J. Biosci., Vol. 2, Number 3, September 1980, pp. 227-233. © Printed in India.
Spectroscopic studies on the denaturation of papain
solubilized and Triton X-100-solubilized glucoamylase from
rabbit small intestine
S. SIVAKAMI and D. CHATTERJI
School of Life Sciences, University of Hyderabad, Hyderabad 500 134
MS received 14 January 1980; revised 2 August 1980
Abstract. Intestinal brush border proteins consist of an enzymatically active hydrophilic moiety
attached to a hydrophobic tail. Papain dissociates the hydrophilic part by cleaving off the hydro-
phobic tail, whereas the detergentTriton X-100 solubilizes the whole molecule. Denaturation by
8 ? urea or 4 ? guanidinium chloride does not alter the structure of the papain-solubilized
enzyme. An appreciable alteration of the structure of detergent-solubilized enzyme was observed
on denaturation. The difference spectra of Triton X-100 (1%)—solubilized enzyme and its urea
denatured form shifts and intensifies, with increase in the concentration of the denaturant with
an isobestic point at 252 nm. A new band at 280 nm also appears at 4 ? urea concentration.
Papain-solubilized glucoamylase has an ? -helical conformation in solution unlike the detergent-
solubilized fraction. An elongated structure for the papain solubilized enzyme is inferred from
the urea denaturation studies and from molecular weight determinations.
Keywords. Rabbit small intestine; glucoamylase; papain denaturation; Triton X-100; difference
Glucoamylase (EC 22.214.171.124) is an exoenzyme that releases glucose residues directly
from starch. Glucoamylase of the small intestine localized in the brush border, has
been purified to varying extents from different animal species (Seetharam et al.
1970, Schlegel-Haueter et al., 1972; Kelly and Alpers, 1973) and to homogeneity
from rabbit (Sivakami and Radhakrishnan, 1973).
In general, the disaccharidases and glucoamylase have been studied after solubili-
zation from the brush border membrane by the proteolytic action of papain which
cleaves the hydrophobic moiety from the solubilized hydrophilic moiety (Maroux
and Louvard, 1976). Recently, detergents have been used for solubilizing the sucrase-
isomaltase (Sigrist et al., 1975) and alkaline phosphatase (Colbeau and Maroux,
1978). The detergent and papain-solubilized forms of sucrase-isomaltase are identical
enzymatically but differ in their tendency to aggregate (Sigrist et al., 1975). Now, it
is well established that the detergent solubilizes the ‘whole’ enzyme by acting on the
membrane-embedded hydrophobic part and this interaction appears to be dependent
on the critical micellar concentration of the detergent (Helenius and Simons, 1975).
In our earlier attempts to determine the molecular weight of papain-solubilized
glucoamylase from rabbit small intestine, an anomalous behaviour of the enzyme
on gel filtration systems was noticed. The estimate of the molecular weight obtained
Sivakami and Chatterji
by gel filtration was at least 5 times larger than the value obtained in the analytical
ultracentrifuge (Sivakami and Radhakrishnan, 1978). An explanation for this dis-
crepancy was not then readily available.
Preliminary experiments have indicated that glucoamylase can be effectively
solubilized using Triton X-100 (Nirmala Murthy, 1978). The Triton-solubilized enzyme
is apparently different from the papain-solubilized enzyme in its stability to heat and
denaturants like urea and guanidinium chloride (Nirmala Murthy, 1978). Spectral
studies on the two forms of the enzyme were therefore initiated with a view to detect
any differences in the state of aggregation and shape and to offer a possible explana-
tion for the observed anomaly in the molecular weight of the enzyme.
Materials and methods
All the reagents used here are of analytical grade. Urea and guanidinium chloride
are of ultrapure grade obtained from Schwarz-Mann, Orangeburg, New York,
USA. Papain was obtained from Sigma Chemical Company, St. Louis, Missouri,
USA and Triton X-100 used was of scintillation grade from Eastman Kodak,
Rochester, New York, USA. Glucoamylase was solubilized using papain or Triton
X-100 and subsequently purified by affinity chromatography on Sephadex G-200
columns (Sivakami and Radhakrishnan, 1973). A 20% homogenate of the intestinal
mucosal scrapings was centrifuged at 12,000 g for 30 min in a refrigerated centrifuge,
model MB 20 (MB Corporation, Bombay) at 4°C. The particulate fraction was
suspended in 0.01 ? potassium phosphate buffer pH 7·0 to get a final protein level
of about 10 mg/ml. This fraction was incubated with crystalline papain at a papain:
pellet protein ratio of 1:100 for 60 min at 37°C, at the end of which it was chilled
and centrifuged at 25,000 g for 3 h in a refrigerated centrifuge at 4°C. Papain was
removed from the enzyme during the specific affinity procedure using Sephadex G-
200 (Sivakami and Radhakrishnan, 1973). For solubilization with Triton X-100, the
homogenate in the phosphate buffer was adjusted to a protein concentration of 6
mg/ml, and incubated at 4°C, for 90 min, in the presence of 1% Triton X-100 and
0.01 mM NaCl. At the end of the incubation, the mixture was centrifuged at 25,000 g
for 3 h. The enzyme in the supernatant was purified by the same affinity technique
under identical conditions as was used for the papain-solubilized enzyme. Throughout
the purification and the subsequent analysis, 1% Triton X-100 was maintained. The
Triton enzyme was apparently free of contamination as judged by polyacrylamide
disc gel electrophoresis (Nirmala Murthy, 1978 and figure 1). Maltase and gluco-
Figure 1. Polyacrylamide gel electrophoresis of Triton X-100 solubilized glucoamylase
Denaturation of glucoamylase from rabbit small intestine 229
amylase activities were determined by measuring the glucose formed by the glucose
oxidase-peroxidase procedure of Dahlqvist (1964), as described earlier (Seetharam
et al., 1970). Protein was estimated by the method of Lowry et al. (1951) using
bovine serum albumin as the standard.
Spectral measurements were carried out in 0.5 ml cuvettes using a Shimadzu UV-
200S double beam spectrometer. The enzyme was allowed to equilibrate with urea
and guanidinium chloride for about 1 h before the spectral measurements were
made. However, the denaturation was found to be completed within 1 min after the
addition of the denaturant. Difference spectra were corrected for the absorption of
the urea and guanidinium chloride under similar conditions. Since Triton X-100
was present in equal amounts in both the reference and sample cuvettes, any contri-
bution by it to the protein absorption spectra was cancelled out. By varying the con-
centration of both Triton-X-100 and the protein, it was also observed that the
spectral properties of free Triton X-100 and protein bound Triton X-100 were the
same. Circular dichroic measurements were carried out in a Jasco J-20 spectro-
Results and discussion
Figure 2 shows the UV absorption spectra in the range 240-300 nm of the papain-
solubilized enzyme (papain-free). The enzyme was characterized by a typical broad
Figure 2. Ultraviolet spectra of papain-solubilized enzyme and the difference spectra of the
enzyme in 8 ? urea and 4 ? guanidinium chloride. During the analysis of difference spectra,
reference cuvette contained the enzyme and the sample cuvette contained enzyme and the
1. Enzyme 40 µg;
2. The difference spectra of the enzyme (40 ? g) + 8 ? urea;
3. Enzyme 20 and 10 ? g + 8 ? urea;
4. Enzyme 40 µ g + 4 M guanidinium chloride.
230 Sivakami and Chatterji
absorption peak at 272 nm contributed by the aromatic amino acid residues present
in the enzyme. Phenylalanine and tyrosine were present to the extent of 69.2
mol/mol of the enzyme (Sivakami and Radhakrishnan, 1978). However, when this
enzyme was subjected to denaturation with 8 ? urea or 4 ? guanidinium chloride,
no significant change was observed as shown in the difference spectra (figure 2).
Change in absorbance was considerable only when a higher concentration of enzyme
was used (40 ?g). The absence of any appreciable change on denaturation suggests
that the aromatic amino acid residues may not be buried in a hydrophobic region as
in a globular protein.
Quite a different result was obtained when the Triton X-100 solubilized enzyme
was denaturated with 8 ? urea (figure 3). It should be mentioned here that the con-
centration of Triton X-100 (1%) in aqueous medium used in these experiments was
higher than its critical micellar concentration value. Therefore, the difference
spectra were obtained by subtracting the contribution of the change in absorbance
arising due to the disruption of the Triton micelle itself by urea (Helenius and
Simons, 1975). With increase in the concentration of the protein, the large negative
Spectra of the Triton X-100 solubilized enzyme denatured with 8 ? urea. Reference
cuvette contained the enzyme (1, 10 µ g: 2, 20 µ g; 3, 40 µ g; 4, 80 ? g protein) and the sample
cuvette contained the enzyme and 8 ? urea.
Denaturation of glucoamylase from rabbi tsmall intestine 231
band at 285 nm shifted towards a higher wavelength. Moreover, at lower concentra-
tions of the protein (10 and 20 µg) a new band appeared at 280 nm. The difference
spectrum was also characterized by a broad band at a lower wavelength which
shifted and intensified with increase in the level of the protein. When the denaturation
of the enzyme (20 µg) was carried out at different concentrations of urea, the band
at 280 nm was found to be distinct at higher concentrations of urea (figure 4). Also,
The difference spectra of the Triton X-100 solubilized enzyme (20 ? g protein) after
denaturation with different concentrations of urea (1, 2 M; 2, 4 M; 3, 6 M; and 4, 8 M urea).
Reference cuvette contained enzyme and the sample cuvette contained enzyme and urea.
an isobestic point at 252 nm was observed indicating that one type of denatured
species is present in the solution (see figure 4). Similar results were also obtained
during denaturation by 4 ? guanidinium chloride (not shown). All these data
suggested that the enzyme in the Triton X-100 medium had a structure, the denatura-
tion of which exposed the aromatic amino acid residues to the solvent. We would like
to point out that most proteins preserve their tertiary structure in the presence of high
concentration of Triton X-100 and it usually does not appear to induce conforma-
ional change in proteins leading to loss of their biological properties (Helenius and
Simons, 1975). It is noteworthy that most of the proteins give a large negative band
in the difference spectra upon denaturation with 8 ? urea (Herskovits, 1967).
However, any change in the spectra arising due to the change in concentration of
the enzyme may be attributed to the aggregation of the molecule which is possible
in the presence of non-ionic detergents like Triton X-100 (Tanford et al, 1974;
Simons et al., 1973).
232 Sivakami and Chatterjee
Hence, a Triton-free form of the enzyme was purified by washing the column
thoroughly with Triton-free buffer before elution of the enzyme. The Triton free-
enzyme was expected to form an aggregate.
However, the addition of 8 ? urea fails to bring about any appreciable change in
the organization of the enzyme aggregate, as noticed by difference spectrophotometry.
Only a broad negative band of low intensity around 290 nm was observed and did
not change with the concentration of the enzyme aggregate. To verify whether the
spectra of the Triton-solubilized enzyme were due to the conformational effects of
Triton X-100 on an enzyme form similar to that solubilized by papain, we carried
out the urea denaturation study of the papain-solubilized enzyme in the presence of
1 % Triton. The difference spectra did not show any band in the region of 250 to 290
nm, although there was a small negative band around 300 nm, The 8 ? urea induced
denaturation did not cause any significant structural alteration of the papain-solubi-
lized enzyme in the presence of 1 % Triton X-100.
All these results indicated that there was a gross structural difference of the
enzyme when solubilized by using either papain or Triton. It is interesting to note
that a small hydrophobic tail of the enzyme might contribute significantly to the
overall conformation of the enzyme. When the difference spectra of the papain-
solubilized enzyme at pH 12 was compared with that at neutral pH, the nature of the
curve was found to be similar to that obtained with free tyrosine. However, no
significant change was observed in alkali-induced difference spectra of Triton-
solubilized enzyme (not shown).
To confirm the above pattern of results we measured the circular dichroic (CD)
spectra of the urea denatured enzyme solubilized by Triton X-100 and papain res-
pectively. The papain-solubilized enzyme had two negative CD bands, one around
218 nm and the other at 205 nm typical for ?-helical conformation (figure 5)
Circular dichroism spectra of papain-solubilized enzyme, (1); 8 ? urea denatured
papain-solubilized enzyme (2); Triton X-100 solubilized enzyme (3); 8 ? urea denatured Triton
X-100 solubilized enzyme (4).
Denaturation of glucoamylase from rabbit small intestine 233
(Greenfield and Fasman, 1969), whereas the Triton-solubilized fraction was charac-
terized by one negative CD band at 222 nm. Urea denaturation caused a greater
disruption of the structure of detergent-solubilized fraction than that of the papain
solubilized enzyme as expected.
It was mentioned earlier that the papain-solubilized glucoamylase had an estimated
molecular weight five times more by gel filtration than that obtained through ultra-
centrifugation. This discrepancy might arise due to the aggregation of the molecule
or if the enzyme has an elongated structure. Urea denaturation data suggested that
the molecule might be elongated without much folding, resulting in the enzyme
eluting on gel filtration system earlier than it should (Sivakami and Radhakrishnan,
1978). One the other hand, the detergent-solubilized glucoamylase appeared to
have a typical three-dimensional folding with buried aromatic amino acid residues
which are vulnerable to urea denaturation. Further studies on the strcuture of the
enzyme above and below the critical micellar concentration of the detergent and
the nature of interaction of the detergent micelle with the enzyme are in progress.
The authors wish to thank Prof. A. N. Radhakrishnan for his kind co-operation and
helpful suggestions during the course of this investigation.
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