, hisg&, was adsorbed on all homoionic M (Table 1) and was bound on those homoionic M on which 457; or more of the peptide was adsorbed. i.e. M-Al, M-Zn and M-Na (Table 2). Hisgl,v was not bound on any homoionic K (Table 4) and was only adsorbed to a limited extent on K-H, K-Na and K-Zn (Table 3). However, hisgl! was adsorbed on K-Ca to the same extent as on M-Ca, suggesting that this peptide complexed with the Ca on the surface of both clays. The order of adsorption of hisgly on homoionic M was Zn = Al > Na > H > Ca = La. The respective bulk pH (pH,) of the homoionic ,M was: 6.7, 4.1. 6.2, 2.7,8.3 and 6.2. These observations indicated that the cation saturating the clay was more important in adsorption than was the pH-pI relation. Alanyllvs is a basic peptide, and although its pI is not known, it probably approaches that of lysine (PI = 9.59) and is greater than that of the acidic dipeptide, uspgly (PI = 3.31). In every instance in which both peptides complexed with the same homoionic clay. more almyllys than mpg/y was adsorbed (Tables I and 3). As the p1 of alun_$~s is probably close to pH 9.0, alun~~llsshould have been readily adsorbed on the acidic clays, M-H, M-AI, K-H and K-AI, which, in fact, it was. However the order of adsorption of almyllys on homoionic M was Zn > H > Al > La > Na > Ca (Table 1). Alun_dl~s was bound only on M-H, suggesting protonation or cation exchange between the acidic M-H and the basic peptide. The order or adsorption of alunyll~s on homoionic K (Ca > H = Al) suggested that alun;l~llwmay have formed a chelate complex with the Ca-- of K-Ca and adsorbed on acidic K-H and K-AI by protonation or cation exchange. Alanyllys was not bound on any of the homoionic K. The order of adsorption of prphgly on the homoionit M was Zn > Al = La > H > Na (prphgly was not adsorbed on M-Ca), which suggested complex formation between the pi-electrons of Zn. Al and La and the phenylalanyl moiety of prphgl:,. The binding of prphgly on M was similar to that of hisgjj,, in that both were preferentially bound on M-Al and M-Zn. This may have been due to the aromatic imidazole moiety of /zisglJ and the phenylalanine of prphgly. Prphgl! vvas adsorbed on all homoionic K and was bound on all K except K-La. There was little or no desorption of prphgl!, from K-.-\I and M-AI (Tables

ldbk

Pepcide added’ f

Prphgl>: (0.37)

tJdriQKS

peprides on m~ntmo~ll~ni~ehomoionic Sodium (pW

~.~--_~-..

i .05)

L-Hiisrid?lgfycine (0.94) L-.Alanyl-L-lysine

Binding of

Hvdroeen (1.01I+,(“,I

ipwmg-.)

L-.Aqmyi&tint

1.

(0.59)

0.16 20.03’

fj

SB

x;B

mg-‘1

xa”

O.M~O.OI

7

0.10 zo.01

25

53

and binding of peptides on clays

Adsorption

10 various cations’

Aluminium (~141mgmL) ilr-ttme-9 ?,I lll_l-i”,) ..____ ___z_... ..Zinc

(*,I NB

SB

NB

XB

Lsnth3num

(pst mg-'i i”,) _

48

XB

3%

il

0.27 r 0.03

19

0.11 to.01

43

NE

NB

N%

ix%

N%

NB

5B

N%

N%

NB

0.02 2 0.02

7

O.i5 ~0.01

42

0.X t0.02

50

0.05 5 0.02

I3

0.10=0.00

“Bindin: *as calculated by subtracting the toraf pcptide recovered in all washings (untit no more pepride was desorbed) from the amount iniilaify added. Digiycine. trigiycine and tettXgiy%Ie were not bound on any of the clays, and none of the peptides was bound on monrmorillonire homoionic to calcium. ‘See Table 1 for molecular wighis iMW) and isoefectric points {pi) of the peptides and bulk pif of homoionic clay suspensions. ‘t-alues are the mean k SE84 of tripiicate anal>scs. “SB = no binding ‘r-Praivf-L-phenglalanyigfycyl-L-lysine.

1 and 3), which suggests that p~p~tgl~ was adsorbed and bound to these clays by the same mechanism. The order of adsorption of prphgl,r on the surface of K was Zn > H = Ca > Na = Al = La, suggesting that prplrg!~ was preferentially adsorbed on K-Zn by a pi-bond complex with the phenyl ring of phenylalanine or by H-bonding with the carbonyl groups of the peptide bonds or by both mechanisms.

DISCUSSlON

The peptides used in this study can be divided &to three groups: monomeric peptides of increasing chain length, the glycyl peptides; acidic and basic peptides, us&x and afu~,~~~~~,respectively; and heterocyclic peptides, hisgfy and prphgly. Each of these groups illustrate properties of the molecules (MW, actd and base functions, and aromaticity, respectively) that may be involved in their equilibrium adsorption and binding to clays. Greenland et al. (1962, 1965a, b) reported that as the MW of glycyl peptides increased, adsorption increased. The results of our study indicated that as the MW of the same glycyl peptides increased, adsorption generally decreased. The discrepancy between our results and those of Greenland et al. (1962) may have been due to differences in the source of the peptides, the preparation of the clay-peptide complexes, or the duration that the peptides were in contact with the clays. In our experiments, the peptides were mixed with the clays for 1 h, as this was sufficient for maximum adsorption of amino acids (Darhman and Stotzky, 1982), whereas Greenland et al. (1962) mixed the clays and peptides for about 16 h. Furthermore, Fripiat et af. (1966) indicated that the apparent degree of retention of glycine, dig& or a-alanine on M-H were significantly different according to the method of analysis of adsorption. Aspartic acid was adsorbed and bound only on M homoionic to Ca or Zn, and more aspartic acid than asp& was adsorbed on these clays (Dashman and Stotzky, 1952). Sieskind and Wey (1959) found that increasing the chain length of glycine to E-amino caproic acid by the addition of methyl groups enhanced its adsorption on M. However, these results showed that increasing the length of aspartic acid (or glycine) to aspg/y did not enhance its adsorption on M, indicating that the mechanisms of adsorption of aspartic acid and aspgiy on M were different. Greater amounts of aspartic acid than of aspg/y

were adsorbed on K-Ca, although aspartic acid was also not bound on K-Ca (Dashman and Stotzky, 1982), suggesting that csrpg!_rwas adsorbed on K-Ca by a mechanism that was aiso different from that For the adsorption of aspartic acid. The adsorption of kisgly on M homoionic to different cations was not dependent upon the pH-pI relationship (Harter and Stotzky, 1971) but rather on the type of cation saturating the clay (Table 1). Dashman and Stotzky (1982) and Harter and Stotzky (1971) obtained similar results with amino acids and proteins, respectively. Heller-Kallai et al. (1972) suggested that histidine forms pi-complexes with M-Al, and therefore, one possible mechanism of the adsorption of hisgiy to IM-AI, and probably to M-Zn, may have been the formation of a pi-complex between the imidazole moiety of the peptide and the clays. Aspgly and hisgly can be considered as acidic and basic analogues, respectively. of cligly. Hisgly was adsorbed to a greater extent than uspgfy on all the homoionic M, except on M-H to which both peptides were adsorbed equally. The peptide, digly, was adsorbed to a lesser extent than hisgly on all homoionic M. Dig& was adsorbed to a greater extent than ctspgly on M-Na and M-AI, to a lesser extent on M-Zn, and equally on M-H and M-Ca. Dig1.v was not adsorbed on M-La. but both aspgly and hisgly, in about equal amounts, were adsorbed on M-La. These data further suggested that adsorption of these analogous dipeptides on the homoionic M was a function of the cation on the clay and the functional groups of the dipeptides. One possible explanation for the high adsorption of ufanyllys on M-Zn is indicated by the work of Jang and condrate (1972a. b), which showed that lysine and I-alanine formed a chelate complex with the CuL* of M-Cu at pH 6 or greater. Consequently, alun,vlf~r may have also formed a chelate complex with the Zn’+ of M-Zn and possibly also with the Ca:+ of M-Ca, which had a pH, of 8.3. Jang and Condrate (1972a) also suggested that lysine was adsorbed as a linear compounds rather than as a bidentate chelate compound, on the inter~ameIlar spaces of M-H, and therefore, afa~~fl~~ may have been adsorbed as a linear compound on the interIamellar spaces of M-H. However, af~n~ll~~ bound on &4-H did not cause expansion of M-H as measured by X-ray diffraction, indicating that the concentration of bound alan$i~s was either too iow to

-.

addedh

Table

(0.59)

L-Alanyl-dysine

pll

are

clay

“_ __

.” .

addedb

0.01 -10.00

“NII

weights

..

-

- -

-

analyses.

.,

0.23 +O.OI

-

NE

NE’

mg .‘)

(6.8)

0.2 I 5 0.03

NE

0.07 * 0.01

NE

0.02* 0.0I

(/‘M

Zinc

homoionic

(until

_ _.. _ _ ,_ _ -

Y

NB

I5

(%I

Zinc

peptide

1,.

1101 bound

no more were

NB

NB

I&‘)

0.0x f 0.00

(/IM

cirlion?

21

Ntt

NB

(‘X)

-

‘)

(jam

NU

22

NB

und none

Ihc amounl

0.08 * 0.00

NB

on any or die clays,

^

lJ.08 * 0.00

2x

01

Nl:

5

(“J

NE

NE

0.01 + 0.01

NE

NE

Aluminium

NB

‘)

0.06 + It.02

(/1 M ,,,x

L:m~h;murn

< I

I

NE

2

(‘%I

(5.1)

(/I M mg

111;~ uklrd.

0. IO * 0.00

0.03 * 0.01

NE

0.01 * 0.00

0.0 I + 0.00

NE

0.03 j: 0.02

(/LM m&t ‘)

Aluminium

was desorhcd)

from

58

NE

8

NE

2

NE

NE

(‘XJ

ro various

the lirsl supernnanc

;md t_-hisGdylglycine

in all washings

0.03 * 0.03

NB

in

34

2x

C;llcium (/LMmg,‘)

ot’ the peptidcs.

10 lm~~hanum. points

*uq~cf~~iou~.

isoelectric

- -

01’ triplicabz

‘L-l’rolyl-L-phenylaI;lnytglycyl-L-lysine.

--

:md

2I rcxwvercd

L-AnyI-LAysine

peplidc

0.08 * 0.03

homoionic

L-aqx~r~ylglycine.

on kaolinire

I5

0.22 * 0.05’
(%)

Sodium

debxted

lJ.12+0.04

26 concenlmlion

0. I6 * 0.02

5 NE

I

20

0.01 fO.OO 0.19~O.tJ1

<
0.01 f 0.00

NE

I

NE

IX

(X.)

ori kaoliniw (X.5)

NE

(pl).


mg

0.28 * 0.00

(I’M

‘)

oentidrs C:dcium

5

21

(‘);)

ot’ various

mg-- ‘)

(/lM

the IOUI

9

2

NB

(?a

pl I <>I‘ lhc clay

are Ihe meim +_ SEM

= IIU hindillg.

lhc buth

I for mdecul;w

.\ tiw

was bound

Triglycine,

by subirxling

“See Table

“Values

ND”

0.13*0.04

‘See ‘I‘uhlc

the peptides

added.

wits calculaled

“Binding

initially

(0.37)

Prphgly’

Hydrogen’

points

the peptidr

0.09 f 0.03

NE

0.05 * 0.00

NE

0.01 * 0.01

0.05 + 0.01

0.12+0.02

rng-.‘)

isoelcwtric

am~lyses.

nnd

(jrM mg-‘)

ot’ lriplicde

(0.88)

(1.51)

mg-I)

Telraglycine

Diglycinc

(jtM

Peptidc

(MW)

sustwnsions.

weights

adsorption.

lhc mean + SEM

= no equilibrium

36

4

5

NE

5

x

7

(%)

(6.2)

adsorntion Sodium

(/IM

3. Euuilibrium

(3.9)’

was calcul~~~ed by sublracGng

molecular

ot’ homoionic

I for

adsorplion

0.13 +0.04

0.02 f 0.00

0.05 f 0.01

NE

0.05 + 0.01

‘L-Prolyl-L-phenylalanylglycyl-Uysine.

‘NE

“V;~lucs

‘Hulk

%ee Table

“Equilibrium

(0.37)

(0.94)

L-Hislidylglycine

Prphgly’

(I .05)

L-Aspartylglycine

(0.8X)

0.09 + 0.02

(I 336)

Triglycine

Tewaglycine

0.1

(I .51)

I kO.07”

(IrMmg-‘)

Hydrogen

Diglycine

(PMmg ‘)

Peptide

22

NI;

NE

NE

I

NE

4

(X

(5.4)

_.

.Adsorption and bindimg of peptides on clays

cause expansion, that the amount of expansion aas below the limit of detection. or that complexes were formed only with the external surfaces of the clay. Regardless of the mechanisms involved. the exchangeable cation on the clays apparently had a greater effect on the adsorption of alan&s than did the pH,. In addition to its greater MW (i.e. almost twice that of the other tetrapeptide used. retragiy). prphg!y possesses moieties that probably contributed to its adsorption, i.e. the phenyl ring of phenylalanine. the imino ring of proline and the terminal amino group of lysine. Aromatic compounds have been shown to form pi-complexes with M (Mortland, 1970: HellerKallai er al., 1972). and therefore, the phenylalanine moiety of prphgiy may have enhanced the adsorption of prphgly, especially on M-Zn and IM-La where it may have been more important than the terminal amino group of lysine. In addition to the formation of pi-complexes, the cationic form of proline or the protonated terminal amino group of lysine may have been involved in cation exchange with the surface of the acidic clays (e.g. M-H, M-Al, K-H and K-Al), and adsorption was thus enhanced. The results of this study showed that the amount of peptide adsorbed and bound varied with the type of clay and with the cation saturating the clay. Peptides with aspartyl, lysyl, phenylalanyl or histidyl moieties adsorbed to the clays used in this study in a manner similar to the adsorption of their respective individual amino acids (Dashman and Stotzky, 1982). These results support the generalization of Weiss (1969) that low MW peptides resemble amino acids in their affinity for clay minerals. The equilibrium adsorption data showed that increasing the chain length of a peptide did not necessarily enhance its adsorption, whereas the addition of functional groups, such as amino or carboxyl, or of histidyl or phenylalanyl moieties did. Peptides containing a heterocyclic amino acid were preferentially bound on M and K homoionic to di- and trivalent cations. Acknowledgement-We thank Miss Karen Schreck for her assistance in preparing the manuscript. REFERENCES

Dashman T. and Stotzky G. (1982)Adsorption and binding

55

of amino acids on homoionic montmorillonite and kaolinite. Soil Biology & Biochemisrrx 14, UXj6. Fripiat J. J.. Cloos P.. Calicis B. and Makay K. (1966) Adsorption of amino acids and peptides by montmorillonite. 11 Identification of adsorbed species and decay products by infrared spectroscopy. Proceedings International Clay Conference. Jerusalem I, X3-232.

Greenland D. J.. Laby R. H. and Quirk J. P. (1962) Adsorption of glycine and its di-. tri-. and tetra-peptides by montmorillonite. Transacrions of fhe Faraday S0cier.t 58, 829-84 1.

Greenland D. J.. Laby R. H. and Quirk J. P. (19658) Adsorption of amino acids and peptides by montmorillonite and illite. Part I. Cation exchange and proton transfer. Transactions of [he Faraday Sociely 61, 2013-2023.

Greenland D. J.. Laby R. H. and Quirk J. P. (1965b) Adsorption of amino acids and peptides by montmorillonite and illite. Part 2. Physical adsorption. Transactions of the Faraday Sociery 61, 2024-2035.

Harter R. D. and Stotzky G. (1971) Formation of clayprotein complexes. Soil Science S0cier.v of America Proceedings 31, 383-389.

Heller-Kallai L., Yariv S. and Reimer IM. (1972) Effect of acidity on adsorption of histidine by montmorillonite. Proceedings 381-393.

International Clay Conference.

.LIadrid

II,

Jang S. D. and Condrate R. A. Jr (1972a) The IR spectra of lysine adsorbed on several cation-substituted montmorillonites. Cla_vs and Clay Minerals 20, 79-82. Jang S. D. and Condrate R. A. Jr (1972b) Infrared spectra of r-alanine adsorbed on Cu-montmorillonitc. Applied Specrroscopy 26, IO2- 104.

Mortland M. M. (1970) Clay-organic complexes and interactions. Advances in Agronomy 22. 75-I 17. Sieskind 0. and Wey R. (1959) Sur l’adsorption d’acides amines par la montmorillonite-H. Influence de la position relative des deux fonctions-NH: et -COOH. Compres Rendus Academic des Sciences, Paris, 248, 1652-1655. Stotzky G. (I 980) Surface interactions between clay minerals and microbes, viruses, and soluble organics, and the probable importance of these interactions to the ecology of microbes in soil. In Microbial Adhesion IO Surfaces (R. C. W. Berkeley, J. M. Lynch, J. Melling. P. R. Rutter and B. Vincent, Eds), pp. 231-249. Ellis Horwood, Chichester. Theng B. K. G. (1974) The Chemistry of CIay-Organic Reactions. Adam Hilger, London. Weiss A. (1969) Organic derivatives of clay minerals, zeolites, and related minerals. In Organic Geochemistry (G. Eglinton and M. T. J. Murphy. Eds). pp. 737-781. Springer, New York.