Production of butyrylcholinesterase

Butyrylcholinesterase is produced in a purity of at least 90% by subjecting plasma fraction IV-4 alone or in admixture with fraction IV-1 to both anion exchange chromatography and affinity chromatography.

The present invention relates to butyrylcholinesterase and more 
particularly to the production and use thereof. 
BACKGROUND OF THE INVENTION 
Butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8, also known as 
pseudocholinesterase) is an enzyme found in the plasma, among other 
tissues, of all vertebrates in which it has been sought (Silver, A. The 
Biology of Cholinesterase, North-Holland, Amsterdam, 1974). The existence 
of this enzyme in human plasma was formally demonstrated 50 years ago 
(Alles, G. A. and Hawes, R. C., J. Biol. Chem., 133:375, 1940), but its 
normal physiological role remains unknown. However, butyrylcholinesterase 
is responsible for the hydrolysis and inactivation of muscle relaxants 
such as succinylcholine and related anaesthetics (LaDu, B. M., Ann. N.Y. 
Acad. Sci., 179:648, 1971), substances currently in clinical use. 
Butyrylcholinesterase is also responsible for degrading the majority of 
the cocaine ingested by a drug abuser (Stewart, D. J. et al., Life Scie., 
20:1557, 1977; Jatlow, P., et al., Anesth. Anag., 58:235, 1979; Stewart, 
D. J. et al., Clin. Pharmacol. Ther. 25:464, 1979). 
The gene for human butyrylcholinesterase exists as a "wild-type" (normal) 
allele and several defective alleles which are present in as much as 5% of 
the population (reviewed in Whittaker, M., Anaesthesia, 35:174, 1980; 
Evans, R. T., CRC Crit. Rev. Clin. Lab. Sci., 23:35, 1985). In 
approximately 1 in 2800 individuals, their genotype results in a severe 
deficiency in butyrylcholinesterase. When these individuals are treated 
with succinylcholine during the induction of general anaesthesia prior to 
surgery, the resulting paralysis is greatly prolonged compared to the 
normal population. During this period the patient is unable to breathe, a 
condition known as apnea, and must be artificially ventilated until the 
succinylcholine is degraded by secondary mechanisms. This is considered to 
be a potentially life-threatening situation. Butyrylcholinesterase 
activity may also be reduced sufficiently from normal levels to induce 
succinylcholine sensitivity during pregnancy (Wildsmith, J. A. W., 
Anaesthesia, 27:90, 1972; Weissman, D. B. and Ehrenwerth, J., Anesth. 
Analg., 62:444, 1983), by certain diseases such as hepatitis (Singh, D. C. 
et al., J. Ind. Med. Assoc.. 66:49, 1976) or as a consequence of various 
medications (Foldes, F. F., Enzymes in Anaesthesiology, Springer-Verlag, 
N.Y., 1978). 
Toxicologically, cocaine is also well tolerated by the majority of the 
population. Nevertheless there is a small incidence of sudden death 
related to acute cocaine abuse see Clouet, D. et al., Mechanisms of 
Cocaine Abuse and Toxicity, NIDA Research Monograph 88; Johanson, C. and 
Fischman, M. W., Pharmacol. Rev. 41:3, 1889). The physiological basis for 
this difference in susceptibility is not known. However, it has been 
argued that a deficiency in butyrylcholinesterase could contribute to an 
individual's sensitivity (Stewart, D. J. et al, supra, 1979; Jatlow, P., 
(supra, 1979; Anton, A. H., Drug Intell. Clin. Pharm., 22:914, 1988; 
Devenyl, P., Ann. Int. Med.. 110:167, 1989). 
A number of compounds of the organonhosphate type are used as pesticides 
(e.g. malathion) or neurotoxic chemical warfare agents (e.g. soman; 
Silver, A., supra, 1974; Aldridge, W. N. and Reiner, E., Enzyme Inhibitors 
as Substrates, North-Holland, Amsterdam, 1972). These compounds exert 
their toxic effects by inhibiting acetylcholinesterase, an enzyme found on 
erythrocytes and at cholinergic synapses where it plays an essential role 
in proper neurological and neuromuscular function. Butyrylcholinesterase 
is also inhibited by these compounds because of the similarity of its 
active site to that of acetylcholinesterase (Soreq, H. and Prody, C. A., 
in: Computer Assisted Modeling of Receptor-Ligand Interactions, Alan R. 
Liss, N.Y., 1989). Therefore, plasma butyrylcholinesterase and erythrocyte 
acetylcholinesterase afford some protection to synaptic 
acetylcholinesterase from these neurotoxins since the toxins themselves 
are inactivated by the reactions that inhibit these enzymes. Only those 
toxin molecules that survive in the circulatory system without reacting 
with the plasma cholinesterases are capable of attacking synaptic 
acetylcholinesterase. It follows that an individual's susceptibility to 
these compounds is determined in part by the amount of cholinesterase 
present in the blood. It has been shown that administration of bovine 
serum acetylcholinesterase to mice increases their resistance to 
organophosphate poisoning (Rauch, L., Ashani, Y., Levy, D., de la Hoz, D., 
Wolfe, A. D. and Doctor, B. P., Biochem. Pharmacol., 38:529, 1989). 
Butyrylcholinesterase is present in human plasma, serum or whole blood. 
Methods have been developed for obtaining butyrylcholinesterase from 
plasma. These can be classified in two groups: those in which the plasma 
is first fractionated by a precipitation method and those in which the 
plasma is chromatographed directly. 
The earliest methods employed ethanol or ammonium sulfate as precipitants. 
Cohn et al. (J. Amer. Chem. Soc., 68:459, 1946) found that the majority of 
"plasma esterase" partitioned into one fraction, designated IV-4, during 
the fractionation of human plasma by ethanol. Subsequently, Surgenor and 
Ellis (J. Amer. Chem. Soc., 76:6049, 1954) extended this method by 
repetitive precipitations to produce human butyrylcholinesterase 
(designated fraction IV-6-4) of about 20% purity with a yield of 7%. An 
intermediate fraction (IV-6-3) obtained by this procedure was further 
purified by chromatography on hydroxylapatite and Dowex anion exchange 
resin (Malstrom et al., Acta Chem. Scand., 10:1077, 1956). While this last 
procedure produced butyrylcholinesterase of high (at least 80%) purity, 
the overall recovery was poor, no better than 3%. 
Several other methods have been developed which employ ammonium sulfate 
precipitation as an early step. These procedures either produced crude 
enzyme (no more than 10% purity; Goedde, H. W. et al., Human Genet., 
1:311, 1965) or incorporated preparative electrophoresis, a technique 
which is impractical for any large scale process, to achieve higher 
degrees of purity with about 10% yields (Svensmark, O. and Kristensen, P., 
Biochim. Biophys. Acta. 67:441, 1963; Haupt, H. et al., Blut, 14:65, 
1966). Because of these drawbacks, these methods have been abandoned for 
any application requiring highly purified butyrylcholinesterase in large 
(commercial) quantities. 
Present methods employ the chromatographic purification of 
butyrylcholinesterase from defibrinated plasma and are based on the 
ability of the enzyme to bind to conventional anion exchange resins under 
acidic (pH 4) conditions (Connell, G. E. and Shaw, R. W., Can. J. Biochem. 
Physiol., 39:1019, 1961). When optimized, anion exchange chromatography at 
pH 4 of human plasma achieves a 400- to 800-fold purification of 
butyrylcholinesterase (i.e. to a purity of 2-4%) in a single step (Das, P. 
K. and Liddell, J., Biochem. J., 116:875, 1970; Meunsch, H. et al., Eur. 
J. Biochem., 70:217, 1976). The subsequent steps used by these groups to 
further purify the enzyme were supplanted by affinity chromatography on 
procainamide-agarose (Lockridge, O. and LaDu, B. N., J. Biol. Chem., 
253:361, 1978) which achieved a two-step purification of 
butyrylcholinesterase to 88% purity with a 70% yield. Further refinements 
of the method added an additional anion exchange step at pH 7 (Lockridge, 
O. and LaDu, B. N., J. Biol. Chem., 287:12012, 1982; Lockridge, O. et al., 
J. Biol. Chem., 262:549, 1987), producing virtually homogeneous enzyme 
with an overall yield of 30-40%. 
SUMMARY OF THE INVENTION 
In accordance with an aspect of the present invention, 
butyrylcholinesterase is recovered from the plasma fraction known as IV-4 
or from a mixed plasma fraction of fractions IV-4 and IV-1 by use of a 
combination of anion exchange chromatography and affinity chromatography. 
In accordance with a preferred aspect, the procedure involves an initial 
anion exchange chromatography, followed by affinity chromatography, with 
the above two steps repeated at least one more time.

DETAILED DESCRIPTION OF THE INVENTION 
The recovery of butyrylcholinesterase from the noted fractions by use of a 
combination of anion exchange chromatography and affinity chromatography 
can produce butyrylcholinesterase in a purity of at least 80%, (preferably 
at least 90%) and in yields of at least 30% based on the average amount of 
butyrylcholinesterase present in human plasma. 
The anion exchange column may be any one of a wide variety of anion 
exchange columns. As representative materials which are effective for 
recovery of butyrylcholinesterase from the noted fractions there may be 
mentioned media comprising amines, tertiary amines or quaternary amines 
covalently bound to a supporting medium such as dextran, agarose, 
polyacrylamide, polystyrene, silica or acrylic or vinyl polymers. A 
preferred column is a crosslinked diethylaminoethyl-agarose column (for 
example a DEAE-Sepharose Fast Flow medium sold by Pharmacia). In a 
preferred embodiment, such anion exchange chromatography is effected at a 
pH of from 4.0 to 4.5. 
The affinity chromatography may be accomplished by any of a variety of 
materials suitable for recovering butyrylcholinesterase by affinity 
chromatography. As representative examples of such materials there may be 
mentioned any substrate or reversible inhibitor of butyrylcholinesterase, 
an antibody specific for butyrylcholinesterase or a lectin particularly 
any of those capable of binding sialic acid residues with high affinity, 
any or all of which could be covalently bound, directly or through a 
"spacer", to a supporting medium suitable for chromatography such as those 
mentioned above. A preferred material is procainamide 
(p-amino-N-(2-diethylaminoethyl)benzamide) covalently coupled to 
aminohexanoic acid-agarose. In accordance with a preferred embodiment, the 
affinity chromatography is run within the range of pH (6-9) in which the 
butyrylcholinesterase activity is optimal. 
The hereinabove noted human plasma fractions are commercially available and 
may be produced by known procedures. In particular as known in the art 
such fractions are obtained from plasma by use of ethanol precipitation. 
Butyrylcholinesterase, whether in plasma or in a highly purified form, is 
not sufficiently stable at elevated temperatures to permit 
heat-inactivation of any residual viruses in a preparation of this enzyme. 
However, we have determined that the enzyme is unaffected by extended 
treatment with tri-n-butyl phosphate and sodium cholate, in concentrations 
sufficient to inactivate viruses such as hepatitis B virus, non-A, non-B 
hepatitis virus and HTLV-III (Horowitz, B, Wiebe, M. E., Lippin, A. and 
Stryker, N. H., Transfusion, 25, 516, 1985; Edwards, C. A., Piet, MPJ, 
Chin, S. and Horowitz, B., Vox Sanquinis, 52:53, 1987). Since the solvent 
and detergents used in this procedure are readily removed by the 
chromatographic methods used to purify butyrylcholinesterase, the viral 
inactivation may be performed at any stage of the purification. Therefore, 
by including this procedure in the purification process and by passing the 
purified enzyme through a filter with an effective pore size not larger 
than 0.22 microns, a sterile and virally-inactive preparation of fully 
active butyrylcholinesterase may be obtained. 
The invention will be further described with respect to the following 
examples; however, the scope of the invention is not limited thereby. 
Unless defined otherwise, all technical and scientific terms used herein 
have the same meaning as commonly understood by one of ordinary skill in 
the art to which this invention belongs. Although any methods and 
materials similar or equivalent to those described herein can be used in 
the practice or testing of the present invention, the preferred methods 
and materials are now described. All publications mentioned hereunder are 
incorporated herein by reference. 
MATERIALS AND METHODS 
Enzyme Assays 
Butyrylcholinesterase is assayed by monitoring the decrease in optical 
density at 240 nm during the hydrolysis of 50 .mu.M benzoylcholine at 
25.degree. C. in M/15 phosphate buffer at pH 7.4 (Kalow, W. and Lindsay, 
A., Can. J. Biochem., 33:868, 1988). The concentration of enzyme is 
adjusted so that the rate of hydrolysis is constant for at least one 
minute. Hydrolysis rates are calculated from the difference in extinction 
coefficients between products and substrate of 6700 M.sup.-1 cm.sup.-1. 
One unit of activity is that amount of enzyme required to hydrolyze 1 
.mu.mol of substrate per minute. Under these conditions 1 mg of 
butyrylcholinesterase is equivalent to 200 units of enzyme activity, 
alternatively expressed a specific activity of 200 units/mg (Lockridge and 
LaDu, supra, 1982). Other substrates or assay methods could also be used. 
Electrophoresis 
Separations in the presence of SDS are performed according to Laemmli 
(Nature, 227:680, 1970). Non-denaturing gels are run according to Juul 
(Clin. Chem. Acta., 19:208, 1968) and stained for esterase activity using 
.alpha.-naphthyl butyrate as a non-specific substrate able to detect 
contaminating esterases if present as well as butyrylcholinesterase 
(Harris, H. et al., Nature, 196:12296, 1862). 
Chromatography Medium 
For anion exchange chromatography, a mechanically and chemically stable 
medium, such as DEAE-Sepharose Fast Flow (Pharmacia), is preferred to 
facilitate cleaning and regeneration. Other anion exchange media, could 
also be used. 
For affinity chromatography, procainamide 
(p-amino-N-(2-diethylaminoethyl)benzamide) is covalently coupled to 
6-aminohexanoic acid-agarose via a condensation reaction mediated by 
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The reaction is run 
in H.sub.2 O in a volume about 5 times that of the swollen agarose which 
contains at least 15 .mu.mol of carboxyl groups per ml of swollen gel. To 
this is added a 4-fold molar excess (with respect to the immobilized 
carboxyl groups) of procainamide and a 40-fold molar excess of EDC. The 
mixture is stirred for 24 hours; during the first 4 hours the pH is 
maintained at about 5.28 by the addition of 0.1 M HCl. The gel is washed 
free of unreacted material and urea and packed into an appropriate column. 
The derivatization is nearly quantitative under these conditions, at least 
15 .mu.mol of procainamide are coupled to each ml of agarose. 
Protein Concentration 
The concentration of protein during the purification is monitored by a 
colorimetric method (Bradford, M. M., Anal. Biochem., 72:248, 1976) or by 
optical absorbance at 280 nm. The concentration of purified 
butyrylcholinesterase is determined by its absorbance at 280 nm assuming 
an extinction coefficient of 1.8 ml.mg.sup.-1 cm.sup.-1 (Lockridge et al., 
supra, 1979). 
Purification Process 
The starting material is human plasma pooled from multiple individuals. The 
plasma may be used as is, either fresh or "outdated", or after 
conventional treatments to produce "cryo-poor" or defibrinated plasma. The 
plasma is then treated by a series of ethanol additions and pH adjustments 
as described (Cohn, E. J. et al., supra, 1946) or by any modification of 
this method designed to effect an equivalent fractionation of the plasma. 
The precipitate designated Fraction IV-4 or the combined precipitates 
designated Fraction IV-1 plus IV-4 are collected. This material may be 
immediately processed further or stored frozen for later use. 
The butyrylcholinesterase and other components of the ethanol precipitate 
are resolubilized in 4-5 volumes (relative to the weight of the protein 
precipitate) of buffer (20 mM acetate, citrate or the like) or of 
deionized water, titrated to pH 4 with the acidic component of the buffer, 
clarified by centrifugation or filtration and dialyzed to near equilibrium 
against 25 volumes of the same buffer, the order of the latter three steps 
being unimportant. In this and all subsequent steps in which the 
composition of the butyrylcholinesterase solution must be adjusted, the 
methods of dialysis, gel filtration or dilution may be used. 
The butyrylcholinesterase solution is then applied to an anion exchange 
column (no larger than 1/2 the volume of the applied solution) 
equilibrated with the same buffer as used for dialysis. The column is 
washed with this buffer to remove the majority of unbound material and the 
butyrylcholinesterase is eluted by increasing the ionic strength of the 
buffer flowing through the column. That part of the eluate containing 
butyrylcholinesterase is titrated to pH 7 and diluted with an 
approximately equal volume of water. This solution is then applied to a 
procainamide-agarose column equilibrated with a moderate ionic strength 
buffer at pH 7.0-7.4 (such as 100 mM NaCl, 20 mM phosphate). The volume of 
this column is about 1/10 the volume of the original protein precipitate. 
The column is washed with about 2 volumes of equilibration buffer and the 
butyrycholinesterase is eluted with a gradient of increasing ionic 
strength, to the equivalent of 1 M NaCl, in a total of 7-10 column 
volumes. That part of the eluate containing butyrylcholinesterase is 
dialyzed to near equilibrium against 15-20 volumes of a low ionic strength 
buffer, such as 20 mM phosphate, at pH 7.4. This solution is applied to a 
second anion exchange column equilibrated with the same buffer used for 
dialysis. The volume of this column is about one tenth that of the first 
anion exchange column. The butyrylcholinesterase is eluted with a gradient 
of increasing ionic strength to the equivalent of 250 mM NaCl in a total 
of 4 column volumes. That part of the eluate containing 
butyrylcholinesterase is diluted with one volume of a low ionic strength 
buffer, such as 20 mM phosphate at pH 7.4. This solution is applied to a 
second procainamide-agarose column of about one tenth the volume of the 
first. This column is washed and eluted as for the first except that all 
volumes are adjusted to maintain the same proportions relative to the size 
of the column. The butyrylcholinesterase eluting from this column is 
sufficiently free of other plasma proteins by the criteria of 
electrophoretic homogeneity and enzyme activity. 
EXAMPLE 
Purification of Butyrylcholinesterase From Human Plasma Fraction IV-4 
The quantitative analyses of each step in this preparation is found in 
Table 1. The starting material was Fraction IV-4 (Lot No. 0208, obtained 
from American Red Cross recovered human plasma and processed by Baxter, 
Hyland Division). This material was frozen as a protein precipitate and 
stored at -70.degree. C. until used. Twelve hours prior to extraction, the 
precipitate was transferred to -20.degree. C. All subsequent steps were 
performed at 4.degree.-6.degree. C. 
1825 grams of Fraction IV-4 was extracted with 9.1 liters of 20 mM sodium 
acetate buffer, pH 4.0, by mechanical stirring for 18 hours. The resulting 
suspension was centrifuged for 90 minutes at 13,700 x g and the combined 
supernatants were dialyzed overnight against 45 liters of the same buffer. 
The following morning the sample was transferred to a fresh 45-liter batch 
of dialysis buffer for an additional 24 hours. Following dialysis, the pH 
required no further adjustment but a flocculence had developed which was 
removed by centrifugation as before. The final volume of this solution 
(EXTRACT) was 10.9 liters. 
The extract was loaded on a column packed with 5 liters of DEAE-Sepharose 
Fast Flow previously equilibrated with the same buffer used for extraction 
and dialysis. The column was washed with 10 liters of this buffer and then 
eluted with 5 liters of 200 mM NaCl in the same buffer. Those fractions 
containing butyrylcholinesterase were pooled in a final volume of 3.3 1 
(DE-I pool). 
The DE-I pool was titrated with 380 ml of 0.8 M sodium phosphate dibasic to 
pH 7.0 and diluted with 4 liters of water. This solution was then loaded 
on a column packed with 200 ml of procainamide-agarose previously 
equilibrated with 20 mM phosphate buffer, pH 7.4, 100 mM NaCl, 1 mM EDTA. 
The column was washed with 400 ml of the same buffer and with 600 ml of 
the same buffer but with 200 mM NaCl. The column was then eluted with a 
1.4-liter linear gradient to 1 M NaCl. Those fractions containing 
butyrylcholinesterase were pooled in a final volume of 1 liter (PAM-I 
pool). 
The PAM-I pool was dialyzed overnight against 18 liters of 20 mM phosphate 
buffer, pH 7.4, and loaded on a column packed with 480 ml of 
DEAE-Sepharose Fast Flow previously equilibrated with the same buffer. The 
column was immediately eluted with a 2-liter gradient from 80 to 250 mM 
NaCl in the same buffer. Those fractions containing butyrylcholinesterase 
were pooled in a final volume of 270 ml (DE-II pool). 
The DE-II pool was dialyzed overnight against 2 liters of 20 mM phosphate 
buffer, pH 7.4, 100 mM NaCl, 1 mM EDTA, and loaded on a column packed with 
20 ml of procainamide-Sepharose equilibrated with the same buffer. The 
column was washed with 120 ml of 200 mM NaCl in the same buffer and eluted 
with a 280 ml gradient to 1 M NaCl. Those fractions containing 
butyrylcholinesterase were pooled in a final volume of 200 ml (PAM-II 
pool). 
The PAM-II pool was concentrated to a final volume of 26 ml in a pressure 
filtration device fitted with a 50,000 nominal molecular weight cut-off 
filter. The solution was then dialyzed overnight against 2 liters of 20 mM 
phosphate buffer, pH 7.4, 184 mM NaCl, 0.8 mM EDTA. The purified 
butyrylcholinesterase was stored at 4.degree. C. 
Characterization of Purified Butyrylcholinesterase 
The principal criterion for evaluating the purified enzyme is its catalytic 
activity. When measured using benzoylcholine as the substrate, the 
specific activity of the preparation described here was 181 Units/mg 
(Table 1). This activity is 91% of the maximum reported for the 
homogeneous enzyme and indicates the presence of no more than 9% 
contaminating proteins and no more than 9% inactivation of the 
butyrylcholinesterase during its purification. Using a second substrate, 
propionylthiocholine, the specific activity of this preparation was 700 
Units/mg, equal to the previously repdrted maximum (Lockridge and LaDu, 
supra, 1982). 
The product of this preparation was also analyzed on SDS gels. By this 
method, purified butyrylcholinesterase is composed of a major polypeptide 
of M.sub.R 90,000, corresponding to the monomeric subunit, and a minor 
band of M.sub.R 180,000. It has previously been shown that purified human 
butyrylcholinesterase includes a M.sub.R 180,000 dimer (presumably 
crosslinked by covalent bonds other than disulfides) of the predominant 
monomeric subunit (Lockridge et al., supra. 1979). Therefore both high and 
low molecular weight bands on the SDS gel comprise butyrylcholinesterase. 
A number of minor contaminating polypeptides account for a small fraction 
of the protein present, consistent with the high purity of the enzyme 
estimated from its specific activity. 
During electrophoresis under non-denaturing conditions, the native 
butyrylcholinesterase migrates as a single band with a M.sub.R 340,000. 
This agrees with the mass of the tetrameric enzyme measured by 
hydrodynamic methods (Haupt, H., et al., supra. 1966). Furthermore, 
histochemical staining of this type of gel for esterase activity 
demonstrates that the enzyme activity is in fact associated with this 
protein and that no other esterases are detectable (not shown). 
The purification method itself appears to be reproduceable. Table II 
compares the results of the purification illustrated here (preparation 1) 
with two others. The yields range from 35% to 43% and the purities from 
91% to virtual homogeneity. The compositional features of all three 
preparations, both on non-denaturing and SDS gels, are similar. 
The buffer selected for the storage of the purified butyrylcholinesterase 
is an isotonic phosphate buffered saline, a suitable vehicle for 
injection. The enzyme is remarkably stable when refrigerated at high 
concentrations (3-6 mg/ml) in the buffer. During a 3 month period, less 
than 8% of the original activity was lost. This indicates that an 
injectable formulation of this enzyme can be stored for prolonged periods. 
The combination of the ethanol-precipitation method to produce Fraction 
IV-4 with the chromatographic techniques herein described affords at least 
a 10-fold scale-up of the previously most efficient procedure for 
purifying butyrylcholinesterase. Specifically, the volumes and amounts of 
protein involved in a procedure starting with Fraction IV-4 are 10% or 
less of those with whole plasma as the starting material. Moreover, the 
size of the first anion exchange column, relative to the amount of enzyme 
produced, has been reduced 20-fold from the original methods referenced 
above. These two improvements and the purity, stability and activity of 
the butyrylcholinesterase isolated by this method make the commercial 
production of this enzyme feasible. 
The butyrylcholinesterase produced in accordance with the present invention 
has a purity of at least 90% and has a wide variety of potential uses. 
One use is to reverse the effects of succinylcholine in patients having a 
deficiency in butyrylcholinesterase to prevent apnea during general 
anaesthesia. Thus, in accordance with an aspect of the present invention, 
a person have a deficiency in butyrylcholinesterase is treated to reverse 
the effects of succinylcholine by administering butyrylcholinesterase 
which has a purity of at least 80% (and preferably at least 90%) in an 
amount effective to reverse the effects of succinylcholine. An effective 
treatment with butyrylcholinesterase could preceed the administration of 
succinylcholine in those cases in which a deficiency in 
butyrylcholinesterase is known or suspected. Alternatively, treatment with 
butyrylcholinesterase could follow administration of succinylcholine when 
an abnormal response to the drug is manifested. 
In general, the butyrylcholinesterase is administered in an amount of at 
least 0.01 mg/kg body weight (and preferably at least 0.1 mg/kg body 
weight). In general, the amount need not exceed 4.0 mg/kg body weight (and 
preferably need not exceed 0.4 mg/kg body weight. 
The butyrylcholinesterase of such purity is employed in combination with a 
pharmaceutically acceptable carrier. The carrier which is selected is 
dependent upon the method of administration. Such methods of 
administration include: intravenous injection, intramuscular injection, 
inhalation of an aerosol form or as eye drops. The preferred form of 
administration could be intravenous injection, for which method the 
aforementioned carrier, phosphate-buffered isotonic saline, would be 
preferred. For other methods of application, the inclusion of carrier 
proteins (such as human serum albumin), anti-oxidants, surfactants, 
anti-foaming agents may be appropriate. 
In general, the butyrylcholinesterase of at least 90% purity is present in 
such pharmaceutical composition in an amount of from 3 mg/ml to 30 mg/ml 
or more. 
The butyrylcholinesterase produced in accordance within the present 
invention may also be employed to inactivate other pharmaceuticals, such 
as chloroprocaine, mivacurium, vecuronium, etc., which are substrates for 
this enzyme, in those individuals with a genetic, induced or acquired 
deficiency in butyrylcholinesterase. Butyrylcholinesterase may also be 
administered in order to inactive and thereby reduce the toxic effects of 
cocaine or of other non-medically and/or illicitly administered compounds 
that are substrates for this enzyme. The butyrylcholinesterase would be 
administered in amounts effective to reduce the toxic effect of cocaine, 
which amounts are similar to those hereinabove noted with respect to the 
treatment of apnea. 
Butyrylcholinesterase may also be administered to increase resistance (in 
both butyrylcholinesterase-normal and butyrylcholinesterase-deficient 
individuals) to any of the carbamates and organophosphates used as 
insecticides or neurotoxins. 
For this last application, the enzyme may be administered prior to an 
anticipated exposure to the toxin, as a prophyllactic, or as a therapeutic 
administered after an exposure to reduce pools of unreacted toxin in the 
body. Butyrylcholinesterase could be used in this application alone or in 
conjunction with a nucleophilic compound (e.g. an oxime). The nucleophilic 
would hydrolyze the initial adduct between toxin and enzyme before the 
adduct is converted to an essentially irreversible form. In this way 
active enzyme would be regenerated from the enzyme-toxin complex. Such a 
nucleophile could be administered together with, prior to or after the 
enzyme. The nucleophilic could be administered by the same route as the 
enzyme or by intramuscular injection. The dosage of the nucleophilic would 
be similar to that in current medical practice (approximately 50 mg/kg of 
body weight) but would be rendered more effective by the presence of 
higher concentrations of butyrylcholinesterase in the plasma. The 
butyrylcholinesterase is administered in amounts effective for reducing 
the toxic effect of a toxin such as a carbamate or organophosphate with 
such amounts generally being the amounts hereinabove described with 
respect to the treatment of apnea. 
The butyrylcholinesterase produced and used in accordance with the present 
invention has a purity of at least 90% and a specific activity which is at 
least 90% of the theoretical maximum specific activity (theoretical 
maximum is 200 .mu.mol min.sup.-1 mg.sup.-1 or 280 s.sup.-1 when assayed 
with 50 .mu.M benzoylcholine as substrate at 25.degree. C.). The 
butyrylcholinesterase is preferably used in a high concentration greater 
than 2 mg/ml in an appropriate pharmaceutical carrier such as an 
injectable carrier. 
Numerous modifications and variations of the present invention are possible 
in light of the above teachings; therefore, within the scope of the 
appended claims the invention may be practiced otherwise than as 
particularly described.