A mutein of an enzyme acceptor polypeptide fragment of .beta.-galactosidase which is resistant to oxidation is provided. The enzyme acceptor fragment has an amino acid other than cysteine located at position 602 of the natural sequence. An especially preferred substitution is serine. Also provided are a method for producing the novel mutein, a reagent composition comprising the novel mutein, and an immunoassay method for determining an analyte in which the novel mutein recombines with an enzyme donor polypeptide fragment to form enzymatically active .beta.-galactosidase.

BACKGROUND 
The present invention relates to modified enzyme acceptor polypeptide 
fragments of .beta.-galactosidase which are resistant to oxidation, to 
processes for the preparation thereof, and to the use thereof as reagents 
in enzyme complementation immunoassays. 
A number of homogeneous immunoassays have recently been described that 
utilize the complementation or reassociation of enzymatically-inactive 
polypeptide fragments to form active enzymes as a step of generating a 
detectable signal which can be utilized to determine the amount of an 
analyte of interest that may be present in a sample such as blood serum. 
Several of these assays propose utilizing the enzyme .beta.-galactosidase 
as the enzyme formed by complementation. 
Enzyme complementation involves the association of two or more inactive 
polypeptides which together provide the structural information required 
for the formation of a biologically active enzyme complex resembling that 
of the native parent enzyme. The enzymatically-inactive polypeptide 
fragments can be obtained as the result of proteolysis, chemical cleavage, 
chemical synthesis, or as the result of a missense or nonsense mutation of 
the gene coding for the active enzyme. Examples of protein complementation 
systems which yield an enzymatically-active complex are the 
ribonuclease-S' complex, the staphylococcal nuclease T complex, various 
two- and three-fragment complexes derived from cytochrome c, and the 
alpha- and omega-complementation complexes of E. coli 
.beta.-galactosidase. The interactions which stabilize these complexes are 
non-covalent in nature and are similar to those involved in the formation 
and maintenance of the three-dimensional structure of the native enzyme. 
Enzyme complementation has been utilized as the underlying basis for the 
development of a novel homogeneous immunoassay technology. Farina and 
Golke, U.S. Pat. No. 4,378,428 issued Mar. 29, 1983, and Gonelli et al., 
(1981, Biochem. and Biophys. Res. Commun. 102:917-923) describe an 
immunoassay based upon the reassociation of S-peptide and S-protein, both 
of which are derived from the proteolytic cleavage of ribonuclease A, to 
generate ribonuclease catalytic activity. Specific components of the assay 
system include an analyte covalently attached to the S-peptide (amino 
acids 1-20), free S-protein (amino acids 21-124), an antibody specific for 
the analyte, and a substrate of ribonuclease which is capable of being 
converted to a reporter molecule. The anti-analyte antibody inhibits the 
association of the analyte:S-peptide conjugate with the S-protein, thereby 
reducing the level of enzymatically-active complex and thus the signal 
generated by the enzymatic reaction. In the presence of a sample 
containing free analyte, a competition for the antigen binding site occurs 
between sample-born analyte and the S-peptide conjugate. The concentration 
of S-peptide conjugate free to participate in complementation with the 
S-protein fragment, and the resulting signal due to the enzymatic activity 
of the ribonuclease A' complex, are directly proportional to the 
concentration of free analyte in the sample. 
A similar immunoassay system based on the alpha-complementation system of 
E. coli .beta.-galactosidase polypeptide fragments is described in 
Henderson, U.S. Pat. No. 4,708,929, issued Nov. 24, 1987, and Henderson, 
PCT Appl. No. PCT/US90/02491, published Nov. 15, 1990, both of which are 
herein incorporated by reference. Galactosidase alpha-complementation 
involves the association of an alpha-acceptor polypeptide fragment and an 
alpha-donor polypeptide fragment and the subsequent formation of an 
enzymatically active .beta.-galactosidase molecule. The alpha-acceptor is 
derived from the internal deletion or chain interruption of consecutive 
amino acids located within the N-terminus proximal segment of the 
.beta.-galactosidase molecule. Specific examples include the lac Z M15 
.beta.-galactosidase deletion mutant lacking residues 11-41 of the 
wild-type sequence, and the lac Z M112 mutant in which residues 23-31 have 
been deleted. The alpha-donor polypeptide can be derived from chemical or 
proteolytic cleavage of the wild-type protein. The cyanogen bromide 
fragment CNBr2 composed of amino acid residues 3-92, or the V8 protease 
peptide spanning residues 3-40, both possess alpha-donor activity. 
Alpha-donor and alpha-acceptor polypeptides can also be generated through 
the application of recombinant DNA technology and peptide synthesis 
techniques. A readily available supply of these molecules and the ability 
to modify the structure of either the alpha-donor or the alpha-acceptor 
polypeptides through these techniques has led to the development of an 
optimized complementation system which has been employed in cloned enzyme 
donorbased homogeneous immunoassays. The alpha-donor molecule can be 
chemically coupled with a specific analyte of interest through the 
modification of either a cysteine or lysine residue which has been 
suitably located within the sequence of the alpha-donor molecule such that 
the conjugation does not interfere with the complementation reaction. 
Complementation between the alpha-acceptor and alpha-donor can be 
modulated by an antigen-antibody reaction between an analyte-specific 
antibody and the alpha-donor to which an analyte has been conjugated. In 
the presence of free analyte, a competition between the free and 
alpha-donor-conjugated analyte is established for the antigen binding site 
of the antibody. Thus, an increase in the level of free analyte results in 
an elevation in the quantity of alpha-donor conjugate which is available 
for complementation with alpha-acceptor. As a result, the concentration of 
the alpha-acceptor:alpha-donor complex and reporter molecule produced from 
the reconstituted enzymatic activity increase and are proportional to the 
concentration of the free analyte present in the sample. A dose response 
curve can be constructed by following the activity, i.e., the slope of the 
rate of the reaction, at several different concentrations of free analyte. 
The enzyme activity observed at an infinite concentration of free analyte 
or in the absence of antibody is defined as the "open rate" and represents 
the maximal signal obtainable from the assay system. 
Krevolin and Kates, European Appl. No. 92304354.1, published Nov. 19, 1992, 
the content of which is herein incorporated by reference, describe enzyme 
complementation assays involving complementation in the omega region of 
.beta.-galactosidase between two polypeptide fragments of the whole 
.beta.-galactosidase molecule formed by a break in the primary structure 
of .beta.-galactosidase in the omega region. As in alpha complementation, 
in some cases the two fragments are not strictly complementary so as to 
form an exact .beta.-galactosidase amino acid sequence without gaps or 
overlaps; both gaps and overlaps are possible as long as the resulting 
fragments can assemble into an active .beta.-galactosidase molecule. Like 
the alpha-acceptor, the omega-acceptor polypeptide is the larger of the 
two fragments and normally contains about two-thirds of the amino acid 
sequence of the natural or modified, full-length .beta.-galactosidase. The 
omega-donor molecule is the smaller fragment containing the remaining 
one-third (approximately) of the amino acid sequence; the omegadonor 
molecule is derived from the C-terminus of the .beta.-galactosidase 
molecule. 
However, the stability of reagent compositions containing these alpha- and 
omega-acceptor polypeptide fragments of .beta.-galactosidase has been 
discovered to be less than optimal. There is a gradual and significant 
loss of activity of the reformed enzyme as storage time of the fragments 
increases. It is well known that enzymes are unusually susceptible to 
thermal denaturation and to proteolytic cleavage. Enzymes also contain 
reactive amino acid side chains located in positions which render them 
particularly susceptible to chemical modification, including oxidation. In 
general, it is not possible to predict from the amino acid sequence the 
extent to which any of the above modifications will occur. Khanna et al., 
U.S. Pat. No. 4,956,274, issued Sep. 11, 1990 addressed this problem by 
the addition of an ionic surfactant or a surfactant derived from a sugar 
residue to the reagent medium containing the peptide fragment. Since the 
presence of surfactant is generally not compatible with the 
complementation of the enzyme acceptor and enzyme donor, excess surfactant 
must be neutralized or removed such as with a cyclodextrin. 
The stability of the major constituents which compose the working reagents 
used in an assay represents an important factor in the overall viability 
of the assay within the commercial market place. The degradation of any 
key component of the assay may drastically alter the performance, and thus 
affect the validity of the results obtained from the assay. Furthermore, 
if the reagents are unstable, the user may be required to perform 
laborious and time-consuming tasks such as daily reagent preparation. 
These repetitive tasks decrease the convenience of the assay to the user. 
An unstable assay system also limits the shelf-life of the working 
reagents and thus decreases the number of tests which can be packaged in 
an assay kit. By increasing the usable number of assays obtainable from a 
given quantity of reagent, the economic value of the assay kit can be 
substantially increased. 
The most labile components of an enzyme-based immunoassay are normally the 
protein constituents. The function of a protein, whether it is the 
catalysis of a chemical reaction or the binding of a specific molecule, is 
intrinsically dependent upon its discrete three-dimensional structure. It 
is generally accepted that the three-dimensional structure of a protein is 
determined by its amino acid sequence. A change in the chemical nature of 
any particular amino acid within the protein sequence may therefore affect 
the folding and/or conformation of the folded molecule. Such 
conformational changes can often lead to a perturbation in the normal 
function of the protein. The difference between the free energy of the 
folded and unfolded states of a protein is relatively small, typically 
only 5-20 kcal/mol. Thus, minor changes in the environment surrounding a 
protein, e.g., pH, temperature, or ionic strength, can also have dramatic 
effects on its conformational state. Changes in the conformational state 
of a protein, particularly to a metastable or partially folded 
intermediate, can lead to the irreversible aggregation or non-specific 
adsorption of proteins to surfaces. 
A number of degradative processes can occur which alter the chemical 
properties, and potentially the conformational integrity, of a protein. 
These include the deamidation of asparagine or glutamine residues to their 
respective acids; the oxidation of cysteine, methionine, or tryptophan 
residues to cysteic acid, methionine sulfoxide, or N'-formyl-kynurenine 
derivatives, respectively; the disruption of disulfide bonds; or the 
hydrolysis of labile peptide bonds. An understanding of the factors which 
contribute to the instability of the protein constituents in any given 
system is a key step in solving protein related stability problems. 
However, most immunoassay systems involve a number of proteins, and the 
complexity of their interactions with each other and with other components 
of the system may limit the number of potential solutions to such 
problems. In the case of cloned enzyme donor-based immunoassays, the 
primary protein components include the analyte-specific antibody, enzyme 
acceptor, enzyme donoranalyte conjugate, and any secondary antibodies 
which may be necessary for optimization of the assay. 
.beta.-Galactosidase is a tetrameric protein having a molecular weight of 
about 540,000 daltons. The four identical monomers consist of 1023 amino 
acids, each with a molecular weight of 116,000 daltons. The monomeric 
protein is divided into three regions: the N-terminus proximal segment 
(the alpha region), a middle region, and a C-terminus distal segment (the 
omega region). 
E. coli .beta.-galactosidase is derived from the Z gene of the lac operon 
and catalyzes the hydrolysis of .beta.-D-galactopyranosides. The catalytic 
mechanism of this enzyme involves the general acid catalysis of the 
glycosidic ester linkage of a substrate molecule by tyrosine-503. This is 
followed by the loss of the aglycon moiety and the stabilization of a 
putative carbonium ion intermediate through an interaction with 
glutamate-461. The final step in the catalytic cycle involves the 
transgalactosylation of an acceptor molecule, usually water, and the 
removal of the product from the active site. The active enzyme is composed 
of four identical subunits with one active site per subunit. Monovalent 
cations, although not required for activity, dramatically enhance the rate 
of enzyme catalysis, whereas divalent cations, e.g., Mg.sup.2+ or 
Mn.sup.2+, are required for activity. 
The E. coli .beta.-galactosidase homotetramer contains 64 cysteine residues 
(16 cysteine residues per subunit), none of which are involved in either 
the enzymatic activity or the maintenance of the quaternary structure 
through intersubunit disulfide bridges, as indicated by the stabilization 
of the molecule in high concentrations of reducing agents. The efficiency 
of the in vitro association of individual monomers to form the active 
tetramer is dramatically increased under conditions in which the cysteines 
are fully reduced. Similarly, reducing agents greatly enhance enzyme 
complementation. The alpha-acceptor polypeptide contains all 16 cysteine 
residues present in a single .beta.-galactosidase subunit. However, 
alpha-acceptor molecules exist as homodimers in solution. Thus, the 
surface area normally buried at the dimer-dimer interface in 
.beta.-galactosidase is exposed in the alpha-acceptor. Chemical 
modification studies of .beta.-galactosidase with iodoacetate lead to the 
identification of cysteine-500 and cysteine-1021 as surface accessible 
residues in .beta.-galactosidase (Jornvall et al., 1978, Biochem. 17, 
5160-64). Carboxymethylation of these two residues did not affect the 
activity of the enzyme to any significant extent. However, when M15, a 
dimeric alphaacceptor molecule, was treated with iodoacetate, three 
additional cysteine residues at positions 78, 389 and 602 were modified. 
Carboxymethylation was found to inhibit the ability of M15 to participate 
in alpha-complementation. This suggests that one or more of these 
additional residues is situated at the dimer-dimer interface, the 
modification of which interferes with alpha-complementation. 
It was surprising, therefore, to discover that substitution by 
site-directed mutagenesis of the cysteine-602 residue on an enzyme 
acceptor polypeptide fragment of .beta.-galactosidase with a conservative 
amino acid, preferably serine, results in substantially increased 
stability of the enzyme acceptor mutein over that of an enzyme acceptor 
polypeptide fragment having cysteine at position 602. 
Predetermined, site-directed mutagenesis of tRNA synthetase in which a 
cysteine residue is converted to serine has been reported (G. Winter et 
al., 1982, Nature, 299, 756-758, and A. Wilkinson et al., 1984, Nature, 
307, 187-188). Estell et al., U.S. Pat. No. 4,760,025, issued Jul. 26, 
1988 describe a cloned subtilisin gene modified at specific sites to cause 
amino acid substitutions of certain methionine residues. Koths et al., 
U.S. Pat. No. 4,752,585 issued Jun. 21, 1988 and U.S. Pat. No. 5,116,943, 
issued May 26, 1992, describe the protection of a therapeutic protein such 
as interleukin-2 or interferon-.beta. against oxidation by substituting a 
conservative amino acid for each methionyl residue susceptible to 
chloramine T or peroxide oxidation. 
Buchwalter et al., European Appl. No. 91106224.8, published Nov. 27, 1991, 
describe an animal somatotropin derivative in which cysteine residues are 
substituted by site-specific mutagenesis techniques for certain serine and 
tyrosine residues and in which glutamic acid has been substituted for 
certain cysteine residues. Breddam et al., PCT/DK91/00103 published Oct. 
31, 1991, describe chemically modified detergent enzymes wherein one or 
more methionines have been mutated into cysteines, and then said cysteines 
are subsequently chemically modified in order to improve stability of the 
enzyme toward oxidative agents. Mattes et el., U.S. Pat. No. 4,963,469, 
issued Oct. 16, 1990, describe a change of an amino acid in the region 
between amino acid 430 and 550 of .beta.-galactosidase to another amino 
acid to produce an enzymatically inactive, immunologically active 
.beta.-galactosidase mutein. Estell et al. (1985, J. Biol. Chem. 260, 
6518-6521) used site-directed mutagenesis to alter the methionine 222 
residue of subtilisin which is a primary site for oxidative inactivation 
of the enzyme. These authors found that mutants containing non-oxidizable 
amino acids, i.e., serine, alanine and leucine, were resistant to peroxide 
inactivation, whereas methionine and cysteine-substituted enzymes were 
rapidly inactivated. 
As used herein, the numbering for the amino acid residues of 
.beta.-galactosidase will be that published by Kalnins et al., 1983, EMBO 
Journal 2, 593-597, the content of which is herein incorporated by 
reference. The nucleotide sequence of the lac Z gene coding for 
.beta.-galactosidase in E. coli was determined and .beta.-galactosidase 
was predicted to consist of 1023 amino acid residues rather than the 1021 
residues previously reported by Fowler and Zabin (1977, Proc. Natl. Acad. 
Sci. U.S.A. 74, 1507-1510 and 1978, J. Biol. Chem. 253, 5521-5525). 
SUMMARY OF THE INVENTION 
The present invention provides novel muteins of enzyme acceptor polypeptide 
fragments of .beta.-galactosidase and processes for producing such 
muteins. In particular, the present invention provides novel muteins of 
enzyme acceptor polypeptide fragments of .beta.-galactosidase in which an 
amino acid other than cysteine is located at position 602. Particularly 
preferred are alpha-acceptor polypeptide fragments of .beta.-galactosidase 
in which serine is substituted for cysteine-602. Also provided are reagent 
compositions comprising these novel muteins and immunoassay methods 
utilizing such compositions in cloned enzyme donor immunoassays involving 
complementation between these enzymatically-inactive donor and acceptor 
fragments to form an enzymatically-active enzyme. The novel enzyme 
acceptor muteins have been found to exhibit substantially increased 
stability and resistance to oxidation over that of the parent enzyme 
acceptor fragment. 
The novel muteins of the present invention are conveniently prepared by 
causing site-directed mutagenesis at the appropriate location on the gene 
coding for the parent enzyme acceptor. Site-directed mutagenesis methods 
(Wallace et al., 1981, Nucleic Acids Res. 9, 3647-3656; Zoller and Smith, 
1982, Nucleic Acids Res. 10, 6487-6500; and Deng and Nickoloff, 1992, 
Anal. Biochem. 200, 81-88) permit the replacement of cysteine-602 of 
.beta.-galactosidase with any amino acid. Chemical synthesis of the 
polypeptide fragment is not beyond the scope of the present invention; 
however, such techniques are generally applied to the preparation of 
polypeptides that are relatively short in amino acid length. 
In an assay according to the present invention, an analyte in a sample such 
as blood serum, i.e., a ligand or receptor, is determined using reagent 
compositions comprising enzyme donor and enzyme acceptor polypeptide 
fragments, wherein the enzyme donor fragment is conjugated to an 
analyte-binding protein specific for the analyte, and wherein the analyte 
is cross-reactive with the conjugated analyte-binding protein or is 
complementary thereto. The enzyme acceptor polypeptide consists 
essentially of a fragment of .beta.-galactosidase which is characterized 
by forming with the enzyme donor an active enzyme complex having 
.beta.-galactosidase activity in the absence of analyte-binding protein 
binding to said conjugate. The reagents are combined with the sample and a 
substrate capable of reacting with the active enzyme complex in an 
appropriate assay medium. The rate of conversion of the substrate by the 
enzyme compared to the rate of conversion of substrate obtained using a 
known concentration of the analyte is used to determine the amount of 
analyte in the sample.

DESCRIPTION OF SPECIFIC EMBODIMENTS 
In accordance with the present invention, the novel enzyme acceptor 
polypeptide fragments of .beta.-galactosidase are prepared by 
site-directed mutagenesis methods, wherein a particular location on the 
gene coding for an enzyme acceptor fragment is mutagenized. In particular, 
site-directed mutagenesis methods are used to cause a mutation at the 
location coding for cysteine at position 602 in the natural sequence, 
thereby causing the substitution of a conservative amino acid for 
cysteine. The preferred amino acid substitution is serine. Other amino 
acids may also be substituted, but conservative substitutions are 
preferred. By conservative substitution is meant replacement of 
cysteine-602 of .beta.-galactosidase by an amino acid which has similar 
characteristics and which is not likely to have an adverse effect on 
either the enzyme acceptor's ability to complement with enzyme donor or on 
the catalytic activity of the reformed .beta.-galactosidase. Examples of 
such conservative amino acid substitutions are glycine, alanine, valine, 
isoleucine, leucine, serine, threonine and methionine. An especially 
preferred substitution is serine, and an especially preferred parent 
enzyme acceptor is EA22, which is described fully in U.S. Pat. No. 
4,708,929. 
The preparation of parent enzyme acceptors can be accomplished using a 
variety of recombinant DNA techniques, including deletion constructions or 
direct synthesis of DNA carrying the desired amino acid sequence followed 
by in frame ligation into the DNA sequence of the m-region of the lac Z 
gene which encodes native .beta.-galactosidase. Such techniques are 
described more fully in U.S. Pat. 4,708,929. 
Organisms producing parent enzyme acceptor polypeptide fragments are also 
publicly available. E. coli strain AMA 1004, In Vitro International, Inc. 
(IVI) (Ann Arbor, Mich.), accession no. 10051, contains a plasmid, pMG22, 
which carries a gene for a .beta.-galactosidase enzyme acceptor with amino 
acids 13-40 deleted (EA22). E. coli strain AMA 1004, IVI 10050, contains a 
plasmid, pMG14, which carries a gene for a .beta.-galactosidase enzyme 
acceptor with amino acids 30-37 deleted (EA14). 
As defined herein, an enzyme acceptor is an enzymatically-inactive 
polypeptide produced by a deletion mutant of the .beta.-galactosidase gene 
which, when combined with an enzyme donor, is capable of forming 
enzymatically-active .beta.-galactosidase by the process of 
complementation. The particular substituted enzyme acceptor muteins 
described herein are produced from EA22, an enzyme acceptor having a 
deletion within the alpharegion of the .beta.-galactosidase gene encoding 
the N-terminus of the .beta.-galactosidase protein. Specifically, EA22 has 
a deletion of amino acid residues 13-40. Other enzyme acceptor fragments 
of .beta.-galactosidase which contain the natural sequence which includes 
amino acid position 602 may also be used to produce muteins according to 
the present invention. Specific examples of enzyme alpha-acceptors are 
disclosed in U.S. Pat. No. 4,708,929 and include EA5, EA11, EA14, EA17, 
EA18, EA20, EA23 and EA24. The distal end of the deletion segment in 
suitable alpha-acceptors will normally fall between amino acid positions 
26 and 54 of the .beta.-galactosidase sequence. In EA22, the distal end of 
the deletion segment is amino acid 40. 
Omega-acceptor fragments are also within the scope of the present 
invention. Omega-acceptors are fully described in European Appl. 
92304354.1, and a specific example of a suitable omega-acceptor is OA721. 
The chief consideration when selecting an enzyme acceptor polypeptide of 
.beta.-galactosidase for stabilization according to the teachings of the 
present invention is that there has been no previous deletion at position 
602. 
As defined herein, an enzyme donor is an enzymatically inactive polypeptide 
comprised of two domains, a donor domain containing a protein sequence 
capable of combining with an enzyme acceptor to form active enzyme, and an 
analyte domain capable of interacting with an analyte-binding protein. The 
analyte domain is either (a) an analyte-coupling domain through which 
attachment to various analytes or analyte analogs can be accomplished or 
(b) a protein domain which itself functions as an analyte analog. An 
especially preferred enzyme donor, ED4, is described in detail in U.S. 
Pat. 4,708,929. 
In the assay method of the present invention, a known amount of an enzyme 
donor of the .beta.-galactosidase system comprising a coupled or fused 
analyte (or analogous analyte derivative) of interest, i.e., the enzyme 
donor conjugate, is combined with a known amount of a specific 
analyte-binding protein or other binding molecule and a known amount of an 
enzyme acceptor capable of complementation with the enzyme donor. 
Competition between the analyte domain of the enzyme donor conjugate and 
free unknown analyte in the sample for the known amount of specific 
analyte-binding protein allows the enzyme donor conjugate to remain free 
so that it binds to the enzyme acceptor. The association of donor 
conjugates and acceptor results in the formation of a catalytically active 
enzyme complex, thus modulating the amount of .beta.-galactosidase enzyme 
activity detectable in the sample. As a result, the amount of free analyte 
in the sample is determined as a direct function of the measurable enzyme 
activity. Enzyme activity is measured by monitoring the rate of substrate 
conversion by the enzyme catalyzed reaction by any of a variety of 
techniques, including but not limited to spectrophotometric and 
fluorometric methods. 
EXAMPLE 1 
Construction of Enzyme Acceptor Mutein 
The site-directed mutagenesis of the alpha-acceptor parent, EA22, was 
carried out according to the method of Deng and Nickoloff, 1992, Anal. 
Biochem. 200:81-88, the content of which is incorporated herein by 
reference. The starting plasmid which contained the structural gene for 
EA22 was p230. Two oligonucleotide primers were synthesized which 
contained twenty uninterrupted bases for hybridization as well as a 
substitution which introduced the cysteine to serine substitution at 
position 602. Additionally, the primers incorporated a new restriction 
endonuclease site and removed a native restriction endonuclease site for 
screening and selection purposes, respectively. 
After the two primers were annealed to the denatured p230, they were 
elongated with DNA polymerase and transformed via electroporation into a 
mut S E. coli strain defective in strand repair, BMH 71-18. A pool of 
plasmid obtained from an overnight culture of these cells was transformed 
again into a lac Z deleted strain, AMA 1004. Plasmids from individual 
colonies were screened for introduction of a new unique restriction 
endonuclease site. Positive clones were sequenced for the incorporation of 
the cysteine-602 to serine change. The final mutagenized product was 
plasmid p230 with a mutagenized amino acid at the cysteine-602 position as 
well as two silent changes, one beside the mutagenized amino acid and one 
at the unique site position elsewhere on the plasmid. 
EXAMPLE 2 
Comparison of Chemical Instability 
In order to determine whether the cysteine-602 to serine substitution had 
improved the resistance of EA37 to oxidation, an experiment was carried 
out in which EA22, EA37 and .beta.-galactosidase were exposed to a 
1000.times. molar excess of H.sub.2 O.sub.2. The proteins were exposed to 
oxidation conditions for variable lengths of time and then assayed for 
residual .beta.-galactosidase activities. 
Assay Buffer 
An assay buffer was prepared having the following composition: 
150 mM Na phosphate, pH 7.2 
400 mM NaCl 
4 mM Mg acetate 
10 mM ethylene glycol tetraacetic acid (EGTA) 
0.05% TWEEN-20 (registered TM of ICI Americas, Inc. for 
polyoxyethylenesorbitan) 
10 mM L-methionine 
Measurement of .beta.-galactosidase Activity 
Measurement of enzyme acceptor .beta.-galactosidase activity was 
accomplished by combining enzyme acceptor in assay buffer with alpha-donor 
ED4 in the presence of the .beta.-galactosidase chromogenic substrate 
o-nitrophenyl-.beta.-D-galactopyranoside (ONPG). The generation of the 
rate of the subsequent enzyme activity was then measured 
spectrophotometrically as a change in absorbance at 420 nm over a fixed 
length of time. This rate was then compared to the rate obtained for a 
control sample of fresh or untreated EA22. 
Oxidation by Hydrogen Peroxide 
Samples were diluted to a concentration of 4.4 .mu.M in assay buffer 
containing 4.4 mM hydrogen peroxide. Samples were removed at various time 
intervals and assayed for residual activity. Inactivation kinetics were 
found to be first-order in all cases. The loss in kinetic activity for 
EA22 (k=0.0443 sec.sup.-1) was found to occur at a rate 11-fold greater 
than that observed for .beta.-galactosidase (k=0.0038 sec.sup.-1). In 
contrast, EA37 (k=0.0149 sec.sup.-1) exhibited a 3-fold decrease in the 
rate of inactivation relative to EA22 but were still more susceptible to 
inactivation than .beta.-galactosidase. The results of these assays are 
shown in FIG. 1. 
EXAMPLE 3 
Comparison of Enzymatic Instability 
In order to compare the shelf-life stability of the EA22 and EA37 fragments 
in a liquid, reagents were made comprising each of the fragments in the 
assay buffer described in Example 2. These reagents were then stored at 
ambient temperature for various lengths of time and assayed for residual 
.beta.-galactosidase activity as described in Example 2. The results of 
these assays are shown in FIG. 2. 
EXAMPLE 4 
Assay for Barbiturates 
In order to demonstrate the ability of EA37 to detect an analyte in a 
sample specimen, varying concentrations of a barbiturate dose 
(secobarbital) were assayed using a monoclonal antibody specific for 
barbiturates as the analyte-binding protein, A dose response curve was 
constructed and is shown in FIG. 4. 
EA Reagent 
100 mM PIPES (1,4-piperazinediethanesulfonic acid), pH 6.9 
700 mM NaCl 10 mM Mg Acetate 10 mM EGTA 20 mM Na Azide 
120 U/ml EA37 10 mM L-methionine 0.5% fetal bovine serum 
1:800 dilution monoclonal barbiturate antibody (ascites) 
ED Reagent 
100 mM PIPES, pH 6.9 
700 mM NaCl 
10 mM EGTA 
1 mM EDTA 
20 mM Na Azide 
2 mg/ml bovine serum albumin fragments 
1 mg/ml CPRG (chlorphenylred-.beta.-D-galactopyranoside) 
93 mM ED28-barbiturate conjugate 
Measurement of Secobarbital 
The assay was performed using a Hitachi 717 automated analyzer (Boehringer 
Mannheim Corp., Indianapolis, Ind.) using equal amounts of ED reagent and 
EA reagent. The secobarbital dose was added to the EA reagent and 
incubated for 5 minutes, following which ED reagent was added. The 
absorbance rate was then measured at 570 nm using a 1-minute read interval 
at 4'00"-5'00" following the addition of ED reagent. In this particular 
experiment, the reagent volumes used were 130 .mu.l each and the sample 
volume was 12 .mu.l. The doses were prepared from an Alltech secobarbital 
calibrator, 10,000 ng/ml.