Journal of Bioscience and Bioengineering

VOL. 120 No. 3, 252e256, 2015

www.elsevier.com/locate/jbiosc

Interaction of carbohydrates with alcohol dehydrogenase: Effect on enzyme

activity
Swati B. Jadhav,1, 2 Sandip B. Bankar,1 Tom Granstrm,1 Heikki Ojamo,1 Rekha S. Singhal,2 and
Shrikant A. Survase1, *
Department of Biotechnology and Chemical Technology, Aalto University, School of Chemical Technology, POB 16100, 00076 Aalto, Finland1 and Food Engineering and Technology
Department, Institute of Chemical Technology, Matunga, Mumbai 400 019, India2
Received 3 July 2014; accepted 7 January 2015
Available online 7 February 2015

Alcohol dehydrogenase was covalently conjugated with three different oxidized carbohydrates i.e., glucose, starch and
pectin. All the carbohydrates inhibited the enzyme. The inhibition was studied with respect to the inhibition rate
constant, involvement of thiol groups in the binding, and structural changes in the enzyme. The enzyme activity
decreased to half of its original activity at the concentration of 2 mg/mL of pectin, 4 mg/mL of glucose and 10 mg/mL of
starch within 10 min at pH 7. This study showed oxidized pectin to be a potent inhibitor of alcohol dehydrogenase
followed by glucose and starch. Along with the aldehyde-amino group interaction, thiol groups were also involved in the
binding between alcohol dehydrogenase and carbohydrates. The structural changes occurring on binding of alcohol
dehydrogenase with oxidized carbohydrates was also conrmed by uorescence spectrophotometry. Oxidized carbohydrates could thus be used as potential inhibitors of alcohol dehydrogenase.
2015, The Society for Biotechnology, Japan. All rights reserved.
[Key words: Alcohol dehydrogenase; Carbohydrates; Inhibition; Thiol groups; Covalent binding]

Proteins and carbohydrates are natural polymers that are

abundantly found in most complex biological systems. These two
polymers interact by forming a complex through covalent and/or
non-covalent binding. Among the various non-covalent interactions, hydrogen bonding and electrostatic interactions play an
important role in enhancing the functional attributes of the polymer complex (1). For covalent interactions, the polysaccharides are
oxidized to get aldehyde groups which then react with the amino
groups of proteins. Enzymes show different kinds of interactions
with bio-macromolecules in the cell. Such interactions affect the
enzyme activity as well as its stability (2). Carbohydrates are one of
the important macromolecules with which enzymes interact.
Hence enzymeepolysaccharide interactions attract attention with
respect to enzyme stability. Covalent interactions between enzyme
and carbohydrate can be either glycosylation or glycation. Although
both the processes involve attachment of glycan to enzyme (protein), glycosylation is an enzymatic process while glycation is a
non-enzymatic process. Glycosylation attaches the glycan part to
nitrogen of asparagine or arginine and hydroxyl oxygen of serine,
threonine or tyrosine. However glycation attaches the glycan part
to nitrogen of lysine via Schiffs base formation (3).
Many authors have reported covalent as well as non-covalent
interactions between the enzymes and polysaccharides (4e6).
These interactions enhance the stability of enzymes at extreme
temperatures and pH (7,8). Non-covalent interactions between

* Corresponding author. Tel.: 358 400368375; fax: 358 9 462 373.

E-mail address: shrikantraje1@gmail.com (S.A. Survase).

enzymes and polysaccharides in a mixture have also been reported

to enhance structural as well as functional properties of proteins
(5). Conjugation of polysaccharide with an enzyme can either
enhance or inhibit the activity and/or stability depending on the
structural changes occurring in the enzyme. Based on the structural
and functional properties, enzymes could respond differently to the
same modication method. For instance, microencapsulation of
glucose oxidase and laccase using poly(ethyleneimine) have shown
completely opposite effects on thermal stability of the said enzymes. At high temperature, poly(ethyleneimine) chelates copper
from active site of the laccase and hence decrease its thermal stability, whereas same microencapsulation enhances the thermal
stability of glucose oxidase (9). Synthetic polysaccharide prepared
using glucal has shown inhibition of amylase (10), whereas basic
polysaccharides are reported to be inhibitory for lipase activity (11).
Alcohol dehydrogenase belongs to the zinc metalloenzymes that
catalyse the oxidation of alcohols to aldehydes and vice versa. Inhibition of alcohol dehydrogenase may prove to be advantageous to
study the alcohol metabolism as well as treatment for methanol
poisoning in humans (12). The susceptibility of humans to methanol toxicity is dependent upon folate metabolism. It is an uncommon but hazardous intoxication characterized by visual
impairment and formic acidaemia. Alcohol dehydrogenase metabolizes methanol to formate which accumulates in body and
thereby causes toxicity. The therapy of methanol toxicity is based
on the inhibition of alcohol dehydrogenase (13,14). Alcohol is
metabolized in the liver by alcohol dehydrogenase to acetaldehyde.
Acetaldehyde is further broken down by aldehyde dehydrogenase
to acetic acid and water. Inhibitors of alcohol dehydrogenase may

1389-1723/$ e see front matter 2015, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2015.01.008

VOL. 120, 2015

INTERACTION OF CARBOHYDRATES WITH ENZYME

help to study this pathway of alcohol metabolism (15). The inhibitors of alcohol dehydrogenase have also been used in cosmetics
to avoid the effect of added alcohol on the skin (16). Many compounds including thiol compound (2), 4,40 dithiodipyridine (17),
avonoids (18), pyrazoles (12) and 2,2,2-triuoroethanol (19) have
been reported to act as inhibitors of alcohol dehydrogenase.
In the present study, we report the inhibitory effect of covalent
binding between carbohydrates (glucose, starch and pectin) and
alcohol dehydrogenase on the enzyme activity. Glucose (monosaccharide), starch (glucose containing polysaccharide) and pectin
(anionic polysaccharides) were selected for the study. The inhibition of alcohol dehydrogenase by oxidized carbohydrates was
thoroughly studied by determining the inhibition rate constant and
evaluating the involvement of thiol groups on the inhibition.
MATERIALS AND METHODS
Materials
Alcohol dehydrogenase from Saccharomyces cerevisiae (46 U/mg
as determined experimentally), sodium metaperiodate, nicotinamide adenine
dinucleotide (NAD) and sodium pyrophosphate were purchased from Sigma Aldrich,
St. Louis, MO, USA. Glucose, starch and pectin (MW 75,000 Da and degree of
esterication 65e70%) were purchased from Himedia, Mumbai, India.
Preparation
of
conjugate
of
carbohydrate
with
alcohol
dehydrogenase
The enzyme-carbohydrate conjugate was formed by using a
previously reported method (8). Sodium metaperiodate (0.1 M) solution was
prepared in 0.1 M sodium acetate buffer of pH 5.0 and used as the oxidizing
solution. Carbohydrates (glucose, starch and pectin) 100 mg each was oxidized in
10 mL of oxidizing solution in dark at room temperature (25  2 C) for 90 min,
after which the oxidation was stopped by adding 0.3 mL of ethylene glycol, and
kept in dark for 1 h. Oxidized carbohydrates (except glucose) solutions were
dialysed against 0.01 M sodium phosphate buffer of pH 7.5 at 4 C overnight.
Alcohol dehydrogenase solution (1 mg/mL) was prepared in 0.01 M sodium
phosphate buffer of pH 7.5 and mixed with equal volume of each oxidized
carbohydrate solution and kept for conjugate formation at room temperature
(25  2 C). These conjugates were used for analysing the activity of alcohol
dehydrogenase.
Alcohol dehydrogenase activity assay
Assay mixture (800 mL) consisted of
0.05 M sodium pyrophosphate buffer of pH 8.8 (260 mL), 50 mM ethanol (220 mL),
15 mM NAD (300 mL) and reaction was started immediately by addition of enzyme
solution (20 mL, 0.1 mg/mL). Reduction of NAD to NADH was followed for 4 min by
taking absorbance at 340 nm. The assay mixture for the blank (800 mL) consisted of
0.05 M sodium pyrophosphate buffer of pH 8.8 (260 mL), 50 mM ethanol (220 mL),
15 mM NAD (300 mL) and distilled water (20 mL). Extinction coefcient () value of
6.22 mM1 cm1 for NADH at 340 nm was used to calculate activity (20). One unit of
enzyme activity was dened as millimoles of NAD reduced per min at room
temperature and pH 8.8. Protein concentration in the sample was analysed using
Folin-Lowry method (21) and it was calculated using bovine serum albumin as the
standard in the range of 40e200 mg/mL. The protein concentration of enzyme was
0.1 mg/mL which was used for calculation of specic activity of an enzyme.
Stability of the enzyme and progress of inhibition
Each oxidized carbohydrate (glucose, starch and pectin) in the range of 2e10 mg/mL was mixed with
equal volume of the enzyme solution (1 mg/mL) and incubated at room temperature. Samples were withdrawn after regular time intervals (5e30 min for glucose
and pectin, 10e60 min for starch), and assayed for the residual enzyme activity as
described above. Control enzyme solution was maintained without addition of inhibitors and used as a reference of the original enzyme activity.
A semi-log plot of the percent residual activity vs time was plotted for each
enzyme-carbohydrate conjugate. The slope of the inhibition rate constant (k) was
calculated, and the time required for the activity to decrease to half of its original
activity (t1/2) in the presence of inhibitor was calculated as 0.693/k.
The effect of pH on the covalent interaction of carbohydrate with alcohol
dehydrogenase
The enzyme solution was prepared in buffer of pH 7, 8 and 9
(sodium phosphate buffer of pH 7 and 8, glycine-NaOH buffer of pH 9). The oxidized
carbohydrates (4 mg/mL) were mixed with equal volume of enzyme solutions (1 mg/
mL) at pH 7, 8 and 9. The enzyme was incubated for 10 min with glucose, 20 min
with starch and 5 min with pectin at room temperature. Each inhibitor had
different inhibition rate constants; hence time of incubation was varied
accordingly to get maximum inhibition for better comparison of residual activity.
The residual enzyme activity was calculated using the assay method described
above. The inhibition rate of enzyme by carbohydrates at each pH was calculated
as percent of the control activity.
Evaluation of thiol groups in free and carbohydrate conjugated alcohol
dehydrogenase
Free thiol content of alcohol dehydrogenase before and after
the conjugation with oxidized carbohydrates was determined using Ellmans
method (22) with slight modications. The enzyme (0.5 mL, 1 mg/mL) was

253

incubated with the oxidized carbohydrate (0.5 mL, 10 mg/mL) for 15 min to
prepare inhibited enzyme sample. The free and inhibited enzyme sample (700 mL)
were added to the mixture of 50 mM 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB)
(50 mL), 1 M Tris buffer of pH 8.0 (100 mL) and distilled water (150 mL). The assay
mixture was incubated for 5 min and absorbance was taken at 412 nm. Blank was
prepared similarly except that distilled water was used instead of sample. The
standard curve of cysteine was prepared using 20e100 mM of cysteine. The thiol
groups present in the enzyme before and after the conjugation with oxidized
carbohydrates were calculated in terms of cysteine equivalent thiol groups.
Fluorescence spectrophotometer analysis
Fluorescence spectrophotometer analysis was done using uorescence spectrophotometer (Cary Eclipse, Agilent
Technologies, Inc., Santa Clara, CA, USA) to investigate structural changes in the
enzyme after its covalent interaction with carbohydrates. The excitation wavelength
was set at 280 nm and the emission spectrum was recorded from 300 to 500 nm. Free
and conjugated enzymes were used as samples. Conjugated enzymes were prepared
by mixing enzyme and oxidized carbohydrates and incubating for 30 min. Fluorescence measurements were carried out in 0.01 M sodium phosphate buffer of pH 7.5.

RESULTS AND DISCUSSION

Stability of the enzyme and progress of inhibition The
covalent interaction of oxidized carbohydrate with alcohol dehydrogenase was studied by preparing enzyme-carbohydrate
conjugates. All the three carbohydrates under study inhibited
alcohol dehydrogenase after binding with it covalently. Inhibition
kinetics was studied by using different concentrations of each
carbohydrate from 2 to 10 mg/mL for various time intervals
(Fig. 1). The inhibition kinetics was evaluated by a semi-log plot
of percent residual activity vs time in terms of inhibition rate
constant, k and half-life t1/2. The inhibition rate constant of
alcohol dehydrogenase in the presence of all oxidized
carbohydrates increased with an increase in the concentration of
carbohydrates. At low concentration (2 mg/mL) of oxidized
carbohydrate, inhibition rate constants of the enzyme were 0.030,
0.014 and 0.052 for glucose, starch and pectin, respectively. At
higher concentration (10 mg/mL) of oxidized carbohydrate,
inhibition rate constants of the enzyme were 0.214, 0.052 and
0.409 for glucose, starch and pectin, respectively. It is evident
from these results that pectin had highest inhibition rate constant
followed by glucose and starch. The half-life of the enzyme after
covalent conjugation with the carbohydrates was calculated using
the inhibition rate constant (Fig. 2). At 2 mg/mL of carbohydrates,
the half-life of enzyme-starch conjugate was approximately four
times higher than that of the enzyme-pectin conjugate and two
times higher than that of the enzyme-glucose conjugate. The
concentration of oxidized carbohydrates at which the enzyme
activity reduced to half of its original activity can be a good
indicator of the efciency of oxidized carbohydrates as an
inhibitor of alcohol dehydrogenase. Pectin (2 mg/mL), glucose
(4 mg/mL) and starch (10 mg/mL) were able to decrease the
activity of enzyme to half of its original activity within 10 min.
These results indicated that oxidized carbohydrates acted as
potent inhibitors of alcohol dehydrogenase. However, pectin was
the most potent inhibitor followed by glucose and starch. The
oxidation of carbohydrate generates reactive aldehyde groups
which can bind to active site of the enzyme. Alcohol
dehydrogenase has binding sites for aldehyde. Alcohol
dehydrogenase can covert aldehyde to alcohol and vice versa.
However, carbohydrate is not a substrate for alcohol
dehydrogenase; it binds to enzyme due to the presence of
reactive aldehyde group. Once the carbohydrate is bound to the
active site of the enzyme, it may act as its inhibitor. Pectin is
structurally very complex which generates more free aldehyde
groups and has additional carboxyl groups which can bind to
imine groups of enzyme forming carbodiimide bond. The
structural complexity and presence of additional carboxyl groups
in pectin as compared to starch may be responsible for strong
binding of oxidized pectin to enzyme, and which resulted in high

254

JADHAV ET AL.

J. BIOSCI. BIOENG.,

FIG. 2. The half-life of alcohol dehydrogenase after covalent conjugation with the
oxidized carbohydrates.

reported to show inhibitory activity on amylase, maltase and

lipase (23).
Alcohol dehydrogenase was not found to be inhibited when it
was mixed with the non-oxidized form of all the above mentioned
carbohydrates. Hence oxidation of the carbohydrates was a critical
step involved in the conjugation and inhibition. Oxidation of carbohydrates generates free aldehyde groups on the carbohydrates
which may be responsible for the inhibition of alcohol dehydrogenase. Formamides are reported to mimic aldehydes and inhibit
liver alcohol dehydrogenase (24).
The effect of pH on the covalent interaction of oxidized
carbohydrate with alcohol dehydrogenase The effect of pH
on the covalent interaction of the oxidized carbohydrates with
alcohol dehydrogenase was studied using buffer of pH of 7, 8 and 9
(Fig. 3). The optimum pH of the enzyme was 8 and it could retain
85% and 60% of the activity at pH 7 and 9, respectively. Hence a
pH range of 7e9 was selected for the study. The inhibition rate of
alcohol dehydrogenase was calculated in the form of percent
inhibition by taking original activity of enzyme without inhibition
as reference. The enzyme showed highest percent of inhibition
(95e97%) at pH 7 for all the three inhibitors. Thus pH proved to
be an important parameter for the inhibition. It may be
hypothesized that this could be due to favourable conditions for

FIG. 1. Kinetics of the inhibition of alcohol dehydrogenase by oxidized form of (A)

glucose, (B) starch and (C) pectin.

inhibition rate constant by oxidized pectin than oxidized starch.

Though starch is more complex than glucose, the inhibition of
alcohol dehydrogenase by oxidized glucose was found to be more
than that by oxidized starch. The oxidized glucose solution was
not dialysed to remove the oxidizing agent due to lower
molecular weight which might have additionally affected the
enzyme activity. This could be the logical reason for obtaining
higher inhibition of alcohol dehydrogenase by oxidized glucose.
Previously, lipase activity was found to be inhibited by basic
polysaccharides (11). The positive charge on the basic
polysaccharide and the degree of substitution was important for
inhibition to occur. The basic polysaccharide is reported to
prevent the hydrolysis of triacylglycerols by retarding the
adsorption of lipase on to the emulsion particles. The
polysaccharides isolated from Ulva lactuca have also been

FIG. 3. The effect of pH on the inhibition of alcohol dehydrogese by the oxidized

carbohydrates.

VOL. 120, 2015

ionic interactions between the two molecules at pH 7. The highest
inhibition of the enzyme was obtained by pectin after 5 min,
glucose after 10 min and starch after 20 min at pH 7.
Evaluation of thiol groups in free and carbohydrate
conjugated alcohol dehydrogenase The thiol groups of
alcohol dehydrogenase were found to be involved in binding with
the inhibitors (25). Alcohol dehydrogenase from yeast is a
tetrameric enzyme containing 9 cysteine residues in each
subunit. Each subunit has 3 reactive thiol (SH) groups present in
active site which helps in catalysis (17). Hence, there is a
possibility of involvement of SH groups during inhibition of
alcohol dehydrogenase. Ellmans method was used for the
detection of thiol groups of alcohol dehydrogenase before and
after binding with oxidized carbohydrate. This method gives free
cysteine equivalent SH groups. While free enzyme had 33 mM of
free cysteine equivalent SH groups, the enzyme-pectin, enzymestarch and enzyme-glucose complexes showed the presence of
30 mM, 22 mM and 21 mM of free cysteine equivalent SH groups,
respectively. It suggested the utilization of 11%, 33 % and 35%
cysteine equivalent SH groups by enzyme-pectin, enzyme-starch
and enzyme-glucose complexes, respectively. The reduction in
the cysteine equivalent SH groups of enzyme after binding with
carbohydrate may suggest the possibility of involvement of SH
groups in the binding of enzyme and carbohydrate (Fig. 4).
Aldehyde groups present on oxidized carbohydrate can react with
SH groups of an enzyme and form hemithioacetal (26). Oxidized
glucose and oxidized starch showed comparable percentage of
the utilization of cysteine equivalent SH groups, whereas oxidized
pectin showed lower utilization of SH groups. Pectin is a complex
molecule as compared to glucose and starch which may make it
difcult to access active site where SH groups are present. This
may be a reason for pectin showing less binding with SH groups
of an enzyme. Though oxidized pectin showed lesser utilization
of SH groups while binding to an enzyme, it was found to be
potent inhibitor. The structural complexity of pectin helped it to
generate more aldehyde groups which may have bound to the
imine group of enzyme to form carbodiimide bonds. Moreover,
pectin has carboxylic groups which also help in binding with
imine groups of enzyme. Thus multiple possibilities of strong
binding of pectin to enzyme may explain potency of oxidized
pectin for being a strong inhibitor of alcohol dehydrogenase.

FIG. 4. The utilization of cysteine equivalent SH groups of alcohol dehydrogenase for

conjugation with oxidized carbohydrates.

INTERACTION OF CARBOHYDRATES WITH ENZYME

255

Binding with SH groups of enzyme is one of the mechanisms of

inhibition of enzyme. Jack bean urease has been reported to be
inhibited by quinone. Quinone has been reported to induce
arylation of thiols in urease to bring about conformational
changes and consequently its inhibition (27).
Fluorescence spectrophotometer analysis Oxidized carbohydrates showed inhibition of alcohol dehydrogenase wherein the
thiol groups of enzyme and the aldehyde groups of oxidized carbohydrates were found to be responsible for inhibition. Further,
structural changes of enzyme after inhibition were investigated by
uorescence spectrophotometer (Fig. 5). Fluorescence intensity
was found to decrease for the inhibited enzyme as compared to
the uninhibited enzyme indicating slight alteration in the
conformation of enzyme after inhibition with oxidized
carbohydrates. Moreover, emission maximum of inhibited
enzyme showed red shift (328 nme337 nm) by 9 nm. Enzymepectin, enzyme-starch, enzyme-glucose conjugates showed a
decrease in uorescent intensity by 116 a.u (arbitrary unit),
110 a.u and 73 a.u., respectively, as compared to free enzyme. Red
shift and decreased uorescence intensity implied that the
microenvironment of aromatic amino acids was changed to more
polar, and hence it can be concluded that structure of the enzyme
was slightly disrupted by the added inhibitors (28). Among all the
conjugates, enzyme-pectin conjugate showed least uorescence
intensity followed by enzyme-starch and enzyme-glucose.
Fluorescence intensity of protein synthase has been reported to
decrease on binding with its potent inhibitor, 2-dithiole-3-ones (29).
Moreover, decreased uorescence intensity has also been observed
on denaturation of HIV-1 protease by urea (30) and streptomycin
acyltransferase by guanidine hydrochloride (29). Decreased uorescence intensity and red shift are good indicators for unfolding of
the enzyme structure. These results suggested that inhibition of
alcohol dehydrogenase by oxidized carbohydrate might be an irreversible inhibition and involve many factors including thiol groups
of enzyme, aldehyde groups of oxidized carbohydrates and structural changes occurring in the enzyme after conjugation. Oxidized
pectin was found to be potent inhibitor of alcohol dehydrogenase
and can be used for treatment of methanol toxicity. Alchohol dehydrogenase converts methanol to toxic formate which can be
avoided using a strong inhibitor such as oxidized pectin. This inhibitor can also be used in cosmetic to avoid the effect of added
alcohol on skin by inhibiting alcohol dehydrogenase present in the

FIG. 5. Fluorescence spectrophotometer analysis of the free and carbohydrate conjugated enzyme.

256

JADHAV ET AL.

cell. Thus the study of potent inhibitor of alcohol dehydrogenase has

its own signicance in many areas.
ACKNOWLEDGMENTS
We would like to thank Sanni Voutilainen (Aalto University,
Finland) and Dr. Harry Boer (VTT Technical Research Center,
Finland) for kind help in uorescence spectrophotometer analysis.
References
1. Shang, S., Zhu, L., and Fan, J.: Intermolecular interactions between natural
polysaccharides and silk broin protein, Carbohydr. Polym., 93, 561e573
(2013).
2. Li, J., Jiang, Z., Wu, H., Liang, Y., Zhang, Y., and Liu, J.: Enzymeepolysaccharide
interaction and its inuence on enzyme activity and stability, Carbohydr.
Polym., 82, 160e166 (2010).
3. Bill, R. M., Revers, L., and Wilson, I. B. H.: Protein glycosylation, pp. 412e413.
Kluwer Academic Publishers, Norwell (1967).
4. Darias, R. and Villalonga, R.: Functional stabilization of cellulase by covalent
modication with chitosan, J. Chem. Technol. Biotechnol., 76, 489e493 (2001).
5. Mc Clements, D. J.: Non-covalent interactions between proteins and polysaccharides, Biotechnol. Adv., 24, 621e625 (2006).
6. Altikatoglu, M. and Kuzu, H.: Improvement of enzyme stability via noncovalent complex formation with dextran against temperature and storage
lifetime, Pol. J. Chem. Technol., 12, 12e16 (2010).
7. Gomez, L., Ramirez, H. L., and Villalonga, R.: Stabilization of invertase by
modication of sugar chains with chitosan, Biotechnol. Lett., 22, 347e350 (2000).
8. Jadhav, S. B. and Singhal, R. S.: Conjugation of a-amylase with dextran for
enhanced stability: process details, kinetics and structural analysis, Carbohydr.
Polym., 90, 1811e1817 (2012).
9. Zhang, Y. and Rochefort, D.: Activity, conformation and thermal stability of
laccase and glucose oxidase in poly(ethyleneimine) microcapsules for immobilization in paper, Process Biochem., 46, 993e1000 (2011).
10. Keki, S., Batta, G., Bereczki, I., Fejes, Z., Nagy, L., Zajacz, A., Kandra, L.,
Kiricsi, I., Deak, G., Zsuga, M., and Herczegh, P.: New types of a-amylase
enzyme-inhibitory polysaccharides from D-glucal, Carbohydr. Polym., 63,
136e140 (2006).
11. Tsujita, T., Takaichi, H., Takaku, T., Sawai, T., Yoshida, N., and Hiraki, J.: Inhibition of lipase activities by basic polysaccharide, J. Lipid Res., 48, 358e365
(2007).
12. Blomstrand, R., Wintzell, H. O., Lof, A., McMartin, K., Tolf, B.-R., and
Hedstrom, K.-G.: Pyrazoles as inhibitors of alcohol oxidation and as important
tools in alcohol research: an approach to therapy against methanol poisoning,
Proc. Natl. Acad. Sci. USA, 76, 3499e3503 (1979).

J. BIOSCI. BIOENG.,
13. Becker, C. E.: Methanol poisoning, J. Emerg. Med., 1, 51e58 (1983).
14. El-Bakary, A. A., El-Dakrory, S. A., Attalla, S. M., Hasanein, N. A., and
Malek, H. A.: Ranitidine as an alcohol dehydrogenase inhibitor in acute
methanol toxicity in rats, Hum. Exp. Toxicol., 29, 93e101 (2010).
15. Weathermon, R. and Crabb, D. W.: Alcohol and medication interactions,
Alcohol Res. Health, 23, 40e54 (1999).
16. Saint-Leger, D.: Use of alcohol dehydrogenase inhibitors for cosmetic treatment of keratinous materials, US patent 2003/0138390 A1 (2003).
17. Zheng, Si-Y., Xu, D., Wang, H. R., Li, J., and Zhou, H.-M.: Kinetics of irreversible inhibition of yeast alcohol dehydrogenase during modication by 4,40 dithiodipyridine, Int. J. Biol. Macromol., 20, 307e313 (1997).
18. Manir, M., Kim, J. K., Lee, B.-G., and Moon, S.-S.: Tea catechins and avonoids
from the leaves of Camellia sinensis inhibit yeast alcohol dehydrogenase,
Bioorgan. Med. Chem., 20, 2376e2381 (2012).
19. Taber, R. L.: The competitive inhibition of yeast alcohol dehydrogenase by
2,2,2-triuoroethanol, Biochem. Educ., 26, 239e242 (1998).
20. Kagi, J. H. R. and Vallee, B. L.: The role of zinc in alcohol dehydrogenase, J. Biol.
Chem., 235, 3188e3192 (1960).
21. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: Protein measurement with the Folin-Phenol reagents, J. Biol. Chem., 193, 265e275 (1951).
22. Ellman, G. L.: Tissue sulfhydryl groups, Arch. Biochem. Biophys., 82, 70e77
(1959).
23. Belhadj, S., Hentati, O., Elfeki, A., and Hamden, K.: Inhibitory activities of Ulva
lactuca polysaccharides on digestive enzymes related to diabetes and obesity,
Arch. Physiol. Biochem., 119, 81e87 (2013).
24. Venkataramaiah, T. H. and Plapp, B. V.: Formamides mimic aldehydes and
inhibit liver alcohol dehydrogenases and ethanol metabolism, J. Biol. Chem.,
278, 36699e36706 (2003).
25. Jin, L., Szeto, K.-Y., Zhang, L., Du, W., and Sun, H.: Inhibition of alcohol dehydrogenase by bismuth, J. Inorg. Biochem., 98, 1331e1337 (2004).
26. Gabriel, L., Bonelli, G., and Dianzani, M. U.: Inhibition of colchicine binding to
rat liver tubulin by aldehydes and by linoleic acid hydroperoxide, Chem. Biol.
Interact., 19, 101e109 (1977).
27. Zaborska, W., Krajewska, B., Kot, M., and Karcz, W.: Quinone-induced inhibition of urease: elucidation of its mechanisms by probing thiol groups of the
enzyme, Bioorg. Chem., 35, 233e242 (2007).
28. Jana, S., Chaudhuri, T. K., and Deb, J. K.: Effects of guanidine hydrochloride on
the conformation and enzyme activity of atreptomycin adenylyltransferase
monitored by circular dichroism and uorescence spectroscopy, Biochemistry,
71, 1230e1237 (2006).
29. He, X., Reeve, A. M., Desai, U. R., Kellogg, G. E., and Reynolds, K. A.: 1,2Dithiole-3-ones as potent inhibitors of the bacterial 3-ketoacyl acyl carrier
protein synthase III (FabH), Antimicrob. Agents Chemother., 48, 3093e3102
(2004).
30. Szeltner, Z. and Polgar, L.: Conformational stability and catalytic activity of
HIV-1 protease are both enhanced at high salt concentration, J. Biol. Chem.,
271, 5458e5463 (1996).