1. CHARACTERIZATIONS OF HORSE SERUM CHOLINESTERASE FOR MOLECULAR
WEIGHT AND ANALYSIS UNDER ENVIRONMENTAL CONDITIONS
Lukas Isenhart, Sage Jordan, Leo Pezzementi
Biology Department
Birmingham-Southern College
Birmingham, Alabama
2 May, 2014

2. 2
Abstract:
After analysis of horse blood serum cholinesterase for substrate specificity, kinetic parameters,
substrate inhibition, molecular weight, and effects of temperature, pH, and [NaCl], we
determined the enzyme inherent in horse serum to be butyrylcholinesterase (BChE). The enzyme
experienced inhibition by substrates too large to bind to an actyl gorge of acetylcholinesterase
(AChE) and displayed responses to pH, temperature, and [NaCl] – these results confirm the data
of previous studies that also suggested the existence of BChE in horse serum.
1. Introduction
Acetylcholine, a neurotransmitter in humans and many other organisms, functions as an
excitatory signal for the contraction of skeletal muscle fibers. If not for cholinesterases (ChEs) –
which are a family of enzymes known to catalyze the hydrolysis of acetylcholine into choline
and acetic acid, thereby impeding the neurotransmitter’s ability to signal contraction – skeletal
muscles would be incapable of relaxation (Pohanka, 2011). Despite their differences in substrate
specificity and susceptibility to inhibitors, butyrylcholinesterase (BChE) and acetylcholinesterase
(AChE) both qualify as cholinesterases (Radic and Taylor, 2006; Monteiro et al., 2005). AChE is
easily observed in the central nervous system and in neuromuscular junctions, functioning as the
primary enzyme involved in the hydrolysis of acetylcholine, but BChE isn’t understood so well.
Though more commonly found in the liver and serum of organisms (Radic and Taylor, 2006),
people completely deficient in BChE can lead generally healthy, disease-free lives (Manoharan
et al., 2007). BChE is known to improve the hydrolysis rates of cocaine (Lynch et al., 1997),
protect mice from cocaine’s toxic effects (Morasco and Goldfrank, 1996), and protect the human
body from organophosphorous AChE inhibitors, suggesting a detoxification role in the body
(Saxena et al., 2006).
AChE is structurally determined to have much narrower substrate specificity than BChE,
as they bind specifically to ACh. BChE has a wider active site than Torpedo californica AChE,

3. 3
with the AChE having a catalytic gorge – lined mostly with aromatic residues – that reaches
halfway into the protein (Sussman et al., 1991). The existence of large aromatic residues from
the volume of the AChE aromatic gorge creates a long and narrow path for substrates, allowing
for higher selectivity of the enzyme at its active site (Sussman et al., 1991). Substrates that bind
to the choline binding site at the base of the AChE are smaller and more positively charged due
to the charge of the many different negatively-charged aromatic residues. The enzyme would
require more positively charged substrates or inhibitors than BChE. BChE’s active site is wider,
it has fewer aromatic residues lining the catalytic gorge and therefore is more voluminous and
more easily accommodating for various substrates, specifically for butyrylcholine (Nicolet et al.,
2003). This difference in substrate specificities can prove to be a valuable tool in the
identification of enzymes within sera. The larger and wider size of BChE’s catalytic gorge also
allows for easier binding of inhibitors that may bind in the gorge and at an active site by the acyl
pocket, there isn’t any substrate inhibition at peripheral site due to BChE’s possession of an
alanine instead of a tryptophan.
Vertebrate sera each display different levels of certain cholinesterases. The dominant
cholinesterase present in plasma of Portugese bird species Morus bassanus, Ciconia ciconia, and
Ardea cinerea is BChE (Santos et al., 2012). The plasma of other bird species, including
Neophron percnopterus, Cincloramphus cruralis, Anthus novaeseelandiae, and Accipiter nisus,
also displayed a dominance of BChE (Strum et al., 2008; Fildes et al., 2009). BChE, as it appears
in human blood serum, has an estimated molecular weight of 366,000 after ultracentrifugation
and gel electrophoresis (Dasan and Liddell, 1969). It is reported that BChE also appears in horse
serum and can be commercially purified, resulting in a molecular weight of 317,000 ± 12,000 in
a diluted phosphate (Lee and Harpst, 1971).

4. 4
With regard to inhibition of cholinesterases, human AChE displays more inhibition
resulting from pyrilium and selenopyrilium salts than does horse BChE while horse BChe is
more easily inhibited by thiopyrilium salts than human AChE, suggesting a method for
identification of the type of enzyme in horse serum by analyzing response to salts (Brestkin et
al., 1988). In rat brain capillaries, BChE displayed longer functionality at higher temperatures
and a higher activation energy at normal body temperatures than did AChE (Hernandez and
Catalan, 1986). Additionally, cholinesterase enzyme activity increased at pH values ranging from
5 to 10 before denaturing at pH of 11 (Eränkö, 1972). Based on the dominant appearance of
BChE in horse serum as suggested by Lee and Harpst (1971), we hypothesize that BChE will
again be the dominant cholinesterase in horse serum after filtration and analysis for molecular
weight. This study will determine the type of cholinesterase inherent in horse serum as well as
address its molecular characteristics and interactions with substrates and environment factors.
2. Materials and Methods
2.1. Preparation for and Execution of Electrophoresis
A sample containing purified cholinesterase was added directly to sample buffer (0.5 M Tris-
HCL, pH 6.8, 10% glycerol, 0.2% SDS, 0.25%bromophenol blue) at the same volume. This
solution and a solution containing molecular weight standards (proteins with standard weights of
250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kD) were heated for 4 minutes at 95ºC. Samples of
15µg for molecular weight standards and 7.5 and 12.5µg of purified cholinesterase (mixed with
sample buffer) were loaded into an electrophoresis chamber above 4-20% polyacrylamide gel.
The samples were electrophoresed in running buffer (0.25 M Tris, 0.2M glycine and 0.1% SDS)
at 200 V for 30 minutes.

5. 5
2.2. Staining SDS-polyacrylamide Gel
After electrophoresis the gel was removed from the gel cassette, incubated in dH2O for 5
minutes, then gently shaken for 1 hour in Bio-Safe Coomassie staining solution. The gel was
then incubated in dH2O overnight.
2.3. Measurement of Sample Migrations
The gel was removed from the dH2O and placed on a light box. Measurements of migration
distances were recorded in mm from the base of each well to the leading edge of each band in all
molecular weight and cholinesterase samples.
2.4. Plotting Data
Using Sigma Plot, log values of standard molecular weights were plotted in relation to distance
(mm). To determine the molecular weight of horse serum cholinesterase the equation
Y=M(X)+B was used and Y was solved for using values of slope, distance migrated, and y-
intercept. We then determined the anti-log of Y to find the molecular weight of horse serum
cholinesterase.
2.5. Effects of Environmental Temperature
Enzyme samples and blanks containing identical pH values and [NaCl] concentrations were
heated for ten minutes at individual temperatures ranging between 4º and 90ºC, where 4ºC acted
as the control temperature and other higher temperatures were experimental values. After
heating, each sample was chilled in an ice bath to preserve the enzyme’s resulting shape.
Spectrophotometric analyses were staggered in five minute intervals; each analysis involved
spectrophotometry for 2 minutes while allowing time for other preparatory processes. After each
2 minute sample-analysis at ice bath temperature, a sample’s resulting slope was converted into

6. 6
percent control activity. Staggered time intervals continued until every blank and sample
experienced analysis by the spectrophotometer. Data were then plotted using Sigma Plot.
2.6. Effects of Environmental pH
Enzyme samples existed in Ellman’s solutions with a pH range of 4.5-8.5,where the pH value of
7 acted as a control. The spectrophotometer was blanked before each measurement, where an
experimental pH value would receive an enzyme and incubate at room temperature for
approximately 10 minutes before receiving a dose of substrate with buffer before
spectrophotometric analysis for two minutes. Experimental analyses were staggered every 5
minutes, resulting slopes were converted to percent control activity, then the converted data was
organized using Sigma Plot software. Complacency
2.7. Effects of Environmental [NaCl]
This procedure is similar to the analysis of pH’s effects on enzyme activity. Aside from replacing
pH addition with the addition of [NaCl] molarities ranging from 0 to 2.5 M, where 0M acted as a
control, the procedure for spectrophotometric analysis of [NaCl] and its effects on horse serum
cholinesterase is virtually identical to that of environmental pH.

7. 7
3. Results
Figure 1. Substrate specificities of horse serum cholinesterases. Data inidicate initial velocities of
ATCh’s and BTCh’s after Ellman esterase assay of serial dilutions. Acetylthiocholine
specificities represented by (●), butyrlthiocholine represented by (○).
Horse serum cholinesterases exhibited higher velocities of cholinesterase activity at higher
substrate concentrations. BTCh exhibited more substrate activity than ATCh, but activity level
out at approximately 1.0mM (Fig. 1.).
Table 1. Kinetic parameters for hydrolysis of ATCh and BTCh by horse serum cholinesterase.
Substrate concentration (Km) calculated at 0.5 maximum velocity; maximum velocity (Vmax) was
also calculated. Standard error suggests N=14.
Substrate Km (mM ± SE) Vmax (µm/mM ± SE)
ATCh 0.34 ± 0.08 43.29 ± 4.24
BTCh 0.28 ± 0.06 59.16 ± 3.20
Lower concentrations of BTCh still managed to produce higher maximum velocities during
hydrolysis than did ATCh, though SE values do suggest overlapping standard errors for
concentrations (Table 1.). Maximum velocity values differed even when including standard error
values, suggesting that BTCh has a higher maximum velocity than ATCh (Table 1.).

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Inhibitor Concentration (M)
10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2
PercentControlChE
0
20
40
60
80
100
120
140
Figure 2. Percent activity of horse serum cholinesterase as a function of inhibitor concentrations.
This depicts data resulting from the addition of inhibitors Iso-OMPA (○), BW284C51 (▼), and
Eserine (●) into Ellman esterase assay. The results analyzed as percent activity in regard to
inhibitor concentration (M). N=4 for Eserine and BW284C51while N=5 for Iso-OMPA.
Ic50 values for inhibitors include 1.7294E-007 (Eserine), 2.5548E-006 (Iso-OMPA), and
3.8881E-005 (BW284c51) (Fig. 2.). As inhibitor concentrations increased, percent control ChE
decreased; Eserine percent control ChE decreased sooner than other inhibitors while BW284C51
decreased at slowest rate (Fig. 2.).

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Table 2. Inhibitor dimensions of molecular modeling by SPARTAN software.
Inhibitor Length ± SE Width ± SE
BW284c51 20.92 ± 0.53 5.24 ± 0.29
Decamethonium 18.37 ± 0.33 3.94 ± 0.28
Ethopropazine 9.69 ± 0.28 8.72 ± 0.35
Iso-OMPA 9.58 ± 0.35 8.09 ± 0.53
Decamthionium and BW284c51 exhibited molecular shapes that were longer and thinner than
the molecular shapes of Ethopropazine and Iso-OMPA, which were shorter and wider (Table 2.).
Distance Migrated (mm)
10 20 30 40 50 60 70
MolecuarWeight
103
104
105
106
Figure 3. Distance migrated (mm) after electrophoresis for 30 minutes at 200V by samples (µg)
of molecular weight standards and purified cholinesterase. Lane 1 of electrophoresis gel
represents molecular weight standard to specified masses (kD), lane 2 contains 12.5µg purified
cholinesterase while Lane 3 contains 7.5µg purified cholinesterase.
Standard molecular weights displayed an inverse relationship with distance migrated – larger
molecular weights (the bands in the gel) travelled shorter distances than smaller molecular
weights (Fig. 1). The molecular weight of horse serum cholinesterase was determined to be
66456 + 2839; N=10 (Fig. 1.).
Lanes
1 2 3
10kD
15kD
20kD
25kD
37kD
50kD
75kD
100kD
150kD
250kD

10. 10

11. 11
Figure 4. Varying Effects of Environmental Factors on Horse Serum Cholinesterase. Graph A
displays effects of pH in a sigmoidal fashion, where percent control activity increased until an
approximate pH value of 8; data normalized to pH of 7. Graph B displays effects of Temperature
as measured in degrees Celsius, lacking dramatic changes resulting from temperature until a
50°C where the enzyme percent activity declines and flatlines at 0; data normalized to 100°C
Graph C displays the negative correlation between increasing molarity of [NaCl] and percent
control ChE activity; data normalized to 0 M. Data are the mean of standard error and 9
determinations.
Horse Serum Cholinesterase activity experienced positive responses to less acidic
environments, with pH values displaying a sigmoidal relationship (Fig. 1.). Activity as a function
of pH, lower values led to less control cholinesterase activity whereas higher function of pH
values led to the higher percent control cholinesterase activity (Fig. 1A). Standard SE values
overlap between data points 8 and 9, suggesting the insignificance of apparent reduction in
percent control cholinesterase activity (Fig. 1.). Cholinesterase activity in horse serum also
experienced a sharp decline in functional ability after relatively constant responses to
environmental temperature, but when temperature values rose above 40°C percent control
cholinesterase activity dipped and flat-lined at 0% around 70°C (Fig. 1.). Horse serum

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cholinesterases additionally experienced a negative relationship with rising molarities of [NaCl],
with the highest molarity of 2.5 displaying the least percent control cholinesterase activity (Fig.
1.).
4. Discussion
Horse serum cholinesterases displayed a highly-accomodating substrate specificity for
larger, wider substrates like Ethopropazine and Iso-OMPA, suggesting that the enzyme in horse
serum possessed an adequately-sized catalytic gorge to allow the entrance of such inhibitors (Fig.
2.). Enzyme activity increased as butyrylthiocholine concentrations increased, also suggesting
that the enzyme had increased binding capabilities with substrates of varying lengths and widths,
a common characteristic of BTChE and an idea common with previous research (Sussman et al.,
1991) (Fig. 1.).
Molecular modeling represents BW284c51and Decamethonium as inhibitors exhibiting
substrate specifity for long and thin catalytic gorges of enzymes while Ethopropazineand and
Iso-OMPA exhibit larger and wider enzyme specificities (Table 2.). It appears that BChE is the
dominant enzyme available in horse serum because the enzyme is accepting of both ACh and
BCh while also displaying more inhibition to varying sizes and shapes of inhibitors. The
existence of 14 large aromatic residues in the aromatic gorge of AChE explains its high
specificity, as the aromatic residues fill the gorge and leave little remaining space for the
entrance of substrates (Sussman et al., 1991). Because of this narrower gorge in AChE , it would
be expected that BCh – a larger, wider molecule – would produce slower kinetic interactions
with that particular enzyme because the substrate simply wouldn’t fit inside the catalytic gorge
(Table 2). But within this experiment the enzyme displayed an increased kinetic relationship with
BCh and ACh, which again suggests the existence of BChE as the enzyme inherent in horse

13. 13
serum due to its more accommodating substrate specificity resulting from ist possession of only
8 aromatic amino acids (Table. 1.). This accomodation also helps explain BChE’s effectiveness
as a detoxifying agent in mice and humans (Morasco and Goldfrank, 1996; Saxena et al., 2006;
Lynch et al., 1997).
Environmental factors affected horse serum cholinesterase in various manners, with
enzymes functioning best at a pH of 8, a temperature of 0 to 40°C, and at low (0 – 0.5M)
concentrations of [NaCl] (Fig. 4.). These data indicate optimum conditions for the horse serum
cholinesterase, further identify the enzyme to be BChE (Brestkin et al., 1988; Hernandez and
Catalan, 1986; Eränkö, 1972). Though data for molecular weight differed from previous
literature – likely a result oft he differences between monomer and tetramer analyses – data in
this study pertaining to environmental factors, substrate specificities, and enzyme kinetics
correspond with a previous studies’ assertions that the enzyme inherent in horse serum is BChE
(Lee and Harpst, 1971; Sussman et al., 1991). The molecular weight of horse serum
cholinesterase in this experiment was deterimined to be (66456 + 2839; N=10) (Fig. 1.).
However, there are multiple issues with this study. All treatments to determine
environmenatal variability and protein function required staggered measurement periods by
spectrophotometry, inconsistent machine-blanking and transfer times between the addition of
substrates and actual measurement of cuvettes in the spectrophotomer likely led to discrepencies
in data. Data measurements were rushed, future studies should eliminate the propensity for
measurement mistakes by individually analyzing test solutions.
5. Acknowledgements
We would like to thank Birmingham-Southern College both for generously supplying materials
and for allowing the use of facilities.

14. 14
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