A D S O R P T I O N OF P R O T E I N S , E N Z Y M E S
AND ANTIBIOTICS BY MONTMORILLONITE
by
L o u i s A. PINCK
Soil and Water Conservation Research Division,
Agricultural Research Service, U.S. Department of Agriculture,
Beltsville, Maryland
ABSTRACT
Proteins interact with montmorillonite forming mono- and poly-layer complexes. About
20 percent of the protein in a monolayer complex undergoes microbial decomposition and
the X-ray pattern remains unchanged, whereas in polylayer complexes the protein undergoes
extensive decomposition and the c-spacings of these complexes shrink from approximately
30 • to 12/~+. Urease is adsorbed completely by H-montmorillonite and only partially
by basic montmorillonite. Initial release of urease from the clay is attributed to urea acting
as a cation. Subsequently the ammonia evolved by the hydrolysis of urea becomes the active
cation. Antibiotic-montmorillonite complexes are classified into three groups. Group I
contains strongly basic antibiotics, II amphoteric, and III acid or neutral. The average
adsorption of antibiotic in mg per g of clay for each group is: I, 186; II, 307; and III, 9.
X-ray diffraction data for groups I and II showed expansion of the c-spacing of 4.4 and 7.6 A,
respectively. Bioassays showed no activity for I and appreciable activity for II. The complexes are incapable of diffusing through agar, but the antibiotics must be released first
by cationic exchange and then diffuse through the agar.
INTRODUCTION
Soil fertility is due i n p a r t to three i m p o r t a n t c o n s t i t u e n t s of the soil;
clays, humus, a n d microorganisms. I t is desirable n o t only to k n o w the
f u n c t i o n of each c o n s t i t u e n t b u t also how t h e y get along with one another.
I n general, this is the m a i n objective of this investigation. Here, a few words
a b o u t h u m u s a n d microorganisms m a y be appropriate. H u m u s is a heterogeneous conglomeration of organic substances derived from plants, animals
a n d microbes. The organic compounds selected for this s t u d y are f o u n d i n
the soft a n d m a n y of t h e m are related chemically b y being proteinaceous.
The soil swarms with microorganisms. A spoonful of rich soil contains more
microorganisms t h a n the t o t a l h u m a n p o p u l a t i o n on the earth. U n d e r
favorable conditions of t e m p e r a t u r e , moisture a n d n u t r i e n t s t h e y m u l t i p l y
very rapidly. As a result of their growth t h e y produce metabolic b y - p r o d u c t s
a m o n g which are a n t i b i o t i c substances. Their m e t a b o l i s m is affected b y
520
ADSORPTION OF PROTEINS
521
a large variety of enzymes, which they possess. B y enzyme action these
organisms decompose certain types of organic matter for growth and energy.
In a laboratory study on the effect o f clays on the retention of organic
matter during humification, it was found that montmorillonite exerted a
marked effect in holding carbon, the increase due to addition of 10 percent
montmorillonite to sand being nearly twofold in certain cases (Allison,
Sherman and Pinck, 1949). In order to elucidate the significance of the
above finding, a number of fundamentalresearches on organic-clay complexes
were carried out. The organic compounds studied were two proteins, the
enzyme urease, and ten antibiotics. The objectives of this investigation
deal with (a) the extent of adsorption and release of the above-named
groups of organic compounds by and from montmorillonite, and (b) the
physico-chemical properties of the organic-clay complexes. In a few cases,
the data will be compared with those of organic-kaolinite complexes.
Since this paper is confined to studies carried on at the Plant Industry
Station in Beltsville, Maryland, a review of the literature is omitted, but
for those interested in previous work done by others, a few pertinent references are cited: for protein adsorption by montmorillonite (Ensminger and
Gieseking, 1939, 1941, 1942; Talibudeen, 1950), for urease in soils (Conrad,
1940, 1942 ; ttofmann and Schmidt, 1953), and for antibiotics in soils (Gottlieb and Siminoff, 1952; Gottlieb, Siminoff and Martin, 1952; Martin and
Gottlieb, 1952, 1955; Siminoff and Gottlieb, ]951).
ACKNOWLEDGMENTS
The author wishes to express his appreciation to Drs. F. E. Allison,
S. B. Hendricks, and D. A. Soulides of this organization and to Dr. J. H. Axley
of the University of Maryland for helpful suggestions.
RESULTS
Protein-Montmorillonite Complexes
This section will be confined to representative data of the results obtained
with protein-montmorillonite complexes. More extensive data and methods
of preparation have been published (Pinck and Allison, 1951; Pinck, Dyal
and Allison, 1954). Fig. ] shows comparative rates of decomposition of
gelatin present to the extent of 5.9 percent in a complex with montmorillonite, in mixture with it and alone in sand culture. In a period of 4 weeks,
the gelatin in the complex, mixture, and gelatin alone decomposed 15, 58
and 80 percent, respectively. The striking difference between the rates of
decomposition of the protein in mixture with the clay and protein alone
indicate that even under natural conditions protein is adsorbed by montmorillonite. The microbial decomposition of protein in a series of organicclay complexes is shown in Fig. 2 (gelatin-montmorillonite complexes) and
CCM 34
522
NINTH NATIONAL CONFERENCE ON CLAYS AND CLAY MINERALS
Fig. 3 (egg albumen-montmorillonite complexes). I n complexes containing
10-13 percent protein about 20 percent of the bound protein decomposed,
whereas about 80 percent decomposed in those complexes containing
35-54 percent protein. A striking difference in the rates of decompositon
of the protein in those complexes containing about 20 percent protein was
observed. I t should be noted t h a t the data in Figs. 1-3 and Table 1 indicate
protein-carbon evolved as COs. I n addition about 10-15 percent of the
decomposed protein goes into microbial growth. Hence the total decomposition is 10-15 percent greater than t h a t reported.
X-ray diffraction data (Table 1) furnish an explanation for the observed
variations in the decomposition of the proteins in the complexes. Those
complexes having about 10 percent protein and a c-spacing of 15 A cor-
,
80
5 9 0 / 0 Gelatin
70
I- Complex
2 - Mixture
3- 6elotin
60
3 ~
z ...-f~
.E2o50
~_40
Eo 3o
c~20
~~
o
I
I0
I
I
2
3
4
T i m e in W e e k s
FIOUR~ 1.--Comparative rates of decomposition of gelatin present to the extent of
5.9 percent in a complex with montmorillonite, in mixture with it, and alone.
80 - o
.~_ 50
ft. 4 0
~ 3o
10
Time
in W e e k s
FlatmE 2.--Deeompesition of gelatin in gelatin-montmorillonite complexes containing 10.6-54.8 percent protein-in sand culture.
523
flkDSORPTION OF PROTEII~S
respond to a monolayer thickness of protein (Talibudeen, 1950). The finding
that the c-spacing remains practically unaltered after decomposition indicates that the proteolytic enzymes furnished by the soil microorganisms
could not penetrate the 4.8 A spacing as they are larger molecules than the
proteins. The 20 percent decomposition that took place in the monolayer
of protein was adsorbed on the outer surface and edges which correspond
to about 20 percent of the total area of montmorillonite (Hendricks, Nelson
and Alexander, 1940). In those complexes containing two or more layers of
protein, the latter decomposed to the same extent as where protein alone
was added. The commonly observed 12.7 A c-spacing in the residual com64
80
-
~ 70
~, 6o
o..
-~
"~40
oE
3o
~2o
7
54. 7"
8
196
134
94
9
~
I0
~0
F
I
I
t
I
2
5
Time in Weeks
FI(UmE 3.-Decomposition of egg albumen in egg albumen-montmorillonite complexes containing 9.4-53.8 percent protein-in sand culture.
plexes (Pinck, Dyal and Allison, 1954) was originally interpreted as an
average spacing of complexes with about half of their layers having a cspacing of 15 A and half having a spacing of 10 ~. Such a mixed-layer complex would not show the regularity of the observed X-ray pattern having
TABLE 1 . - - X - R A Y
DIFFRACTION DATA FOR PROTEIN--l%IONTMORILLONITE
COMFLEXES BEFORE AND AFTER DECOMFOSITIO~ BY SOIL I~ICROORGANISMS
FOR 4 W E E K S
Protein in Complex
Kind
Gelatin
Egg Albumen
Gelatin
Egg Albumen
34*
Percent
I0.6
9.4
54.8
53.8
Protein-c~rbon
Evolved as COs
c-Spacing
Before
Decomposition
After
Decomposition
Percent
(h)
(A)
18.4
17.4
82.0
86.4
15.0
15.4
29.2
30.0
14.6
15.0
12.7
11.8
524
NINTH NATIONAL CONFERENCE ON CLAYS AND CLAY MINERALS
a sharp p e a k corresponding to t h e o b s e r v e d spacing. I t is more l i k e l y t h a t
t h e 12 J(-b spacing m a y be d u e to t h e presence of N ~ H in t h e c r y s t a l
l a t t i c e resulting from d e c o m p o s i t i o n of t h e proteins.
Urease-Clay Complexes
The enzyme urease was f o u n d to be a d s o r b e d c o m p l e t e l y b y h y d r o g e n m o n t morillonite, for the s u p e r n a t a n t liquid gave a negative t e s t for urease activi t y , whereas i n c o m p l e t e a d s o r p t i o n was o b t a i n e d w i t h u n t r e a t e d m o n t morillonite (pH 8.4) a n d h y d r o g e n k a o l i n i t e (Pinck a n d Allison, 1961). D a t a
on urease activities of several complexes f o r m e d b y t h e a d d i t i o n of 5, 10
a n d 20 m g samples of commercial urease to 0.5 g samples of H - c l a y s a n d
u n t r e a t e d clays are shown in Table 2. Using urease a c t i v i t y in aqueous
TABLE 2.--ACTIVITY OF UREASE--CLAu COMPLEXES
H-Clay
Kind of Clay
I
Untreated Clay
Urease Additions (rag)
5 110t201
110120
Total ammonia formed (mg) 1
lHontmorillonite
Kaolinite
9.1
17.6
16.5
30.3
23.5
43.0
22.7
19.9
39.3
37.2
56.6
60.9
1 Amounts of ammonia formed by 5, 10, and 20 mg urease in water solution were 33.1,
56.2, and 98.2 mg NH,~ respectively.
s o l u t i o n as a s t a n d a r d , it was c a l c u l a t e d t h a t H - m o n t m o r i l l o n i t e i n a c t i v a t e d
a b o u t t h r e e - f o u r t h s of the a d d e d urease, a n d u n t r e a t e d m o n t m o r i l l o n i t e
a b o u t one-third. The a c t i v i t y of urease a d s o r b e d on H - m o n t m o r i l l o n i t e was
a b o u t half as g r e a t as t h a t a d s o r b e d on H - k a o l i n i t e . The activities of urease
a d s o r b e d on u n t r e a t e d clays were similar.
A n e x p e r i m e n t was designed to show w h e t h e r urease is active in t h e ads o r b e d s t a t e or w h e t h e r i t has to be released f r o m t h e c l a y to become active.
T h e p r o c e d u r e i n v o l v e d t h e a d d i t i o n of urea to t h e first s u p e r n a t a n t liquid
o b t a i n e d b y centrifugation of t h e u r e a s e - H - m o n t m o r i l l o n i t e complex a n d
to t h e r e s u s p e n d e d complex. The suspension was s h a k e n for a s h o r t time,
a n d t h e second s u p e r n a t a n t liquid o b t a i n e d b y c e n t r i f u g a t i o n a n d filtration
was d i v i d e d i n t o two equal aliquots, each of which was t i t r a t e d w i t h s t a n d a r d i z e d a c i d for evolved a m m o n i a a t different t i m e intervals. The first
s u p e r n a t a n t l i q u i d g a v e a n e g a t i v e t e s t for urease a c t i v i t y , whereas t h e
aliquots of t h e second s u p e r n a t a n t gave positive tests increasing w i t h t i m e
(Table 3). The results i n d i c a t e t h a t some urease is released from t h e H - c l a y
owing to t h e a c t i o n of u r e a as a cation. A l t h o u g h u r e a is n e u t r a l it is k n o w n
ADSORPTION OF PI~OTEINS
525
to form salts with acids. Consequently, in acid solution it behaves as a cation.
After the initial release of some urease the evolved ammonia resulting from
the hydrolysis of urea raises the p i t of the solution, thereby eliminating the
cationic action of urease. Ammonia then takes over in releasing more of
the bound urease. Such a mechanism explains why the resuspended ureaseH-montmorillonite complex exhibits urease activity in contrast to the inactivity of the first supernatant liquid.
TABLE 3.--HYDROLYSIS OF UREA BY UREASE
RELEASED FRON UREASE--]!C~ONTMORILLONITE
COMPLEXES
Time (hr)
Ammonia Formed (rag) in
Equal Aliquots of Second
Supernatant Liquid
a
0-1.5
0-3.5
1.5-24
3.5-24
2.6
24 (total)
9.7
b
3.6
7.1
6.2
9.8
Antibiotic-Montmorillonite Complexes
Data on the adsorption of ten antibiotics by montmorillonite are shown
in Table 4, arranged in three groups according to their reaction characteristics: Group I consists of complexes of strongly basic antibiotics (streptomycin sulfate, dihydrostreptomycin sulfate, neomycin sulfate and kanamycin sulfate), Group I I deals with amphoteric antibiotics (chlortetracycline hydrochloride, oxytetracycline hydrochloride and baeitracin) and
Group I I I contains acid (penicillin) and neutral (chloramphenieol and eycloheximide) antibiotics. The amounts of the antibiotics adsorbed are expressed
in terms of dry weight of the materials in the forms received. Average values
in mg of antibiotics adsorbed by 1 g of montmorillonite in groups I, I I
and I I I are 185, 307 and 9, respectively (Pinck, Holton and Allison, 1961).
X-ray diffraction determinations were run on the first two groups of
complexes. Studies on Group I I I were omitted because these antibiotics
were adsorbed only to a minor extent. The average expansions (Table 4)
of the crystal lattices of Groups I and I I were 4.4 and 7.6 A. On the basis
of atom models of representative antibiotics, their amounts adsorbed, and
the expansion of the crystal lattices, it was concluded that the complexes
of Group I contained monolayers and Group I I dilayers of antibiotics.
526
~N~I~TH NATIONAL CONFERENCE ON CLAYS AND CLAY MINERALS
TABLE 4.--ADSORPTION OF ANTIBIOTICS BY MONTMORILLONITEAND
EXPANSION OF c-SPAcINGS
Antibiotic in Complex
),'Ig Antibiotic
per g of Clay
Expansion
(/~)
Group I--Strongly Basic
Streptomycin sulfate
Dihydrostreptomycin sulfate
Neomycin sulfate
Kanamycin sulfkte
Average
209
194
160
178
185
4.4
4.7
4.3
4.3
4.4
Group II--Amphoteric
Bacitracin
Chlortetracycline hydrochloride
0xytetrgcycline hydrochloride
Average
315
300
306
307
7.6
7.5
7.7
7.6
Group III--Acidic or Neutral
PeniciHin
Chloran~phenicol
Cyeloheximide
Average
10
8
9
9
Biological assays for measuring the extent of activity of complexed antibiotics were made by the cylinder-plate method (Grove and Randall, 1955),
the results of which are illustrated in Plate 1. Plate 1A shows the effect of
20ttg streptomycin in 0.2 ml buffer, in 25 mg m0ntmorillonite complex
and in 25 mg kaolinite, each wetted with 0.2 ml buffer. Plate 1B shows
results with chlortetracycline in solution and complexes. The basic streptomycin-montmorillonite complex failed to show any inhibition of bacterial
growth whereas the amphoteric chlortetraeyeline-montmorillonite showed
appreciable inhibition. Experimental evidence was obtained indicating that
an antibiotic is first released from the clay by an exchange reaction with the
cation of the buffer and then diffuses through the agar (Pinek, Soulldes and
Allison, 1961).
With the object of determining the smallest quantity of an antibiotic
that could be detected in 1 g of clay, bioassays were made on antibioticmontmorillonite complexes containing minimal quantities of antibiotics.
For comparison bioassays were also made on buffer solutions of the antibiotics, and on antibiotic-kaolinite complexes (Table 5). The previously
mentioned division of the complexes into groups depending upon the
reaction of the antibiotics applies equally well to their bactericidal activities.
The montmorillonite complexes of the strongly basic antibiotics failed to
show any activity, whereas those containing amphoterie antibiotics gave
positive tests. Although kaolinite complexes of Groups I and I I gave positive
PLATE 1.--(A) Activity of streptomycin-clay complexes. Control (top), montmoril-
lonite ([eft) and kaolinite (right).
(B) Activity of chlortetracycline-clay complexes. Control (top), montmorillonite
(right) and kaolinite (left).
527
ADSORPTION OF PROTEINS
tests there was a marked difference in the sensitivity of the two groups
when compared with their respective controls. The differences in activities
in the kaolinitic and montmorfllonitic complexes of the basic antibiotics
are attributed to differences in Coulomb forces of the two clay minerals,
for montmorillonite has a much greater degree of substitution in the tetrahedral and octahedral layers than does kaolinite.
TABLE 5.--~IINIMUM DETECTABLE AI~OUNTS OF ANTIBIOTICS RELEASED EROI~I
~r
AI~D KAOLINITE COMPLEXES 1
Group I - S t r o n g l y Basic Antibiotics Group II-Amphoteric Antibiotic~,
Clay
Montmorfllonite
Kaolinite
Control
DihydroStrepto- strepto- Neomycin
mycin mycin
Kanamycin
h
25
1
25
1
800
2.5
2OO
0.5
Baeitracin
800
1
0.25
Chlortetracycline
Oxytetracycline
0.5
0.05
0.02
2.0
0.4
0.1
1 Figures for complexes and controls in fig of antibiotic per g and ml respectively.
DISCUSSIONS
AND
CONCLUSIONS
The adsorption of proteins and antibiotics b y montmonllonite involves
a base exchange reaction. The slight degree of adsorption of acidic and
neutral antibiotics by montmorillonite in comparison with the extensive
adsorption of basic and amphoteric antibiotics and of proteins supports
this conclusion.
Monolayers of "proteins and antibiotics are more rigidly held to montmorillonite than polylayers. This m a y be attributed to Coulomb and van der
Waals forces (Hendricks, 1941).
The results indicate a common mechanism of release and activity of
urease and amphoterie antibiotics complexed with montmorillonite,
implying that enzymatic and antibiotic activities take place in aqueous
solution and not on clay surfaces,
In decomposition studies of protein- and urease-montmorillonitc complexes, the p H rose considerably above the isoelectric points of the organic
compounds as a result of the evolution of ammonia, and in the bioassays
of the eomplexed amphoteric antibiotics, the p H was adjusted to a reaction
above the isoeleetric point by means of buffers. Since amphoterie compounds
are dependent upon low p H to maintain their cationic state, any reaction
which causes a rise in p H lowers the cationic concentration with a concurrent rise in concentration of anions. Hence the electrostatic forces
originally present are greatly weakened, thereby permitting a release of
528
I~INTH NATIONAL CONFERENCE ON CLAYS AND CLAY MINERALS
t h e o r g a n i c m a t t e r . I n t h e c a s e of t h e b a s i c a n t i b i o t i c s , a c h a n g e i n p H
h a s n o effect o n t h e C o u l o m b f o r c e s ; c o n s e q u e n t l y t h e y a r c n o t r e l e a s e d
f r o m t h e clay.
I n c o n c l u s i o n t h e r e s u l t s of t h i s s t u d y s h o w a c l e a r - c u t r e l a t i o n s h i p i n
t h e b e h a v i o r of p r o t e i n s , u r e a s e a n d a n t i b i o t i c s w i t h m o n t m o r i l l o n i t e ;
t h e y also s h o w h o w e x t e n s i v e l y t h e m u l t i f a r i o u s f u n c t i o n s of t h e m i c r o o r g a n i s m s a f f e c t t h e a d s o r p t i o n a n d r e l e a s e of o r g a n i c m a t t e r b y a n d
f r o m t h e clay.
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ADSORPTION OF PROTEINS
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