Physical properties of homoionic montmorillonite and kaolinite complexed with amino acids and peptides

003%0717/85 $3.00+ 0.00 CopyrightQ 1985Pergamon Press Ltd

Soil BioI.Biochem.Vol. 17,No. 2, pp. 189-195,1985 Prmted in Great Britain. All rights reserved

PHYSICAL PROPERTIES OF HOMOIONIC MONTMORILLONITE AND KAOLINITE COMPLEXED WITH AMINO ACIDS AND PEPTIDES T. DASHMAN* and G. STOTZKY Laboratory of Microbial Ecology, Department of Biology, New York University, New York, NY 10003, U.S.A. (Accepted 20 June 1984) Summary-X-ray diffractometry was used to investigate whether various amino acids and peptides expanded montmorillonite (M) homoionic to different cations, and the electrophoretic mobility (EPM) was measured to determine whether the binding of these amino acids and peptides influenced the net surface charge of M and kaolinite (K). Neither aspartic acid, adsorbed at equilibrium at concentrations of 20-250 nMmg-’ M homoionic to Ca or Zn, nor cysteine, at 50-830n~mg-’ M homoionic to Zn or Al, caused expansion, whereas proline, at 90-870 nM mg-’ clay, caused expansion of M homoionic to Ca or Zn, and both proline and arginine, at 60-l 150 nM mg-’ clay, caused expansion of M homoionic to H or Al. The extent of expansion caused by proline and arginine was related to the concentration of the amino acids and the exchangeable cation on the homoionic M. Only M-Al complexed with arginine remained expanded after extensive washing of the clay-amino acid complexes. The expansion of M was apparently dependent on both the type of exchangeable cation on the clay and the characteristics of the amino acid (i.e. the functional groups, dielectric constant increment, dipole moment and concentration). M homoionic to H, Na, Zn, Al or La was not expanded by a series of di- to tetrapeptides bound on these clays and none of the homoionic K-amino acid or peptide complexes was expanded. The EPM of the homoionic M and K and of the corresponding clay-amino acid or peptide complexes was identical, indicating that the concentration of the amino acids or peptides bound was not sufficient to cover enough of the surface of the clays to alter significantly their net charge. The lack of differences in the bulk pH of the clays after binding with amino acids or peptides (with the exception of complexes with argmine) also indicated limited coverage of the clays by these organics.

INTRODUaION Amino acids (Dashman and Stotzky, 1982), peptides (Dashman and Stotzky, 1984) and proteins (Harter and Stotzky, 1971, 1973) have been shown to be adsorbed and bound on montmorillonite (M) and kaolinite (K) homoionic to various cations. These compounds may have been adsorbed on the external surfaces of M and K and also on the interlayer spaces of M. When the complexes of clay and amino acid, peptide or protein were washed until no more amino acid, peptide or protein was desorbed, the compounds were considered to be bound on the clays (Chassin, 1969; Harter and Stotzky, 1971). Gerard and Stotzky (see Stotzky, 1972) showed that some proteins were unavailable as nutrients for microorganisms when bound on M, and Dashman and Stotzky (1985) found that some amino acids and peptides were also unavailable as nutrients for microbes when bound on M or K. One purpose of this study was to determine, by X-ray diffraction (XRD) analysis and electrophoretic mobility (EPM) measurements, whether the amino acids and peptides were located, after equilibrium adsorption and binding, on the external surfaces or in the interlayer *present address: Department of Pharmacokinetics, Biopharmaceuticsand Drug Metabolism, Hoffmann-La Roche Inc., Nutley, NJ 07110, U.S.A.

spaces of the homoionic M. As previous studies have indicated that factors such as the molecular weight of the organic compound and the cation saturating the clay (Barrer and Perry, 1961; Barshad, 1952; Greenland ef al., 1962; Harter and Stotzky, 1971. 1973: Theng, 1974) are important in the adsorption and binding of organic compounds by clays and in their intercalation of M, the role of these factors in interactions between homoionic clays and amino acids and peptides was also studied. MATERIALS AND

METHODS

Wyoming bentonite was obtained from Fisher Chemical Co. and kaolin from Merck & Co. Aspartic acid. free acid (asp), cysteine free base (cys) and arginine. free base (arg) were obtained from Calbiochemical Corp. Proline. free base (pro) was obtained from Eastman Chemical Co. L-NAspartylglycine, L-histidylglycine, glycylglycine (diglycine), glycylglycylglycine (triglycine) and glycylglycylglycylglycine (tetraglycine) were obtained from Schwartz-Mann Co. L-Alanyl-L-lysine and L-prolyl-L-phenylalanylglycyl-L-lysine were obtained from Miles Laboratories. These compounds were used as received, and all other chemicals were of reagent grade. The preparation of the homoionic M and K and of the clay-organic complexes (in unbuffered systems) 189

190

T. DASHMAN and G. STOTZKY

has been described (Dashman and Stotzky, 1982, 1984; Harter and Stotzky, 1971). The amount of clay used was 50mg for the peptide studies and IOOmg for the amino acid studies. Equilibrium adsorption was calculated by subtracting the amount of amino acid or peptide detected in the first supernatant from the amount added. Binding was calculated by subtracting the total amount of amino acid or peptide recovered in all washings (i.e. until no more amino acid or peptide was desorbed) from the amount added. Samples for XRD analysis were prepared by spreading approx. 20mg of a clay-amino acid or clay-peptide complex (after either equilibrium adsorption or binding) over 2.5 x 2.5 cm of a 2.5 x 3.75 cm glass slide. The slides were either dried at ambient temperature and humidity or at 150°C for 48 h in a vacuum oven. The oven-dried samples, while still hot, were placed in a desiccator containing Drierite, and the XRD spectra were determined within several hours after cooling. To avoid rehydration of the oven-dried samples, the scatter shield of the diffraction unit was covered with a plastic film (Saran wrap), and a container of Drierite was placed in the shield (Harter and Stotzky, 1973). Despite this precaution, there was some variability in the amount of rehydration and, therefore, in basal spacings, as a result of differences in the ambient humidity at different times of measurement. Each time that an XRD analysis of a complex was made, a sample of the homoionic clay, washed concurrently and as often as the complex, was analyzed. Although drying the clay-amino acid or clay-peptide complexes at 150°C for 48 h may have altered the amino acids (e.g. oligomerization), all samples were treated uniformly and any effects of heating were assumed to be similar in all systems. Moreover, the relative expansion of complexes dried at ambient temperature or at 150°C were similar. The XRD spectra were obtained with a Norelco diffractometer equipped with a Xenon proportional detector and a pulse height analyzer, using Ni-filtered CuK, radiation at 35 kV and 15 mA. A l/2o-O.OO6 in -l/Z’ slit arrangement was used, and the scans were started at 2” 20, using a scan rate of 1” 20 min-’ (Harter and Stotzky, 1973). The d spacings (in nm) for angles 20 were obtained from standard conversion tables. Only first order reflections are re-

Table

1 Expansion

Ammo acid None Cysteine Proline Arginine

ported, although higher order reflections were evaluated to confirm the validity of the first order reflections. For measurements of the EPM, bound clay-amino acid or clay-peptide complexes were diluted with 4 x distilled water to yield approx. 0.1 mg clay 100 ml-’ water. The EPM was calculated from migration rates of the complexes in an electric field of 200 V in a cell that was 1Ocm long (Zeta Meter, New York). The migration rate ivas determined by following the movement of obliquely-lighted colloidal particles under a binocular microscope at 90 x magnification. At least 20 particles of each sample of clay-amino acid or clay-peptide complex were tracked, and the migration rates were averaged (Harter and Stotzky, 1973). The bulk pH (pH,) of the homoionic clays alone and in complex with the amino acids or peptides was measured, with continuous stirring, with a Photovolt Digicord, Model 120, pH meter. RESULTS

X-ray d$iiaction analysis of cla_v-amino acid and clay-peptide complexes

To evaluate the relative importance of concentration, dipole moment and dielectric constant increment of an amino acid to the expansion of M, M-AI was suspended in solution of amino acids that differed in these characteristics (Table 1). M-AI suspended in pro at a concentration that was 18 x greater than the maximum amount used in the adsorption and binding studies (Dashman and Stotzky, 1982) expanded to 2.10 nm. However, when M-Al was suspended in arg at a concentration that was only 6 x greater than the maximum amount used in the adsorption and binding studies (Dashman and Stotzky, 1982) the d spacing was 2.32 nm. After one water wash of the complexes, the d spacing of M-Al suspended in pro and arg was 1.40 and 1.88 nm, respectively. A concentration of cys that was 5 x greater than that used in the equilibrium adsorption and binding studies (Dashman and Stotzky, 1982) may have resulted in a slightly greater expansion of M-AI (compare Table 1 and 2). There was no reduction in the d spacing of the expanded clay-cys complex after one wash (Table 1).

of montmorillonite homoiomc to aluminum (M-AI) after suspension concentrated solutions of cysteine, proline or argmine

Dipole

Concentration (no mg-’ clay)

Dielectric constant increment”

moment2 (x lO’*esu)

4100 15.600 7200

NA’ 21 62

NA I6 ;4

d spacing

__________ Equhbrlum suspension’

1.22 143 2 10 2.32

“Values from Greenstein and Win& (1961). %Zlay-amino acid complexes dried on slides at amblent temperature ‘M-Al and amino acids were mlxed, and aliquots were taken for X-ray equilibrium adsorption (I.e. not centrlfiged or washed). dThe equilibnum adsorption mixture was centrifuged, the supernatant was suspended in 1.0 ml dlstilled water. Aliquots of the suspension diffractIon analysis. ‘NA = Not avadable

(nm)b

m

~~~

Resuspended suspenslond 1.22 I 43 I 4n 1.88

and humldlty. diffraction analysis after discarded, and the pellet were prepared for X-ray

Physical properties of clay-amino acid/peptide complexes Table 2. Expansion of homoiomc montmonllonites of amino acids Saturating cation H

Amino acid _c Proline

Arginme

Ca

Aspartic acid

Equilibrium concentration (nM mg-‘)

90 220 430 370d 60 290 570 1150 25od

after equilibrium adsorption

Equilibrium adsorption” (nM mg-‘)

A

40 * 1 280 + 1 400*9 360 + 19

1.07 1.07 1.07 1.18 1.36 100 I .oo 1.19 1 32 1 40

0 0 0.11 0.31 0 0 19 0.31 0.40

80 + 5

1.07 1.06

60 + 3 160+8 34Of7 710 + 19

20 + 1 100 * 7 90 + 7 100+7 80 5 1

830d

810 i 1

Proline

90 220 430 870d

30* 1 80 + 2 150*4 370 f 31

Cysteine Proline

830d

150+ 1

90 220 430 870’

30 * 1 80 + 5 80 f 1 loo&4

140 290 570 1150d

140+1 280 + 1 5SOf 1 570 + 1

Zn Asparttc acid Cyst&e

Al

Arginine

d spacmgb (nm)

-

90 220 430 870d 250d

Proline

191

-0.01

1.00 1.oo 1.21 1.21 1.36

0 0.21 0.21 0 36

1.05 1.05

0

1.05 1.02 1.08 1.05 1.25 1.38 1.47

-0.03 -0.03 0.17 0.30 0.37

1.34 1.36 1.19 1.21 1.30 1.38 1.47 1.22 1.34 1.45 145 1.52

0.02 0.02 0.11 0.19 0.28 0.12 0.23 0.23 0.30

“Mean f SEM of tnplicate analyses. bd spacing was detetmmed after drying at 150°C for 48 h in uacuo. ‘Homoionic clays were suspended in a volume of 4 x distilled water equal to that of the amino acid solution. dMaxtmum amount used in the equilibrium adsorptton and binding studies (Dashman and Stotzky, 1982).

Asp, at the concentration used in this study, did not cause an expansion of M-Ca or M-Zn, either after equilibrium adsorption (Table 2) or after binding (Table 3). When the equivalent of 250 nM asp or 220 nM pro was added to 1 mg M-Ca or M-Zn, the amounts adsorbed at equilibrium were similar: 80 nM asp and 100 nM pro mgg’ on M-Ca and 80 nM asp and 80 nM pro mg-’ on M-Zn (Table 2). However, only pro caused an increase in the d spacing of M-Ca and M-Zn. The cyclic structure of pro and the carboxyl groups of asp were probably responsible for expansion or lack of expansion, respectively, of M-Ca and M-Zn. Asp, a dicarboxylic acid, probably formed a chelate with Ca or Zn on the surface of M. The dielectric constant increment of asp and pro is 28 and 21, respectively (Greenstein and Winitz, 1961), indicating that the structure of the amino acids was a major characteristic in the expansion of M-Ca and M-Zn.

Pro, after equilibrium adsorption, expanded M homoionic to H, Ca, Zn or Al (Table 2, Fig. 1). The amount of expansion, as shown by the change in d spacing (A), induced by pro at 870 nM mg-’ clay was in the order of M-Zn = M-Ca > M-H > M-Al, and at 430nMmg-’ clay, it was in the order of M-Zn > M-Ca > M-Al > M-H, even though the at equilibrium order of pro adsorbed was M-H + M-Zn > M-Ca = M-Al and M-H $ MZn > M-Ca > M-Al, respectively (Table 2). These data emphasize the importance of the type of exchangeable cation on the clay in the intercalation of polar organic compounds (Theng, 1974). After extensive washing, the bound M-pro complexes showed no significant expansion (Table 3), indicating that pro was only weakly adsorbed to the interlayer surfaces of the homoionic M. The equilibrium adsorption of arg caused a 0.3 and 0.4 nm increase in the d spacing of M-Al and M-H,

and G. ST~TZKY

T. DASHMAN

192

Table 3. Bulk pH (pHb), electrophoretxc mobility (EPM) and expansion (d spacing) of homoionic montmonilonite (M) and kaohmte (K) and of bound homoiomc clay-ammo acid complexes Homoionic clay

Amino acid

-5 Praline Arginme Asp&c acid Prohne Aspartic acid Cyst&e Proline Arginine Cysteme Prolme Cyst&e

M-H

M-Ca

M--2n

M-AI K-H

K-Zn

Bound’ mgg’

(nM

)

390+2

240+20 -

pHbb 3.3 3.4 6.6

d spacmg” (urn) __1 02

EPM’ (pm -- s-I V-‘cm-‘) -2.6 _t 0.31 -29kO.29 -26F0.33

1.04 1.oo

8.8

-19+025

1.00

8.6 89

-1.710.19 -2.0 * 0.30

1.oo

70

-2.5 + 0 30

69 7.0 7.0

-2.5 * 0 28 -2.5+027 -2.6ItO.31

1.03

5.3 69

- 1 9 F 0.28 -21+020

I .22 1.40

5.3 5.4 54

-2 420.30 -2.3 * 0 28 -24+027

0.72 0.71 0.73

-

6.6

2IOf28

6.5

-2.4*029 -2.5 k 0.30

0 73 0 13

70 i 3 50f 12

1052 660 f 74 2.50+3 46Oi3 160+8 lo& 1

I 00 1.03 1.00 100

‘Mcan i SEM of triplicate anaiyses. bBulk pH of the homoionic clays and the complexes CMean + SEM of the migratmn of 20 particles. “d spa&g was determmed after drymg at 150°C for 48 h in wcuo. %omoionic clays were washed as many times as the clay-ammo and

respectively, even though Iess arg was adsorbed on M-H than on M-AI when 1150 IIM mg-’ arg was added (Table 2). In contrast, the order of adsorption of 430 nM pro mg-’ clay was M-H $ M-AI, but the order of expansion was M-Al > M-H. These data again emphasize the importance of the exchangeable cation and the structure of the amino acid in the intercalation of M by amino acids. If the exchangeable cation had no role in the mechanism of expansion, then both the order of expansion and of adsorption would probably have been M-H > M-Al. As the concentration of arg was increased from 570 to 1150 IIM mg-’ M-Al, there was no significant increase in the amount adsorbed (Table 2), suggesting that the increase of 0.07nm in d spacing (Fig. 2) was the result of the increased dielectric increment or the reorientation of arg rather than of the formation of a second layer of arg. Although the d spacing of M-H increased as the concentration of arg was increased

complexes

(Fig. 2), this expansion was probably also not the result of the formation of multilayers, as the amounts of arg adsorbed at 570 and 1150 nM mg-’ clay were not significantly different (Table 2). Therefore, this increase in d spacing by arg may also have been the result of the increase in the dielectric constant increment or the rearrangement of the amino acid in the interlayer space. The bound M-Al-arg complex was the only complex of the clay-amino acid systems studied that remained expanded after extensive washing. None of the amino acids remained bound to M homoionic to Na or La after extensive washing (Dashman and Stotzky, 1982). Argmne

15

l /

3.4

/

”

13

tk

Probe

/------’

.-•

XIX M-AL

$

14

l

% b i3

i ;~i.i_i/;!;H 090 430 220 Equlllbrlum

I

870

090 430 220

M-H

11

1 ;l;i_,. 87O--

concentratron (nM mg-’ cloy)

Fig. 1. Changes in basal spacing with increasing amounts of proiine added to M-H, M-Ca, M-Zn or M-AI.

KJ-( 50

’

300

I

I

570

fl50

Equihbwm concentrotlon (nM mg-‘clay

)

Fig. 2. Changes in basal spacing with increasing amounts of arginine added to M-H or M-Al (@----a) and of arginine bound on M-AI ( x __ x ).

Physical

properties

Table 4. Bulk pH (pH,), homotonic montmorillonite

M-Al

M-La

K-Na

K-Ca

K-Zn

-2.6 i: 0.29 - 2 4 & 0.30 -2.5 + 0.28 -2.6 + 0.27

1.02 1.01 1.00 1.02

6.1 6.1 61

-2.8 k 0.27 -29*030 -2.9 +_0.25

1.oo 101 I 02

7.0 7.0 70

-2.5 + 0.22 -2.5 k 0 25 -2.3 5 0.23

I .oo

5.3 5.3 5.3

-1.9*0.15 -1.8+0.18 -2.0 + 0.19

1.20 1.21 1.22

64 6.4

-1.6+0.16 -1.5+0.18

1.15 1.17

5.3 5.0 5.1

- 2.4 + 0.25 -24TO.27 -2.5+023

0.72 0.71 0 72

53 0 34

6.2 6.2 6.2 6.3

-23kO.22 -2.3 + 0.24 -24kO.21 -2.3 +0.19

0.70 0 70 0.71 0 70

12 33

8.1 8.0 81

-2.4 ? 0.23 -2.5 +O 26 -24kO.25

0.70 0.71 0.71

19

6.6 6.6

-2.5 f 0.24 -2 5 kO.26

071 0.72

5.5 5.5

-2.5 &-0.30 -2.5+028

0.71 0 70

160+30 40*7 100 + 8

L-Histtdylglycine Prphgly t_-Histtdylglycme Prphgly L-Histidylglycme Prphgty Prphgty

K-H

3.3 33 3.4 33

_e

L-Aspartylglycine L-Alanyl-t_-lysine Prphgly’

M-Zn

Tetraglycine Prphgty Diglycine Tetraglycme Prphgly Diglycme Prphgly Prphgly

K-Al

bkb

193

d spacingd

Bound” (nM mg-‘)

M-Na

complexes

EPM’ (rem s-’ V-’ cm-‘)

Peptide

M-H

acidipeptide

electrophoretic mobility (EPM) and expansion (d spacing) of (M) and kaolinite (K) and of bound homoiomc clay-peptide complexes

Homoionic Clay

of clay-amino

40+7 20 + 20 270 f 32 150+8 410 + 18 180+28 50+21 10 + 20 30 f 30 220 + 0+ 80 + 230 f 40 + SO*

80 + 2

~IH,.~

(nm)

1.00 1.01

‘Mean + SEM of triphcate analyses. ‘Bulk pH of the homoiomc clay and the complexes. ‘Mean t SEM of the migration of 20 partmles. “d spacing was determined after drying at 150°C for 48 h m VLICUO. ‘Homotonic clays were washed as many times as the clay-pepude complexes rt,-Prolyl-r.-phenylalanylg,lycyl-t-lysine.

Glycine peptides adsorbed at equilibrium have been shown to form single- and double-layer complexes with M-H and M-Na (Chassin, 1969; Fripiat et al., 1966; Greenland et al., 1962). Only the bound complexes between M and the glycine and other peptides used in this study were analyzed by XRD. None of the peptides expanded M (Table 4), suggesting that any peptides that intercalated M during equilibrium adsorption were desorbed by the washings used to prepare the bound complexes. Consequently, the mechanisms of binding of peptides on the surfaces of the clays were probably different from those involved in their adsorption in the interlayer spaces of the clays (Dashman and Stotzky, 1984). K was not expanded by any of the amino acids or peptides that were adsorbed or bound on this clay (Tables 3 and 4). K was only intercalated under pressures ranging from 1 to 15 kbars (e.g. Range et al., 1969). Electrophoretic mobility (EPM) and pH, of bound clay-amino acid or clay-peptide complexes

There were no significant differences in EPM between clays complexed with amino acids or peptides and the comparable non-complexed homoionic M and K washed as many times (ranging from 10 to 46

washings, depending on the clay-amino acid or clay-peptide complex) with the same volume of water as the complexes (Tables 3 and 4). The absence of an effect of the bound amino acids or peptides on the EPM of the clays was reflected in the general lack of difference between the pH, of the clay-amino acid or clay-peptide complexes and the pH, of the comparable non-complexed homoionic clays (Tables 3 and 4). Only arg caused a significant increase in the pH, of M-H and M-Al, but this was not accompanied by changes in EPM. Apparently, the amount of arg bound was sufficient to alter the pH, but not the EPM nor, with the exception of the M-Al-arg complex, the expansion of the clay (Fig. 2). After extensive washing, the EPM of M-H, M-Ca, M-Zn and M-Al did not change significantly (Table 5), suggesting that leaching of cations from the clays had not occurred, as had been shown with K saturated with inorganic ions (Buchanan and Oppenheim, 1968). However, the pHs of M-H increased from 2.4 to 3.3, that of M-Al increased from 4.5 to 5.3 and that of M-Ca increased from 8.3 to 8.8 (the increase in the pH, of M-Zn was not significant) (Table 5), indicating that although their net charge was unaffected by the washings, there was a decrease in the number of protons on these clays.

T. DASHMAN and G. STOTZKY

194 Table 5 Bulk pH (pH,)

and electrophoretlc mobility (EPM) of washed and unwashed montmorillonite Washed

Not washed Saturating catlon

PH,

H Ca Zn Al

2.4 8.3 6.8 45

EPM” (pms-‘V-‘cm-‘) -2.9 * 0.21 -2.oio 19 -2.5 + 0.24 -1.9+0.19

homoiomc

Number of washing&’ 10 5 11 9

EPM* (~ms~‘V~‘crn~‘) pH, ____.____ 3.3 -2.6 + 0 29 8.8 - 1.9 + 0 25 7.0 -2.5 + 0 23 53 -1 9io.15

‘Mean & SEM of the migration of 20 particles. blOO mg clay washed in S ml of 4 x distilled water for each washmg.

DISCUSSION

The expansion of M is dependent on the amount and type of exchangeable cations, the solvent (magnitude of the dipole moment) and the solute (dipole moment, dielectric constant increment and concentration) (Barrer and Perry, 1961; Barshad, 1952; Greenland et al., 1962; Theng, 1974). When M-Al was suspended in pro and arg at concentrations that were 18 and 6 x greater, respectively, than those used in the adsorption and binding studies (Dashman and Stotzky, 1982), arg caused a greater expansion of M-Al than did pro (Table 1). As the dipole moment and dielectric constant increment of arg are greater than those of pro, these characteristics of the amino acids were apparently more important than their concentration in the expansion of M-Al. Pro caused an expansion of M-Ca and M-Zn, whereas asp (as the free acid) adsorbed at comparable concentrations did not (Table 2), indicating that, at least at these concentrations, the structure (i.e. the ring imino moiety) of pro rather than the COOH groups of asp may have been a factor in its ability to expand M. Also, if the dielectric increment constant of asp was more important than that of pro in the expansion of M-Ca and M-Zn, then asp, which has a greater dielectric increment than pro, should have caused expansion of M-Ca and M-Zn. The effects of concentration and use of either the salt or the free acid or base of an amino acid on expansion are illustrated by comparing the results of this study and those of Greenland ef al. (1965), who reported that asp. HCl, at a concentration of asp that was only about 2x that used in this study (460 nM mg-’ clay), caused an expansion of M-Ca. The dissociated HCl apparently contributed to the acidity and dipole moment of the solution and, thus, enhanced the expansion of the clay. In contrast, Heller-Kallai et al. (1972) reported that M homoionic to Ca or Cu was expanded more by histidine. free base than by histidine . HCl, presumably as a result of the greater adsorption of histidine. free base and of different interlayer associations with the two types of histidine. Another example of the effects of concentration and type of amino acid on the expansion of M was the expansion of M-Al by the sulfhydryl amino acid, cys. At a high concentration, cys caused the expansion of M-Al, but at a lower concentration, which was not significantly different from that of pro (830 and 870 nMmg-’ clay, respectively), there was no significant expansion of M-Al or M-Zn after equilibrium adsorption (Table 2). Either this concentration of cys was too low to cause expansion, the

amount of expansion was below the limit of detection or complexes were formed only with the external surfaces of M-Al and M-Zn. Greene-Kelly (1956a, b) showed that saturated ring compounds were intercalated either parallel or perpendicular to the silicate surfaces. The increase in d spacing concomitant with increased adsorption of pro on M-H and M-Zn (Fig. 1, Table 2) suggests that pro was intercalated parallel to the silicate surfaces and that the dielectric increment of pro enhanced expansion. Because there was no significant increase in adsorption of pro on M-Al as the concentration of pro was increased from 220 to 870nMmgg’, the increased d spacing probably resulted from factors other than adsorption. The break in the d spacing curve of M-Ca-pro suggests the possibility that pro may initially have been oriented parallel to the interlayer silicate surfaces of M-Ca, but that at the highest concentration, it was oriented perpendicular to the silicate surfaces. This possibility was suggested by the lack of difference in the amounts of pro adsorbed over the equilibrium concentration range of 220 to 870nMmg-’ M-Ca, whereas there was an increase of 0.15 nm in d spacing at 870 nM rng-’ over that at 430 nM mg-’ (Fig. 1, Table 2). Jordan et al. (1950) suggested that the first plateau in the basal spacing curve of M as a function of increasing amounts of the Cl8 ion, n-octadecyl ammonium, was probably the result of the formation of parallel monolayers and that the second plateau was the result of the formation of parallel double layers. The plateau in the d spacing curve of M-Al-arg (Fig. 2) suggests that arg filled the interlayer space as a monolayer. The increase in d spacing of M-Al as the concentration of arg was increased from 570 to 1150nMmg-’ was apparently not caused by the formation of a second layer of arg, because there was no significant increase in adsorption over this concentration range. The increase in expansion may have been the result of a change in the orientation of arg from a planar to a twisted configuration. Harter and Stotzky (1973) reported changes in the EPM of some homoionic M-protein complexes, even though the amounts of proteins added (0.4 to 117.8 I’tM mg-’ clay) were, in general, considerably less than the amounts of amino acids or peptides used in this study. They suggested that the proteins, although possibly shielding or masking some negatively-charged sites on the clays because of their large size, disrupted tactoids during adsorption, thereby exposing more negatively-charged sites on the clays, and that the ionizable groups of the pro-

Physical properties of clay-am lino acidlpeptide complexes

teins contributed to the EPM. As the smaller amino acids and peptides may not have been able to break tactoids, covered less of the surface of the clays and the number of ionizable groups on the amino acids and peptides was considerably less than on the proteins, the amino acids and peptides were not able to influence the EPM. Our results showed that the expansion of several homoionic M by amino acids was dependent on the valence of the exchangeable cation on the clay and on the concentration, dielectric constant increment, dipole moment and functional groups of several of the amino acids. Some amino acids with different dielectric constant increments were able or unable to expand the same or different homoionic M. For example, asp, a dicarboxylic amino acid, did not expand M-Ca or M-Zn, whereas pro, an imino acid, caused the expansion of these clays. Cys, a thiolcontaining amino acid, did not expand M-H or M-Al, whereas arg expanded these clays. Peptides bound to M did not cause any discernible expansion, probably as a result of the small amounts complexed and of their functional moieties (e.g. the oxygen of the peptide bond, terminal amino or carboxyl groups). For example, the amount of histidylglycine and arg bound on M-Al was 400 and 460 nM mg-‘, respectively, but only arg caused expansion, indicating again that the characteristics, such as conformation or ring structures, of the organic compound are important in expanding M. The net surface charge of the clays and the corresponding clay-amino acid or peptide complexes was the same, as was the pH,, with the exception of complexes with arg, suggesting that the concentrations of the organic molecules bound on the clays were not sufficient to cover enough of the surface of the clays to alter their net surface charge and pHb. The relevance of these observations to the utilization of amino acids and peptides bound on the surface of M or K or intercalated in M as nutrient and energy sources by microorganisms has been discussed (Dashman and Stotzky, 1985). Acknowledgements-We

thank Miss Karen Schreck for assistance in preparing this manuscript.

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