Indian Journal of Biochemistry & Biophysics Vol. 37, October 2000, pp. 341-346

Adsorption of glycine and alanine on montmorillonite with or without coordinated divalent cations

Sippy Kalra, C K Pant, H D Pathak* and M S Mehtat

*Department of Chemistry and toepartment of Physics, D S B Campus, Kumaon University, Nainital 263 002

Received 20 January 1 999; revised 13 August 1999

Adsorption of glycine and alanine on montmorillonite and on Ca2+- and Mg2+- exchanged montmorillonite clay over a range of pH (4.0-9.0) and temperature (20-35"C) under possible abiotic conditions have been investigated UV­spectrophotometrically. Adsorption of both the amino acids was considerable on all the three adsorbents used. Maximum adsorption was observed at 25 <>C and neutral pH. ci+-montmorillonite exhibited relatively better adsorption as compared to Mg2+-exchanged form or montmorillonite. The values of KL and Xm were determined using Langmuir isotherm characteri stics.

Ever since Bernal' suggested that clay surfaces might have played a significant prebiotic role in selection and concentration through adsorption processes of abiotically formed biomonomers as well as protection against hydrolytic fission in remote prebiotic envi­ronments, a new era of investigations opened up in the field of chemical evolution2

·9

. It has been empha­sized that clays and metal ions were most widely spread over the earth's surface, at the site of sea shores and at the bottom of the sea before and during prebiotic and organic evolution, presumably after the formation of early hydrosphere 10

-12

. In early prebiotic times, the earth's hydrocarbon, ammonia and water vapours laden environment was acted upon by violent lightning thunders, electric discharges, ionizing ra­diations, volcanic heat and vast amount of UV -flux of solar radiations as there was no ozone layer to cut it off13

-20

. During the period of intense activity of high energy radiations on the heavy blanket of CH4 • NH3

and H20, vapours wrapped the earth, large amount of biomonomers formed might have been trapped by clays, metal ion coordinated clays and silica through d . d d . 2 1"22 u d fl . a sorption an con ensat1on . n er uctuatmg

dehydrating periods of wetting and drying cycles they gave rise to large biomolecules like protein and nu­cleic acid polymers. Clays might have acted as natu­ral ion exchangers and adsorption of amino acids on them led to the formation of amino acid metal com­plexes . Recently, work of Lawless and Edelson23 on adsorption and clay initiated synthesis reported by

* Author to whom correspondence may be addressed.

Ferris and co-workers24-27 has shown that montmoril­

lonite works as rrii.dticatalytic system in the initiation of reaction of prebiotic significance. Probably clay and mineral sedimentation occurred far and wide across the earth along with adsorption of building blocks of biopolymers.

In our search to retrace the probable abiotic path­ways of chemical evolution, occurrence of several amino acids as chemical fossils either of extinct or­ganisms or geochemically formed peptides and pro­teins in the stromatolites formed from the organic matter of biogenic origin, probably 1350-950 million years ago from now, has been reported28

. This has provided evidence of existence of ancient sediments possessing adsorbed biomonomers. Kamaluddin and

k 29"10 h d h d . f . co-war ers · ave reporte t e a sorptiOn o ammo acids and nucleotides on transition metal ferrocya­nides as possible adsorbents in primitive lifeless era.

Apart from the efficient adsorption and catalytic capabilities shown by the clays in the process of amino acid and nucleotide oligomerization, a few

k 11 -12 h I d h b. . wor ers· - ave a so propose t at pre Jotlc reac-tions might have occurred on the surfaces of water soluble minerals during dehydrating periods of wet­ting and drying cycles of a fluctuating environment. Bodenheimer and coworkers3

', Hedges and Hare34

have investigated the adsorption of amino acids on clays and Cu-ion exchanged clays. Lawless and Levi35 were able to synthesize peptides by adsorption of monomeric building blocks using clays with diva­lent coordinated cations.As a remarkable increase in the catalytic activity has been noticed with clays ex-

342 INDIAN J. BIOCHEM. BIOPHYS., VOL. 37, OCTOBER 2000

chang.t?d with divalent metal ions, the metal ions with clays ipve been suggested as natural mineral matrices capable of adsorbing monomeric biomolecules and releasl?g the resulting products back to aqueous me­diumijl order to preserve their catalytic activity.

Ad~Jrption of amino acids under a range of pH, temperature and concentration on montmorillonite with or without Ca2+- and Mg2+ cations was investi­gated using the Langmuir characteristics36 of glycine and alanine and the results are reported in the present communication.

Materials and Methods The montmorillonite was from Aldrich Chemical

Co. All other chemicals used were of analytical grade. For preparation of solutions/ reagents and ex­perimentation, deionised water was used after double distillation in an all glass assembly. All containers, measuring equipments and assemblies were of boro­silicate glass. A Jasco, V-550, UVIVIS spectropho­tometer was used for determination of absorbance of the amino acids.

Montmorillonite was repeatedly washed with dis­tilled water, its aqueous suspension was centrifuged and the solid material thus isolated was dried in an oven at 35°C. Ca2+- and Mg2+-exchanged forms of the clay were prepared37 by saturation of montmorillo­nite separately with calcium chloride and magnesium chloride and excess of the salts were ]eached out by washing with distilled water until it was freed of chloride ion. The divalent cation exchanged forms thus obtained were dried at room temperature and kept in vacuum desiccator. Before use, all the glass­ware after through cleaning, washing and drying was autoclaved alongwith double distilled water at 20 lbs/sq. inch steam pressure for 20 min . Portions of montmorillonite and its Ca2+- and Mg2+-exchanged forms (50 mg each) were used in each investigation.

The adsorption of amino acids on clay or cation exchanged clays in aqueous medium was studied as a function of pH and concentration of adsorbate. There­fore, adsorption of glycine and/or alanine in varying concentration (7 .0 x I o·5 M - 0.5 x I o-5 M) over a pH range 3.6-5.5 and 6.8-9.2) was studied in order to obtain saturation point by adding relevant buffer to the amino acid solution (5 ml) containing mont­morillonite/Ca or Mg-montrnorillonite (50 mg) keep­ing in mind that the buffer should be a very poor li­gand so that stable complex formation with clay could be avoided . Acetate buffer (0.2 N acetic acid and 2 N

sodium acetate) and borax buffer (0.2 M boric acid and 0.05 M borax) were used to maintain pH in the range 3.6-5 .5 and 6.8-9.2 respectively. This was veri­fied by conductivity measurements as no change in the inflexion point of buffer with or without any type of clay was noticed . The inflexion point was deter­mined by titrating buffer against KaOH. Glycine and alanine concentration in solution was determined UV !VIS spectrophotometrically after adding clay to it.

Buffered solutions of glycine and alanine (5 ml) at different pH were added to montmorillonite, Ca-and Mg- montmorillonite kept in separate conical flasks (50 ml), stirred mechanically for 20 min. and allowed to stand at room temperature for 8 hrs. Similar sets of different concentrations of glycine and alanine with different adsorbents were incubated over a tempera­ture range 20° to 35°C for varying periods and pH to find out the conditions of maximum adsorption. The study of amino acid adsorption as a function of tem­perature showed maximum adsorption at 25°C. After about 8 hrs the solutions were centrifuged at 3000 rpm for 15 min. Then supernatant was decanted leaving the clay as residue and the pH of the liquid was found unchanged . The suspen3ions were used for a separate study on the formation of peptides under wetting and drying cycles for various periods. The concentrations of glycine and alanine in the super­natant were determined by recording their absorban­

ces at Amax (Amax Gly=202 nm and Amax Ala=l97.6 nm). The amount of amino acid adsorbed under dif­ferent colflditions was calculated from the difference between the initial amino acid concentration and after adsorption in each case. The equilibrium concentra­tion of amino acids and the amount adsorbed were used to obtain the adsorption isotherms25

.

Results lllnd Discussion The adsorption of glycine and alanine on mont­

morillonite and on Ca2+- and Mg2+ -co-ordinated montmorillonite have been studied as a function of pH ( 4.0-9.0) and temperature (2:5" to 35°C) These data should help to find out the possible catalytic role of alumino-silicate and cation exchanged clays in the abiotic formation, adsorption of amino acids and their subsequent linking to larger biopolymers under con­ditions comparable to those which might have existed over clay, silica and mineral deposits at the bottom of sea, on sea-shores and under dehydrating-wetting cy­cles of environment.

KALRA eta/.: ADSORPTION OF GLYCINE AND ALANINE ON MONTMORILLONITE 343

Table !-Percent binding and Langmuir constants of glycine and alanine on montmorillonite clay with or without divalent cation

Type of clay

Montmorillonite Ca2

+- Montmorillonite Mg2

+- Montmorillonite

100

Binding (%) Glycine Al anine

80.0 95.7 83.3

42.9 56.3 52.9

~Q 90

a 80 E 70

.... -·-·-.il::r.::::::: ::::::::::::::::: · :a~~ ....... ~

I 80

I 60 40

f 30

3 20

10

0

.......

5 5.5 6 6.5 7 7.5 8 6.5 9 9.5 pH

Fig. !-Adsorption of glycine on montmorillonite clay (-0 -) and montmorillonite with Mg2

+ (-D-) and with Ca2+ (-to.-) as

a function of pH ; temp 25°C.

80~--------------------------------~

"en 70

~ 60

i 50

~40 ::i!30

~ 20

8 10

~~::~=: ::=: == :~ ==~= ::;: : = :::~_: ::' ..

5 5.5 6 6.5 7 7.5 8 6.5 9 9.5 pH

Fig. 2-Adsorption of alanine on montmorillonite clay (-0-) and montmorillonite with Mg2

+ (-0-) and with Ca2+ (-to.-) as

a function of pH; temp 25°C.

Our pre liminary studies show that the amount of amino acids adsorbed is dependent on the pH of the solution and it was maximum at 25°C and neutral pH in all studies. Subsequent studies were therefore car­ried out at pH 7 .0 . Neutral pH is phys iologically of great value as biochemical reactions in living systems take place in neutral medium. The variation in the amount of amino ac id adsorbed with varying pH on different adsorbents have been illustratred in F igs . 1 and 2. It is seen that glyc ine and alanine show in­crease in adsorption at pH 7.0 and uptake decreases with increase of pH of the solution. The isoelectric point of glycine and alanine are 5.97 and 6.1 respec­tively . Their large adsorption at pH above isoelectric

59.9 90.9 73.0

Glycine Langmuir Constants

24.6 12.5 15.2

54. 1 69 .4 66.7

Alanine

11.9 11.8 11.3

point shows that adsorption of amino acids is in their anionic form. However, interaction of clay and metal ion co-ordinated clays with biomonomers is depend­ent on other factors also like amino and carboxyl groups as sites for interaction with clay surfaces. In acidic medium glycine and alanine occur as cation and as neutrality approaches they acquire the zwitte­rion form. Percent binding of glycine and alanine are shown in Table 1. This has been calculated from opti­cal densities of glycine and alanine solution before and after adsorption corresponding to saturation point on the curves. It was found that additional incubation time did not appreciably change the degree of ad­sorption . The adsorption isotherms (Figs 3 and 4) and Langmuir adsorption isotherms (Figs 5 and 6) show that the amount of amino acids adsorbed increase in their equi librium concentration in solution upto a certain limit i.e. saturation point. Above this adsorp­tion became independent of glycine or alanine con­centration . The asymptotic nature of the curves (Figs 3 and 4) suggest Langmuir type adsorption . Initially the plots show linear relationship between amounts adsorbed and equilibrium concentration of amino acid whereas at higher concentration saturation point oc­curs and no more amino acid is adsorbed on mont­morillonite surface. The adsorption of both the amino ac ids on clays with or without metal ions follows Langmuir type of adsorption as outlined below:

Where C eq is equilibrium concentration of glycine or alanine, KL is constant re lated to energy i.e. enthalpy

f d . ff" . (K .c, HIRT) X . o a sorptton coe tc tent L oc e ; c ts amount of amino acid (mg) adsorbed per gram of absorbent i.e. montmorillonite or ci+ -and Mg2

+- exchanged forms; Xm is the amount of glyc ine or a lanine re­quired for the weight of montmorillonite for forming a complete mono layer on the surface. The adsorption parameters Xm and KL were calculated form the slope

34+ }NDIAN J. BIOCHEM. BIOPHYS., VOL. 37, OCfOBER 2000

80~--------------------------------~

~ 70

reo 1: ~30

1:

12~---------------------------------,

10

:., r 8

'b 8 .... J(

~ 4 u

2

5 6.5 8 6.5 7 7.5 8 8.5 9 9.5 0 +-"r-"-'"T"""""T"""""T""~--r-..--..--.-..---r--r---r-"""T""--r--i

pH

Fig. 3--Adsorption of glycine on montmorillonite clay (-0-) and montmorillonite with Mg2

+ (-[J-) and with Ca2+ (-~-) pH 7.02; temp 25°C.

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7

Equilibrium cone. of alanine (x 10 .. Ill)

Fig. 4--Adsorption of alanine on montmorillonite clay (-0-) and montmorillonite with Mg2

+ (-D-) and with Ca2+ (-D.-),

pH 7.02; temp 25°C.

and intercept obtained from the graph of Ceq I Xe ver­sus Ceq. These values for glycine and alanine are summarised in Table I. The value of Xm can be cal­culated asymptotically from Figs 3 and 4 on extrapo­lating the adsorption curve towards Y-axis when satu­ration occurs. It has been observed that adsorption trend (% of binding, Table 1) of both glycine and ala­nine on the adsorbants used, though differ in percent binding from each other, was in the following order.

Ca-montmorillonite > Mg-montmorillonite > mont­morillonite

Xm values recorded in Table I obtained from Langmuir adsorption isotherms for both amino acids though differ among themselves, show the identical trend as given below:

Ca .. montmorillonite > Mg-montmorillonite > mont­morillonite

Addition of Ca2+ and Mg2+ increases the adsorption

of both the amino acids throughout the entire con­centration range of the isotherms. This shows that added divalent metal ion first gets involved in com-

0.5 1 1.5 2 2.5 3 3.5 4 4.5 tl 5.5 6 6.5 7 7.5 8 EqulllbtMn cone. ot glycine (X 1 O"' Ill}

Fig. 5-Langmuir adsorption for glycine on montmorillonite clay (-0-) and on montmorillonite with Mg2

+ (-D-) and with Ca2

+ (-~-). pH 7.02; temp 25°C.

14r----------------------------------,

12

:., 10

r s 1:. -; 6

5 4

2

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 Equilibrium cone. of •Lantne (x 1 ~ M)

Fig. 6-Langmuir adsorption for alanine on montmorillonite clay (-0-) and on montmorillonite with Mg2

+ (-D-) and with Ca2+ (-~-).pH 7.02; temp 25°C.

plex formation with glycine/ alanine and then amino acids get adsorbed on clay surface or- the increase in ionic strength of clay surface lowers the repulsive force between the amino acid and the clay surface or that both these mechanisms may operate. In normal condition in montmorillonite structure [(Al-Mgh OH2]'

1• [(Na-Ca)n H20 ]'1 ,the lattice silica and alumina

lC).yers appear to be sandwiched together with some vacant sites . The adsorption of amino acids most likely proceeded via carboxyl and the amino group is either hydrogen bonded to the structural oxygen or as such are tightly fixed with silica structure. The car­boxyl grolllps are generally fixed with positively charged Al-oxy-hydroxy element by means of ionic bridges. The surface of montmorillonite is entirely comprised of si lica and in interlayer position where oxygen colllfront each other, metal cations and polar molecules are located causing lattice expansion. Montmorillonite is thus capable of forming intercala-

KALRA et al.: ADSORPTION OF GLYCINE AND ALANINE ON MONTMORILLONITE 345

tions with amino acid which forms the basis of ad­sorption property of clays. Earlier Egon T. Degens and Johann Matheja38 have also drawn similar con­clusions relating to montmorillonites and kaolinite's more effective adsorption behaviour. Lawless and Edelson 16 explained the value of Xm as a measure of the accessibility of adsorption sites and KL as a meas­ure of the affinity of clay for adsorbate. The values of KL for glycine and alanine on montmorillonite, Ca2

+­

and Mg2+~ coordinated montmorillonite (Table I) are related to the enthalpy of adsorption. This observation shows that added metal ion increases the ionic strength of montmorillonite surface and thus lowers the repulsive forces between organic molecule and clay surface so that a lower value of KL is observed when adsorbent is exchanged with divalent cation.

Conclusion The adsorption of glycine and alanine on mint­

morillonite with or without metal ion subsitution is maximum at neutral pH and 25°C.These values are of physiological importance as they also correspond to the ideal conditions for various biochemical reactions in living systems.

The adsorption of both the amino acids on all the three forms of montmorillonite follows Langmuir type of adsorption, which clearly indicate monolayer formation of adsorbates (i .e, glycine and alanine) on clay surface. The percent binding and Xm values for both glycine and alanine on the clays depend on the type of metal ion substitution. Results reported in this communication show that Ca2

+- exchanged montmor­rilonite clay has better adsorption properties as com­pared to Mg2

+- exchanged form and montmorillonite without cation. Further investigations on adsorption of short peptides of glycine and alanine on mont­morillonite clay with or without divalent cations may be considered based on the aforesaid studies .

Acknowledgement The authors are grateful to Dr C S Mathela, De­

partment of Chemistry, Kumaon University, Nainital for providing necessary research . Facilities ; to Dr H B Tripathi , Department of Physics of this University, for allowing us to carry out UV -spectrophotometric studies. Authors gratefully acknowledge the financial support given by the University Grants Commission New Delhi, in carrying the research work .

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