86
METHODS FOR THE STUDY OF ANTIBIOTICS
[6] I m m u n o l o g i c a l
Techniques
[6]
for
Studying/~-Lactamases
By M. H. :RICHMOND I. Introduction . . . . . . . . . . . . . . . . . . II. Preparation of Antisera . . . . . . . . . . . . . . . A. Using Purified Enzyme Preparations . . . . . . . . . . B. Using Crude Enzyme Preparations . . . . . . . . . . . C. Isolation of ~-Lactamase-Less Mutants . . . . . . . . . D. Inoculation Program for Crude Preparations . . . . . . . . III. Examination of ~-Lactamases with Specific Antisera . . . . . . . A. Neutralization Analysis . . . . . . . . . . . . . . B. Precipitation Analysis . . . . . . . . . . . . . . .
86 86 86 87 89 89 90 90 98
I. Introduction I n principle, fl-lactamases are no different from other enzymes in respect to their action with antisera, and m a n y of the techniques to be described below are capable of much wider application t h a n has occurred in the past. The reason for describing t h e m here, however, is t h a t they have a particular relevance to fl-lactamases since m a n y of the minor variants of this enzyme t h a t are found in naturally occurring strains have been characterized b y these immunological methods.
I I . P r e p a r a t i o n of Antisera
A. Using Purified Enzyme Preparations For the preparation of specific antisera, and those for reaction with enzymes are no exception, it is preferable to have the antigen as pure as possible. I n this w a y the sera are likely to have their highest titers and to be as free as possible of nonspecific side reactions. In practice, preparation of a pure antigen m a y be more exacting than normal protein purification since small amounts of impurity m a y be disproportionately immunogenic. On the other hand, these considerations do not often invalidate the use of sera for analytical purposes with fl-lactamases. Indeed all the studies on staphylococcal and Escherichia coli B-lactamases described in this section were carried out with enzymes purified by the methods described elsewhere in this volume. 1,2 1This volume [53c]. This volume [53d].
[6]
IMMUNOLOGICAL TECHNIQUES FOR STUDYING ~-LACTAMASES
27
A number of different animal species and dosage regimes have been used to raise fl-lactamase sera. All the most effective, however, use inoculation of rabbits with purified enzyme preparations treated in various ways2 ,4 For antistaphylococcal penicillinase,4 sandy lop rabbits are each injected in alternate thighs at 3-day intervals with a total of four 0.2-ml portions of a solution of purified staphylococcal penicillinase made by suspending about 5 mg of purified enzyme per milliliter of Freund's adjuvant2 After a further 2 weeks, each rabbit is injected intravenously through the ear vein with three successive 0.2-ml samples of an alum-precipitated preparation of purified enzyme (approximately 5 mg/ml) at 2-day intervals. Samples of blood are withdrawn from the rabbit's ear vein 6 days after the end of the second phase of treatment, the serum is separated by conventional techniques, and the antibody titer is determined. In practice, once a serum of suitable titer has been obtained, it is better to kill and exsanguinate the rabbit since this procedure provides maximum quantities of a serum of uniform properties. In a typical example with staphylococcal penicillinase, about 100 ml of a serum, with titer about 104 units per milliliter of undiluted serum, was obtained from a single sandy lop rabbit. After separation of the serum, the material is best stored by dividing into ampules in about 5-ml quantities and freezedrying. Ampules may then be reconstituted for use as required by adding 5 ml of distilled water. If diluted serum is needed, the reconstituted serum should be diluted for use in physiological saline.
B. Using Crude Enzyme Preparations The above method is the one that should be followed when reasonable supplies of purified enzyme are available. It is possible, however, to prepare adequate specific sera by inoculating relatively crude enzyme preparations in Freund's adjuvant, and then absorbing out the unwanted antibodies before use. For this procedure to be successful, it is vital to have a mutant of the producer strain that makes no fl-lactamase. Before going on to describe the actual techniques for preparation of fl-lactamase from crude ])reparations, the purification procedure for crude enzyme and the methods of isolating fl-lactamase-less mutants will be described. Preparations of fl-lactamase from most gram-negative bacteria may be prepared as follows. The method is based on the one described elsewhere in this volume for the purification of the fl-lactamase from Escherichia coli (Rr+~M).2 A culture of the producer strain growing exponena M. R. Pollock, J. Gen. Microbiol. 14, 90 (1956). 4 M. H. Richmond, Biochem. J. 88, 452 (1963). 5 Difco Bacto-adjuvant, Complete (Freund), 0638-60-7.
88
METHODS FOR THE STUDY OF ANTIBIOTICS
[5]
tially in 1% CY medium 1 is centrifuged and the bacteria are collected. These organisms are resuspended in 0.1 M Na2HPO4/KH2P04 buffer, pH 7.0, to a density of about 30 mg dry weight of bacteria per milliliter and disrupted in an ultrasonic disintegrator. ~ The broken bacteria are stored at 2 ° until disruption of the whole batch is complete; they are then centrifuged, first at 5000 g for 15 rain at 4 ° (to remove large cell debris), then at 40,000 g for 4 hr at 4 ° (to remove smaller fragments), and last at 105,000 g for 2 hr at 4 ° to sediment ribosomes and minute pieces of comminuted membrane. After centrifugation the supernatant containing the enzyme is dialyzed against 100 volumes of 0.1 M Na~ HPO4/KH2P04 buffer, pH 7.0, for 24 hr at 2 °. During this process a precipitate often forms, and this is removed by centrifugation at 5000 g for 15 min at 4 °. After dialysis and any necessary centrifugation the enzyme is run through a Sephadex column. Adequate preparations are obtained if a Sephadex G-75 column equilibrated against 0.1 M Na~HPO~/KH2P04 buffer, pH 7.0, is used, but cleaner enzyme preparations may be obtained by using either DEAE-Sephadex or CM-Sephadex, as appropriate. The choice of Sephadex depends on the nature of the enzyme, and to decide this it is useful to know something of the ionic properties of the protein. In general all fl-lactamases from gram-negative species with a predominant activity against cephalosporins are positively charged at neutral pH values 6 and therefore should be purified on CM-Sephadex. The remaining fl-lactamases are likely to be more acidic 6 and in this case DEAE-Sephadex may be more appropriate./~lthough a general tendency, however, this correlation between charge andsubstrate profile should not be taken as absolute, and it is always necessary to carry out some preliminary test absorptior~ experiments with the enzyme and various substituted Sephadexes when an enzyme is being examined for the first time. As a guide it is always helpful to know the electrophoretic properties of the enzyme before doing the experiments. When G-75 Sephadex is used, the column (150 X 1.5 cm) is equilibrated against 0.1 M Na2 HPO4/KH2P04 buffer, pH 7.0, and the material is eluted with similar buffer. After elution from the Sephadex column the enzyme preparation is dialyzed against distilled water to remove salts and then stored at 2 ° until required for use. In general it seems better not to freeze dry enzyme preparations destined for raising antisera since the freezing and thawing process is likely to denature some of the enzyme molecules, and this in turn is likely to broaden the specificity of the resulting antiserum. M. H. Richmond and R. B. Sykes, Recent Adv. Microb. Physiol. 9, 36 (1973).
[6]
IMMUNOLOGICALTECHNIQUES FOR STUDYING ~-LACTAMASES
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C. Isolation of/~-Lactamase-Less Mutants The fl-lactamase producing culture is grown exponentially in any convenient growth medium and then treated with N-methyl-N-nitro-Nnitrosoguanidine (NMG) as described elsewhere in this volume. 1 After treatment and the necessary period of growth following removal of the mutagen, the bacteria are plated as single colony-forming units on 1% CY agar l and incubated overnight at 37 °. In the morning the colonies are flooded on the plate with a solution (5 mg/ml) of the chromogenic cephalosporin 87/312. 7 In this example, the great majority of the colonies will turn red (indicating production of fl-lactamase), but a few (probably not more than 1/50,000) will produce no color. It is therefore necessary to do this experiment on a large scale. About 100 plates with 1000 colonies per plate are commonly employed; but it may be necessary to work on an even larger scale. Any colorless colonies are picked onto fresh agar and purified by restreaking. The isolates are next checked for their production of fl-lactamase, and any producing no detectable enzyme by quantitative methods are retained for use. The process of isolating mutants in this way is tedious, since methods for selection of fl-lactamaseless mutants are not available, but fl-lactamase-less mutants of this type must be available for specific sera to be obtained when crude enzyme preparations are used as immunogens.
D. Inoculation Program for Crude Preparations As with the program for purified enzyme, rabbits are the most convenient animal for raising sera in the conventional laboratory. The larger the rabbit the better, since the final yield of serum will depend to some extent on the blood volume of the animal; and absorbed sera are likely to have lower titers than those prepared against purified antigens. A typical program for raising an antiserum using a crude enzyme preparation is as follows. Samples of crude enzyme, purified as described above, are injected after mixing with equal volumes of Freund's adjuvant into alternate thighs of a sandy lop rabbit. In all, a sequence of six injections at 2-day intervals is used. Two weeks after the last injection, the animal is injected through the ear vein with 0.2 ml of alum-precipitated crude enzyme. Four injections of this material are made at 2-day intervals. About 10-14 days after the end of this sequence of injections, the animal is test bled, and the antiserum titer is determined against the appropriate enzyme (see below). When two test bleeds 2 days apart show the same titer, the animal is killed and exsanguinated, and serum is prepared from TC. H. O'Callaghan, A. Morris, S. M. Kirby, and A. H. Shingler, Antimicrob. Ag. Chemother. 1, 283 (1972).
90
METHODS FOR T H E STUDY OF ANTIBIOTICS
[6]
the whole blood by conventional techniques. Titers of about 5000 units of neutralizing ability are not uncommon by this technique, but it must be stressed that not all anti-fl-lactamase sera are of the neutralizing type (see below). Once an active serum has been obtained by this method it is then treated with crude protein prepared from the fl-lactamase-less mutant strain. Ideally the serum should be treated with material made in an identical manner to the crude enzyme, protein being collected from the Sephadex G-75 column at the point at which the relevant fl-lactamase is known to elute. However, it is equally effective to use some of the total cell protein obtained after ultrasonic disruption of the fl-lactamaseless mutant. However, if material that is the product of ultrasonic disintegration without centrifugation is used, losses in the titer of the serum are likely to be high, but if the material at the stage at which it would normally be loaded onto the Sephadex column is employed, absorption is more effective and losses of specific anti-fl-lactamase activity is minimized. The adsorption technique involves adding the absorbing protein to the serum in a series of 0.2-ml quantities until precipitation ceases. Ideally this should lead to no loss in titer of the antiserum, but in practice some loss seems difficult to avoid, and it is usually necessary to compromise between having an absolutely specific serum and one of such low titer that it is virtually useless. In a typical absorption experiment, 0.2-ml quantities of absorbing protein were added at intervals to 5 ml of undiluted serum. The serum was then incubated at 30 ° for 2 hr before the precipitate was centrifuged off at 5000 g for 15 min. In one experiment about 0.75 ml of absorbing protein had to be added to the 5-ml batch of serum before precipitation ceased. In order to sterilize the serum during this procedure, 0.1% (w/v) sodium thiomersalate is added to the serum. Once absorption is complete, the serum is transferred to ampules and freeze-dried in the normal way. III. Examination of fl-Lactamases with Specific Antisera Once antisera to a B-lactamase have been obtained, they can be used to study the enzymes in a number of ways. These include precipitation analyses both in solution and by gel diffusion. But perhaps the most effective is by neutralization of enzyme activity since this can be studied with as little as 0.1 ~g of enzyme in impure preparations.
A. Neutralization Analysis When antibodies interact with enzymes, the activity of the preparation may be altered. However, interaction with an antibody molecule
[~]
IMMUNOLOGICAL TECHNIQUES FOR STUDYING ~-LACTAMASES
91
need not affect activity, and it is impossible to predict the manner in which an antiserum preparation raised against either a crude or a purified preparation of fl-lactamase will act. Indeed, antisera obtained with samples of the same enzyme preparation in different rabbits may v a r y greatly in their effect, even when the immunization program is identical and carried out in parallel. It follows, therefore, that when antisera are being raised it is important to obtain as large a quantity as possible so that a large series of tests may be made with a serum of uniform properties. Provided an antiserum affects enzyme activity, this interaction m a y be used to provide much information about the nature of a fl-lactamase. Most sera reduce the activity of B-lactamases, 3,~ and this neutralizing effect will be discussed first. However, stimulatory sera, ~ or even sera that stimulate and then inhibit at higher concentration are known, 9 but their use will be discussed later. The most common way to use a neutralizing serum is to study the enzyme/antiserum interaction as a titration of a constant amount of antigen. Increasing amounts of antiserum are added to a series of vessels each containing a fixed amount of antigen. After a period for the antigen/antibody reaction to occur (usually about 10 rain at room temperature) the residual enzyme activity is assayed by a convenient method (usually the iodometric method of Ferret TM as modified by N o v i c k ' ) . With E. coli fl-lactamase, where a serum with titer about 104 units/ml is available, the usual experimental approach is to put about 50-75 units of enzyme in each of a series of conical flasks and immediately add increasing amounts of serum, leaving the first without addition to act as a measure of the untreated enzyme activity. The reaction is allowed to continue for 10 rain. Then 10 ml of a prewarmed solution of benzyl penicillin (1%, w/v, in 0.1 M Na~ H P O J K H 2 P O 4 buffer, pH 5.9) is added. After 6 rain further incubation, 10 ml of standard iodine solution in 2 M sodium acetate buffer, pH 4.2, is added and the excess iodine back-titrated against sodium thiosulfate." Figure 1 shows a typical neutralization curve obtained in this way2 There is a linear decrease in residual enzyme activity with increasing antiserum concentration up to a point (the equivalence point: E--see Fig. 1) at which no further inactivation occurs however much serum is added. The activity remaining after the equivalent amount of serum has been added is known as the "residual activity" and probably represents the activity of the enzyme/antibody complex. The slope of the neutralization curve G. W. Jack and M. H. Richmond, J. Gen. Microbiol. 61, 43 (1970). 9 M. R. Pollock, Immunology 7, 707 (1964). ~oC. J. Perret, Nature (London) 174, 1012 (1954). See also this volume [5]. ~ R. P. Novick, Biochem. J. 83, 229 (1962).
92
METHODS FOR THE STUDY OF ANTIBIOTICS
[6]
F
100~-,,~
'~: .I- \ . 6o-
\o
\o
--
'~EO--~O
20 I 0.02
I 0,04
I 0,06
I 0.08
Antiserum
I 0.1
I 0.12.
: ml
FIG. 1. Neutralization of type I I I a ~-lactamase by anti-type I I I a fl-lactamase serum [G. W. Jack and M. I-I. Richmond, J. Gen. Microbiol. 61, 43 (1970)]. E, equivalence point.
gives the titer of the serum; and if the turnover number of the fl-lactamase is known, this titer m a y be expressed as the number of enzyme molecules neutralized per milliliter of antiserum. In practice 10 min is normally allowed for the enzyme/antibody complex to forIn at room temperature, but a study of the time course of enzyme/antiserum interaction shows that the reaction occurs very rapidly (Fig. 2). Neutralization with more than the equivalent amount of serum is more than 90% complete in 2 min at room temperature. Figure 1 illustrates a typical neutralization curve obtained with an anti-fl-lactamase serum. However, all sera do not neutralize. Figure 3
~
c t00 ]uu .2
6{ E
E E
•
O
/
°~° 2C I 2
I 4
I 6
I 8
I 10
minutes
FIG. 2. Time course of reaction between type I l i a /~-lactamase and a specific antiserum.
[6]
IMMUNOLOGICAL TECHNIQUES FOR STUDYING /~-LACTAMASES
93
241 20(
//
ID
/* /* 40,;I 0.05
i 0.1 Antiserum
| 0.15
I 0-2
: ml
FIG. 3. Stimulation of staphylococcal ~-lactamase activity by a specific antiserum [M. H. Richmond, Biochem. J. 88, 452 (1963)]. E, equivalence point.
shows the effect of addition of increasing amounts of anti-staphylococcal serum to staphylococcal fl-lactamase type A. In this case this particular batch of serum stimulates the activity of the enzyme to a maximum about 4.5 times higher than that of the enzyme without serum. ~ Once again the response is linear with increasing amounts of antiserum, and once more there is an equivalence point (E) above which further addition of serum produces no change in activity. As with neutralization effects, stimulation by antiserum can be used to characterize an enzyme. The slope of the curve (Fig. 3) gives a measure of the titer of the serum, and the extent of stimulation is constant for a given type of fl-lactamase. At present it is uncertain how combination with an antiserum increases the activity of an enzyme. Intuitively one feels that the process must hold the enzyme in a more favorable conformation, but convincing experimental evidence for this sort of mechanism is lacking. Nevertheless one thing seems certain: The binding of stimulatory sera cannot block the active center of the enzyme. Certain sera show even more complex effects with fl-lactamases than the examples shown in Figs. 1 and 3. Pollock has studied the interaction of specific serum with purified fl-lactamase from Bacillus licheni]ormis2 In this example (Fig. 4) addition of antiserum causes stimulation and then inhibition. Such a curve has two equivalence points (El and E.~) but is difficult to use for analytical purposes, although the shape of the curve has great value as a qualitative means of identifying an enzyme type. This complex curve is certainly due to the interaction of the enzyme with an antiserum containing both inhibitory and stimutatory compo-
94
METHODS FOR THE STUDY OF ANTIBIOTICS
[5]
3001 /\,E1 .. 200 >
0
i ,°0,
\,
o
E2
I
I
O.2
o.1 ml
of
I
O.3
antiserum
FIG. 4. Interaction of fl-lactamase from Bacillus licheni]ormis 6346 with specific antiserum [M. R. Pollock, Immunology 7, 707 (1964)]. E,, stimulatory equivalence point; E~, neutralizing equivalence point.
nents. Partial absorption of the serum with enzyme removes the stimulatory component and gives a residual serum which is purely neutralizing2
I. Characterization o] Variants in Naturally Occurring Strains Since the effect of antisera on fl-lactamase activity can be studied with such small amounts of enzyme and since contaminating protein does not interfere significantly with the interaction, tests in solution are an ideal way of looking for molecular variants of fl-lactamase whether of natural or laboratory (i.e., mutational) origin. An example of the detection of natural variants comes from studies that have been made on staphylococcal fl-lactamase. 12,13 The reaction of anti-staphylococcal fllactamase serum with the fl-lactamase found in most clinical isolates of Staphylococcus aureus gives a stimulatory curve already discussed (Fig. 1). However, analysis of a large number of clinical isolates shows that four types of response are to be found (Fig. 5). The majority show the standard stimulation by a factor of about 4.5-fold---known as A-type response--while a substantial minority show no stimulation at all. Indeed in this group (C type) there is no evidence that the serum (which was raised against purified A-type enzyme) interacts with the fl-lactamase at all until a precipitation analysis is carried out. Under these conditions an enzyme/antiserum precipitate is obtained, but this has the expected activity of the amount of enzyme combined in the precipitate. Two other ~2M. It. Richmond, Biochem. J. 94, 584 (1968). ~3V. T. Rosdahl, J. Gen. Microbiol. 77, 229 (1973).
[{)]
IMMUNOLOGICAL TECHNIQUES FOR STUDYING ~-LACTAMASES
95
20o
"~160 •;
120
o / / -
• ~
• ~
• ~ OA
o
o
o
I
I
/+
i40, ~-~ I
0.1 0.2 0.3
I
Od
Co
I
0.5
ml of antiserum
Fro. 5. Response of the four variants of staphylococcal f~-lactamase after interaction with a serum raised against type A [M. H. Richmond, Biochem. J. 94, 584 (1968)]. A, B, C, D: fl-lactamase types based on their response to serum.
types of response are also found. In the first (type B), the antiserum stimulates, but the titer is about one-fifth of that expressed with A-type fl-lactamase. In fact, further analysis shows that this response is due to expected antiserum binding to a fl-laetamase molecule with about onefifth the turnover number of A-type fl-lactamase. The fourth type of response (type D) gives a lower maximum degree of stimulation (about 1.5-fold at most) with a lower apparent titer. 13 In this case, the turnover number of the fl-lactamase variant is the same as that of A-type enzyme, and therefore the serum interacts with the enzyme with lower affinity. Subsequent experiments have shown that types A, B, and C fl-lactamase from S. a u r e u s (and probably type D as well) are minor variants of a common structure probably with one or a few amino differences in their primary sequence. Similar experiments have been done with fl-lactamases from other bacterial species, and in general these experiments have led to the view that these enzymes tend to be species specific, but that within a given species a relatively large number of minor variants are to be found. 6''4 The cross reaction found between the minor variants within a given species, but the absence of cross reaction between the fl-lactamases from different species suggests that perhaps interaction between enzyme and antiserum is particularly sensitive to changes in the primary sequence of the enzyme protein, and that a change in only one or two residues can be tolerated before the binding of the serum becomes suSstantially impaired. Nor is such a view incompatible with the normal high specificity of antibody molecules. ~ N. Cirri and M. R. Pollock, Advan. Enzymol. 28, 237 (1966).
96
METHODS FOR THE STUDY OF ANTIBIOTICS
[5]
2. The Use o] Antisera to Study Laboratory-Induced Mutations The main objective here, since one starts with the information that any changes in reaction are due to changes in amino acid sequence, is to try to discover whether the mutation has affected only the turnover number of the enzyme, or whether there has also been some alteration in affinity for antibodies. A typical example is the examination of the pen P2 mutant of staphylococcal fl-lactamase. 1~ Bacterial cultures synthesizing this mutant enzyme express about 5% of the normal wild-type level of activity. Comparison of the serum interaction of this mutant enzyme with the wild type shows that the total stimulation obtained is the same (i.e., about 4.5-fold), but that the antiserum titer with the mutant enzyme is about ½oth of that found with the wild type. These results suggest strongly, therefore, that the mutational change in P2 has resulted in a protein with about 5% the turnover number of the wild-type enzyme but with no alteration in the affinity of interaction between serum and enzyme. This result is only an indication and must be confirmed by purification of the enzyme; and indeed in the case of mutant P2 the turnover of the purified mutant enzyme is found to be about 5% of the unmutated parental fl-lactamase. As yet this approach to the study of fl-lactamases is largely unexploited since no systematic analysis of the effect of primary sequence changes on enzyme activity and reaction with antiserum has yet been made. Once this approach is developed, however, the use of sera in an appropriate way will give much useful preliminary information about the properties of mutant enzymes, even if the final situation can be confirmed only by the painstaking isolation and purification of each mutant.
3. Use o] Antisera to Analyze One Component in a fl-Lactamase Mixture The one group of strains where the general rule that fl-lactamases tend to be species specific breaks down in that group of gram-negative bacteria susceptible to R factor infection. In this group it is possible to find bacterial isolates that express two distinct types of fl-lactamase,8 and it can therefore be valuable to have methods for determining the relative amounts of the two enzymes expressed by such strains. One typical example is the use of anti-type I I I a fl-lactamase serum to analyze the enzyme produced by a strain of E. coli which can be shown by electrophoresis of a crude enzyme preparation to make two enzymes, one a cephalosporinase of type Ib and the other an R factor-mediated enzyme (type IIIa). The type Ib enzyme does not react with the anti-type IIIa serum ~ See footnote 4, and also unpublished experiments b y M. It. Richmond.
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IMMUNOLOGICAL TECHNIQUES FOR STUDYING ~-LACTAMASES
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whereas the t y p e I I I a e n z y m e - - b e i n g the enzyme against which the serum was raised in the first p l a c e - - g i v e s a neutralization reaction with a residual activity of about 35% (see Fig. 1). About 100 units of the enzyme mixture was titrated with anti-type I I I a serum as a constant antigen titration. The serum partially neutralized the activity of the mixture, and the residual activity was about 67% of the initial activity (Fig. 6). Since (a) only the type I I I a enzyme reacts with the antiserum, and (b) the presence of type I b enzyme does not interfere with the reaction, it is possible to calculate the relative amounts of the two enzymes in the original mixture using the fact that the residual activity obtained with type I I I a enzyme and this antiserum is 35% of the initial activity. I n this particular example, in fact, the calculation shows that the original mixture was made up of about 50 units of type Ib enzyme and 50 units of t y p e I I I a . This approach to analyzing the composition of mixtures of fl-lactamases is very useful, but it is subject to two limitations. First, the antiserum must be specific to one of the two component enzymes; and second, the amount of the two components must be approximately equal. Clearly the total activity of small amounts of type I I I a enzyme in the presence of large amounts of a nonreactive fl-lactamase will show relatively little decrease of activity in the presence of antiserum, and any subsequent calculations must be subject to large errors. 120
\ 8( .>
E
'esidual activity
40
0.1 ml
0.2
I
of a n t i s e r u m
FIG. 6. Neutralization of a mixture of type Ib and type IIIa fl-lactamase with anti-type IIIa serum. E, equivalence point. Type Ib enzyme does not react with anti-type IIIa serum. Moreover this serum neutralizes type IIIa fl-lactamase to 35% residual activity. The actual neutralization observed in Fig. 6 is 35 units out of a total of 103 present; and this corresponds to the neutralization of 54 units of type IIIa fl-lactamase. The type Ib component therefore amounts to 49 units, by difference.
98
METHODS FOR THE STUDY OF ANTIBIOTICS
[6]
B. Precipitation Analysis ~6
Even if antisera to fl-lactamases do not affect enzyme activity, they will precipitate enzyme protein provided the concentration of the reactants is high enough. However, since most enzymes have such high specific enzyme activities it is unusual to have sufficient enzyme protein in a test system to produce an effective precipitate. It follows, therefore, that quantitative precipitation analysis of fl-lactamases is usually confined to rather special circumstances; and it is normally necessary to label the enzyme protein with radioactivity in order to follow the process accurately. Quantitative precipitation analysis has been used effectively to discover whether fl-lactamase I induction in Bacillus cereus was due to the biosynthesis of enzyme de novo, or whether conversion of an inactive precursor was involved.16 In these experiments a culture of Bacillus cereus growing exponentially was induced by adding 6 gg of benzyl penicillin per milliliter, and at the same time a source of 14C-labeled amino acids was added to the culture. Incubation was continued, and samples of the culture were withdrawn at intervals for precipitation analysis. The amounts of fl-lactamase I present in the growth medium in this experiment (the enzyme is an extracellular protein in B. cereus) were far too low for precipitation to occur without the addition of carrier fllactamase. Accordingly, each sample from the culture supernatant was treated with enough purified nonradioactive B. cereus fl-lactamase I to bring the total activity of the sample to 9000 units, followed by a 50% excess of specific antiserum. The precise experimental sequence was carried out as follows.16 Samples of whole bacterial culture (4.0 or 5.0 ml) were pipetted directly into a centrifuge tube containing a few crystals of oxine (8-hydroxyquinoline) and sufficient solid sodium chloride to bring the final concentration to molar strength. After shaking to dissolve the solids, the tubes were cooled rapidly and the bacteria centrifuged down. The penicillinase activity in the supernatant was assayed and enough carried fl-lactamase added to 3.0 ml of the total sample to bring the total penicillinase activity to 9000 units. This amount of enzyme is equivalent to about 3 mg of pure enzyme. The carrier supplemented samples were then dialyzed overnight at 2 ° against 1 mM KH2P04/K~HPO~ buffer, pH 7.0. After overnight dialysis, the carrier-supplemented enzyme preparations were transferred to centrifuge tubes and 0.15 ml of a suspension of "fine-mesh" glass powder (100 mg of glass per milliliter) added to each. The preparation was left at room temperature for 3 min, then the glass powder was centrifuged off and washed once with 5 ml of dis~6M. R. Pollock and M. Kramer, Biochem. J. 70, 665 (1958).
[6]
IMMUNOLOGICAL TECHNIQUES FOR STUDYING ~-LACTAMASES
99
tilled water. The enzyme was then eluted from the glass with two successive washes (2.5 ml and 1.0 ml, respectively) of alkaline 1 M NaC1 at pH 8.5. These two washings were combined and filtered through an Oxoid membrane filter to sterilize them and made up to 5.0 ml with distilled water; their fl-lactamase content was assayed. Enough carrier fl-lactamase to return the total activity of the preparation to 9000 units was then added to 4.0 ml of the eluate. Anti-exo-penicillinase serum was then added to exceed the equivalence point by 50%, and the mixture was incubated overnight at 35% The next morning the precipitate was centrifuged down after cooling to 2 °, resuspended in 2 ml of distilled water, and centrifuged down once more. This precipitate was then prepared for radioactivity measurements in the conventional way. If this procedure is followed meticulously, at least 90% of the fl-lactamase activity is precipitated as an antigen/antibody complex; and the application of these precipitation techniques allowed Pollock and Kramer TM to show conclusively that B-lactamase induction was not due to the activation of an inactive precursor, but was the result of de novo fl-lactamase synthesis. Gel Precipitation and Other Qualitative Procedures The main objective in this article has been to describe the preparation of specific anti-fl-lactamase sera and to show how they may be used for the quantitative study of penicillinases and cephalosporinases. Once the sera have been obtained, however, they may be used for the full range of gel precipitation and immune-electrophoresis procedures available for other enzymes and proteins. These have frequently been reviewed, and the reader is referred to them. 17 One special point must be borne in mind when applying these techniques to fl-lactamases. Several of the enzymes, but notably all the B-lactamase variations synthesized by plasmid carrying strains of Staphylococcus aureus are relatively basic proteins and adsorb strongly to acidic polymers such as agar. Gel diffusion experiments involving these enzymes should therefore be carried out in the presence of 1 M NaC1. Without this no diffusion of enzyme occurs and precipitation, if it occurs at all, is either on the edge of the well or even in the enzyme well itself. Unfortunately, it is impossible to add such high salt concentrates to electrophoretic systems, and so far immune electrophoresis of staphylococcal fl-lactamase has been unsuccessful. These enzymes do not adsorb to starch gels, but the precipitation bands are difficult to locate in this medium unless they are visualized by staining the gels for enzyme activity. The best method of doing this is to treat starch gels after electro1, C. A. Williams and M. W. Chase, Eds., "Methods in Immunology and Immunochemistry," Vol. 3, pp. 118 and 234. Academic Press, New York, 1971.
100
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
phoresis with the reagent used to detect fl-lactamase by colonies growing on starch agar. 1 This method is less effective than might be expected, however, since the reaction spreads rapidly and the precipitation bands appear clearly only for a short time.
[7] Paper Chromatography of Antibiotics By VLADIMIRBETINA I. II. III. IV.
V.
VI.
VII.
VIII.
IX.
X. XI. XII.
Introduction . . . . . . . . . . . . . . . . . . General T e c h n i q u e s . . . . . . . . . . . . . . . . Bioautography . . . . . . . . . . . . . . . . . Paper C h r o m a t o g r a p h y of fl-Lactam Antibiotics . . . . . . . A. Penicillins a n d 6-Aminopenicillanic Acid . . . . . . . . . B. Cephalosporin C F a m i l y , 7-ACA, a n d C e p h a m y c i n s . . . . . C a r b o h y d r a t e Antibiotics . . . . . . . . . . . . . . A. Aminoglycosidic Antibiotics . . . . . . . . . . . . B. Streptothricin Group . . . . . . . . . . . . . . C. Oligosaccharides w i t h C h r o m o p h o r e . . . . . . . . . . D. E v e r n i n o m i c i n a n d L i n c o m y c i n Group . . . . . . . . . E. Other Sugar D e r i v a t i v e s . . . . . . . . . . . . . Macrocyclic Lactone Antibiotics . . . . . . . . . . . . A. N o n p o l y e n e Glycosidic Macrolides . . . . . . . . . . B. Polyene Macrolides . . . . . . . . . . . . . . . C. Other Macrocyclic Antibiotics . . . . . . . . . . . . Quinone Antibiotics . . . . . . . . . . . . . . . A. Tetracyclines . . . . . . . . . . . . . . . B. A n t h r a c y c l i n e s a n d A n t h r a c y c l i n o n e s . . . . . . . . . Amino Acid a n d Peptide Antibiotics . . . . . . . . . . A. Diketopiperazine D e r i v a t i v e s . . . . . . . . . . . B. H o m o p e p t i d e s . . . . . . . . . . . . . . . C. H e t e r o m e r Peptides . . . . . . . . . . . . . . D. Sideromycins . . . . . . . . . . . . . . . . E. Bleomycin Group . . . . . . . . . . . . . . F. A c t i n o m y c i n s . . . . . . . . . . . . . . . G. E c h i n o m y c i n - T y p e Antibiotics . . . . . . . . . . N i t r o g e n - C o n t a i n i n g Heterocyclic Antibiotics . . . . . . . A. M i t o m y c i n Group . . . . . . . . . . . . . . B. P y r i m i d i n e Nucleosides . . . . . . . . . . . . . C. P u r i n e Nucleosides . . . . . . . . . . . . . . O x y g e n - C o n t a i n i n g Heterocyclic Antibiotics . . . . . . . . Alicyclic Antibiotics . . . . . . . . . . . . . . . A r o m a t i c Antibiotics . . . . . . . . . . . . . . A. Chloramphenicol a n d I t s Derivatives . . . . . . . . . B. Novobiocin . . . . . . . . . . . . . . . .
101 102 105 110 110 116 119 119 123 126 128 128 129 129 134 136 137 137 141 144 144 145 146 146 148 150 150 152 152 152 153 154 156 159 159 160
METHODS FOR THE STUDY OF ANTIBIOTICS
[6] I m m u n o l o g i c a l
Techniques
[6]
for
Studying/~-Lactamases
By M. H. :RICHMOND I. Introduction . . . . . . . . . . . . . . . . . . II. Preparation of Antisera . . . . . . . . . . . . . . . A. Using Purified Enzyme Preparations . . . . . . . . . . B. Using Crude Enzyme Preparations . . . . . . . . . . . C. Isolation of ~-Lactamase-Less Mutants . . . . . . . . . D. Inoculation Program for Crude Preparations . . . . . . . . III. Examination of ~-Lactamases with Specific Antisera . . . . . . . A. Neutralization Analysis . . . . . . . . . . . . . . B. Precipitation Analysis . . . . . . . . . . . . . . .
86 86 86 87 89 89 90 90 98
I. Introduction I n principle, fl-lactamases are no different from other enzymes in respect to their action with antisera, and m a n y of the techniques to be described below are capable of much wider application t h a n has occurred in the past. The reason for describing t h e m here, however, is t h a t they have a particular relevance to fl-lactamases since m a n y of the minor variants of this enzyme t h a t are found in naturally occurring strains have been characterized b y these immunological methods.
I I . P r e p a r a t i o n of Antisera
A. Using Purified Enzyme Preparations For the preparation of specific antisera, and those for reaction with enzymes are no exception, it is preferable to have the antigen as pure as possible. I n this w a y the sera are likely to have their highest titers and to be as free as possible of nonspecific side reactions. In practice, preparation of a pure antigen m a y be more exacting than normal protein purification since small amounts of impurity m a y be disproportionately immunogenic. On the other hand, these considerations do not often invalidate the use of sera for analytical purposes with fl-lactamases. Indeed all the studies on staphylococcal and Escherichia coli B-lactamases described in this section were carried out with enzymes purified by the methods described elsewhere in this volume. 1,2 1This volume [53c]. This volume [53d].
[6]
IMMUNOLOGICAL TECHNIQUES FOR STUDYING ~-LACTAMASES
27
A number of different animal species and dosage regimes have been used to raise fl-lactamase sera. All the most effective, however, use inoculation of rabbits with purified enzyme preparations treated in various ways2 ,4 For antistaphylococcal penicillinase,4 sandy lop rabbits are each injected in alternate thighs at 3-day intervals with a total of four 0.2-ml portions of a solution of purified staphylococcal penicillinase made by suspending about 5 mg of purified enzyme per milliliter of Freund's adjuvant2 After a further 2 weeks, each rabbit is injected intravenously through the ear vein with three successive 0.2-ml samples of an alum-precipitated preparation of purified enzyme (approximately 5 mg/ml) at 2-day intervals. Samples of blood are withdrawn from the rabbit's ear vein 6 days after the end of the second phase of treatment, the serum is separated by conventional techniques, and the antibody titer is determined. In practice, once a serum of suitable titer has been obtained, it is better to kill and exsanguinate the rabbit since this procedure provides maximum quantities of a serum of uniform properties. In a typical example with staphylococcal penicillinase, about 100 ml of a serum, with titer about 104 units per milliliter of undiluted serum, was obtained from a single sandy lop rabbit. After separation of the serum, the material is best stored by dividing into ampules in about 5-ml quantities and freezedrying. Ampules may then be reconstituted for use as required by adding 5 ml of distilled water. If diluted serum is needed, the reconstituted serum should be diluted for use in physiological saline.
B. Using Crude Enzyme Preparations The above method is the one that should be followed when reasonable supplies of purified enzyme are available. It is possible, however, to prepare adequate specific sera by inoculating relatively crude enzyme preparations in Freund's adjuvant, and then absorbing out the unwanted antibodies before use. For this procedure to be successful, it is vital to have a mutant of the producer strain that makes no fl-lactamase. Before going on to describe the actual techniques for preparation of fl-lactamase from crude ])reparations, the purification procedure for crude enzyme and the methods of isolating fl-lactamase-less mutants will be described. Preparations of fl-lactamase from most gram-negative bacteria may be prepared as follows. The method is based on the one described elsewhere in this volume for the purification of the fl-lactamase from Escherichia coli (Rr+~M).2 A culture of the producer strain growing exponena M. R. Pollock, J. Gen. Microbiol. 14, 90 (1956). 4 M. H. Richmond, Biochem. J. 88, 452 (1963). 5 Difco Bacto-adjuvant, Complete (Freund), 0638-60-7.
88
METHODS FOR THE STUDY OF ANTIBIOTICS
[5]
tially in 1% CY medium 1 is centrifuged and the bacteria are collected. These organisms are resuspended in 0.1 M Na2HPO4/KH2P04 buffer, pH 7.0, to a density of about 30 mg dry weight of bacteria per milliliter and disrupted in an ultrasonic disintegrator. ~ The broken bacteria are stored at 2 ° until disruption of the whole batch is complete; they are then centrifuged, first at 5000 g for 15 rain at 4 ° (to remove large cell debris), then at 40,000 g for 4 hr at 4 ° (to remove smaller fragments), and last at 105,000 g for 2 hr at 4 ° to sediment ribosomes and minute pieces of comminuted membrane. After centrifugation the supernatant containing the enzyme is dialyzed against 100 volumes of 0.1 M Na~ HPO4/KH2P04 buffer, pH 7.0, for 24 hr at 2 °. During this process a precipitate often forms, and this is removed by centrifugation at 5000 g for 15 min at 4 °. After dialysis and any necessary centrifugation the enzyme is run through a Sephadex column. Adequate preparations are obtained if a Sephadex G-75 column equilibrated against 0.1 M Na~HPO~/KH2P04 buffer, pH 7.0, is used, but cleaner enzyme preparations may be obtained by using either DEAE-Sephadex or CM-Sephadex, as appropriate. The choice of Sephadex depends on the nature of the enzyme, and to decide this it is useful to know something of the ionic properties of the protein. In general all fl-lactamases from gram-negative species with a predominant activity against cephalosporins are positively charged at neutral pH values 6 and therefore should be purified on CM-Sephadex. The remaining fl-lactamases are likely to be more acidic 6 and in this case DEAE-Sephadex may be more appropriate./~lthough a general tendency, however, this correlation between charge andsubstrate profile should not be taken as absolute, and it is always necessary to carry out some preliminary test absorptior~ experiments with the enzyme and various substituted Sephadexes when an enzyme is being examined for the first time. As a guide it is always helpful to know the electrophoretic properties of the enzyme before doing the experiments. When G-75 Sephadex is used, the column (150 X 1.5 cm) is equilibrated against 0.1 M Na2 HPO4/KH2P04 buffer, pH 7.0, and the material is eluted with similar buffer. After elution from the Sephadex column the enzyme preparation is dialyzed against distilled water to remove salts and then stored at 2 ° until required for use. In general it seems better not to freeze dry enzyme preparations destined for raising antisera since the freezing and thawing process is likely to denature some of the enzyme molecules, and this in turn is likely to broaden the specificity of the resulting antiserum. M. H. Richmond and R. B. Sykes, Recent Adv. Microb. Physiol. 9, 36 (1973).
[6]
IMMUNOLOGICALTECHNIQUES FOR STUDYING ~-LACTAMASES
89
C. Isolation of/~-Lactamase-Less Mutants The fl-lactamase producing culture is grown exponentially in any convenient growth medium and then treated with N-methyl-N-nitro-Nnitrosoguanidine (NMG) as described elsewhere in this volume. 1 After treatment and the necessary period of growth following removal of the mutagen, the bacteria are plated as single colony-forming units on 1% CY agar l and incubated overnight at 37 °. In the morning the colonies are flooded on the plate with a solution (5 mg/ml) of the chromogenic cephalosporin 87/312. 7 In this example, the great majority of the colonies will turn red (indicating production of fl-lactamase), but a few (probably not more than 1/50,000) will produce no color. It is therefore necessary to do this experiment on a large scale. About 100 plates with 1000 colonies per plate are commonly employed; but it may be necessary to work on an even larger scale. Any colorless colonies are picked onto fresh agar and purified by restreaking. The isolates are next checked for their production of fl-lactamase, and any producing no detectable enzyme by quantitative methods are retained for use. The process of isolating mutants in this way is tedious, since methods for selection of fl-lactamaseless mutants are not available, but fl-lactamase-less mutants of this type must be available for specific sera to be obtained when crude enzyme preparations are used as immunogens.
D. Inoculation Program for Crude Preparations As with the program for purified enzyme, rabbits are the most convenient animal for raising sera in the conventional laboratory. The larger the rabbit the better, since the final yield of serum will depend to some extent on the blood volume of the animal; and absorbed sera are likely to have lower titers than those prepared against purified antigens. A typical program for raising an antiserum using a crude enzyme preparation is as follows. Samples of crude enzyme, purified as described above, are injected after mixing with equal volumes of Freund's adjuvant into alternate thighs of a sandy lop rabbit. In all, a sequence of six injections at 2-day intervals is used. Two weeks after the last injection, the animal is injected through the ear vein with 0.2 ml of alum-precipitated crude enzyme. Four injections of this material are made at 2-day intervals. About 10-14 days after the end of this sequence of injections, the animal is test bled, and the antiserum titer is determined against the appropriate enzyme (see below). When two test bleeds 2 days apart show the same titer, the animal is killed and exsanguinated, and serum is prepared from TC. H. O'Callaghan, A. Morris, S. M. Kirby, and A. H. Shingler, Antimicrob. Ag. Chemother. 1, 283 (1972).
90
METHODS FOR T H E STUDY OF ANTIBIOTICS
[6]
the whole blood by conventional techniques. Titers of about 5000 units of neutralizing ability are not uncommon by this technique, but it must be stressed that not all anti-fl-lactamase sera are of the neutralizing type (see below). Once an active serum has been obtained by this method it is then treated with crude protein prepared from the fl-lactamase-less mutant strain. Ideally the serum should be treated with material made in an identical manner to the crude enzyme, protein being collected from the Sephadex G-75 column at the point at which the relevant fl-lactamase is known to elute. However, it is equally effective to use some of the total cell protein obtained after ultrasonic disruption of the fl-lactamaseless mutant. However, if material that is the product of ultrasonic disintegration without centrifugation is used, losses in the titer of the serum are likely to be high, but if the material at the stage at which it would normally be loaded onto the Sephadex column is employed, absorption is more effective and losses of specific anti-fl-lactamase activity is minimized. The adsorption technique involves adding the absorbing protein to the serum in a series of 0.2-ml quantities until precipitation ceases. Ideally this should lead to no loss in titer of the antiserum, but in practice some loss seems difficult to avoid, and it is usually necessary to compromise between having an absolutely specific serum and one of such low titer that it is virtually useless. In a typical absorption experiment, 0.2-ml quantities of absorbing protein were added at intervals to 5 ml of undiluted serum. The serum was then incubated at 30 ° for 2 hr before the precipitate was centrifuged off at 5000 g for 15 min. In one experiment about 0.75 ml of absorbing protein had to be added to the 5-ml batch of serum before precipitation ceased. In order to sterilize the serum during this procedure, 0.1% (w/v) sodium thiomersalate is added to the serum. Once absorption is complete, the serum is transferred to ampules and freeze-dried in the normal way. III. Examination of fl-Lactamases with Specific Antisera Once antisera to a B-lactamase have been obtained, they can be used to study the enzymes in a number of ways. These include precipitation analyses both in solution and by gel diffusion. But perhaps the most effective is by neutralization of enzyme activity since this can be studied with as little as 0.1 ~g of enzyme in impure preparations.
A. Neutralization Analysis When antibodies interact with enzymes, the activity of the preparation may be altered. However, interaction with an antibody molecule
[~]
IMMUNOLOGICAL TECHNIQUES FOR STUDYING ~-LACTAMASES
91
need not affect activity, and it is impossible to predict the manner in which an antiserum preparation raised against either a crude or a purified preparation of fl-lactamase will act. Indeed, antisera obtained with samples of the same enzyme preparation in different rabbits may v a r y greatly in their effect, even when the immunization program is identical and carried out in parallel. It follows, therefore, that when antisera are being raised it is important to obtain as large a quantity as possible so that a large series of tests may be made with a serum of uniform properties. Provided an antiserum affects enzyme activity, this interaction m a y be used to provide much information about the nature of a fl-lactamase. Most sera reduce the activity of B-lactamases, 3,~ and this neutralizing effect will be discussed first. However, stimulatory sera, ~ or even sera that stimulate and then inhibit at higher concentration are known, 9 but their use will be discussed later. The most common way to use a neutralizing serum is to study the enzyme/antiserum interaction as a titration of a constant amount of antigen. Increasing amounts of antiserum are added to a series of vessels each containing a fixed amount of antigen. After a period for the antigen/antibody reaction to occur (usually about 10 rain at room temperature) the residual enzyme activity is assayed by a convenient method (usually the iodometric method of Ferret TM as modified by N o v i c k ' ) . With E. coli fl-lactamase, where a serum with titer about 104 units/ml is available, the usual experimental approach is to put about 50-75 units of enzyme in each of a series of conical flasks and immediately add increasing amounts of serum, leaving the first without addition to act as a measure of the untreated enzyme activity. The reaction is allowed to continue for 10 rain. Then 10 ml of a prewarmed solution of benzyl penicillin (1%, w/v, in 0.1 M Na~ H P O J K H 2 P O 4 buffer, pH 5.9) is added. After 6 rain further incubation, 10 ml of standard iodine solution in 2 M sodium acetate buffer, pH 4.2, is added and the excess iodine back-titrated against sodium thiosulfate." Figure 1 shows a typical neutralization curve obtained in this way2 There is a linear decrease in residual enzyme activity with increasing antiserum concentration up to a point (the equivalence point: E--see Fig. 1) at which no further inactivation occurs however much serum is added. The activity remaining after the equivalent amount of serum has been added is known as the "residual activity" and probably represents the activity of the enzyme/antibody complex. The slope of the neutralization curve G. W. Jack and M. H. Richmond, J. Gen. Microbiol. 61, 43 (1970). 9 M. R. Pollock, Immunology 7, 707 (1964). ~oC. J. Perret, Nature (London) 174, 1012 (1954). See also this volume [5]. ~ R. P. Novick, Biochem. J. 83, 229 (1962).
92
METHODS FOR THE STUDY OF ANTIBIOTICS
[6]
F
100~-,,~
'~: .I- \ . 6o-
\o
\o
--
'~EO--~O
20 I 0.02
I 0,04
I 0,06
I 0.08
Antiserum
I 0.1
I 0.12.
: ml
FIG. 1. Neutralization of type I I I a ~-lactamase by anti-type I I I a fl-lactamase serum [G. W. Jack and M. I-I. Richmond, J. Gen. Microbiol. 61, 43 (1970)]. E, equivalence point.
gives the titer of the serum; and if the turnover number of the fl-lactamase is known, this titer m a y be expressed as the number of enzyme molecules neutralized per milliliter of antiserum. In practice 10 min is normally allowed for the enzyme/antibody complex to forIn at room temperature, but a study of the time course of enzyme/antiserum interaction shows that the reaction occurs very rapidly (Fig. 2). Neutralization with more than the equivalent amount of serum is more than 90% complete in 2 min at room temperature. Figure 1 illustrates a typical neutralization curve obtained with an anti-fl-lactamase serum. However, all sera do not neutralize. Figure 3
~
c t00 ]uu .2
6{ E
E E
•
O
/
°~° 2C I 2
I 4
I 6
I 8
I 10
minutes
FIG. 2. Time course of reaction between type I l i a /~-lactamase and a specific antiserum.
[6]
IMMUNOLOGICAL TECHNIQUES FOR STUDYING /~-LACTAMASES
93
241 20(
//
ID
/* /* 40,;I 0.05
i 0.1 Antiserum
| 0.15
I 0-2
: ml
FIG. 3. Stimulation of staphylococcal ~-lactamase activity by a specific antiserum [M. H. Richmond, Biochem. J. 88, 452 (1963)]. E, equivalence point.
shows the effect of addition of increasing amounts of anti-staphylococcal serum to staphylococcal fl-lactamase type A. In this case this particular batch of serum stimulates the activity of the enzyme to a maximum about 4.5 times higher than that of the enzyme without serum. ~ Once again the response is linear with increasing amounts of antiserum, and once more there is an equivalence point (E) above which further addition of serum produces no change in activity. As with neutralization effects, stimulation by antiserum can be used to characterize an enzyme. The slope of the curve (Fig. 3) gives a measure of the titer of the serum, and the extent of stimulation is constant for a given type of fl-lactamase. At present it is uncertain how combination with an antiserum increases the activity of an enzyme. Intuitively one feels that the process must hold the enzyme in a more favorable conformation, but convincing experimental evidence for this sort of mechanism is lacking. Nevertheless one thing seems certain: The binding of stimulatory sera cannot block the active center of the enzyme. Certain sera show even more complex effects with fl-lactamases than the examples shown in Figs. 1 and 3. Pollock has studied the interaction of specific serum with purified fl-lactamase from Bacillus licheni]ormis2 In this example (Fig. 4) addition of antiserum causes stimulation and then inhibition. Such a curve has two equivalence points (El and E.~) but is difficult to use for analytical purposes, although the shape of the curve has great value as a qualitative means of identifying an enzyme type. This complex curve is certainly due to the interaction of the enzyme with an antiserum containing both inhibitory and stimutatory compo-
94
METHODS FOR THE STUDY OF ANTIBIOTICS
[5]
3001 /\,E1 .. 200 >
0
i ,°0,
\,
o
E2
I
I
O.2
o.1 ml
of
I
O.3
antiserum
FIG. 4. Interaction of fl-lactamase from Bacillus licheni]ormis 6346 with specific antiserum [M. R. Pollock, Immunology 7, 707 (1964)]. E,, stimulatory equivalence point; E~, neutralizing equivalence point.
nents. Partial absorption of the serum with enzyme removes the stimulatory component and gives a residual serum which is purely neutralizing2
I. Characterization o] Variants in Naturally Occurring Strains Since the effect of antisera on fl-lactamase activity can be studied with such small amounts of enzyme and since contaminating protein does not interfere significantly with the interaction, tests in solution are an ideal way of looking for molecular variants of fl-lactamase whether of natural or laboratory (i.e., mutational) origin. An example of the detection of natural variants comes from studies that have been made on staphylococcal fl-lactamase. 12,13 The reaction of anti-staphylococcal fllactamase serum with the fl-lactamase found in most clinical isolates of Staphylococcus aureus gives a stimulatory curve already discussed (Fig. 1). However, analysis of a large number of clinical isolates shows that four types of response are to be found (Fig. 5). The majority show the standard stimulation by a factor of about 4.5-fold---known as A-type response--while a substantial minority show no stimulation at all. Indeed in this group (C type) there is no evidence that the serum (which was raised against purified A-type enzyme) interacts with the fl-lactamase at all until a precipitation analysis is carried out. Under these conditions an enzyme/antiserum precipitate is obtained, but this has the expected activity of the amount of enzyme combined in the precipitate. Two other ~2M. It. Richmond, Biochem. J. 94, 584 (1968). ~3V. T. Rosdahl, J. Gen. Microbiol. 77, 229 (1973).
[{)]
IMMUNOLOGICAL TECHNIQUES FOR STUDYING ~-LACTAMASES
95
20o
"~160 •;
120
o / / -
• ~
• ~
• ~ OA
o
o
o
I
I
/+
i40, ~-~ I
0.1 0.2 0.3
I
Od
Co
I
0.5
ml of antiserum
Fro. 5. Response of the four variants of staphylococcal f~-lactamase after interaction with a serum raised against type A [M. H. Richmond, Biochem. J. 94, 584 (1968)]. A, B, C, D: fl-lactamase types based on their response to serum.
types of response are also found. In the first (type B), the antiserum stimulates, but the titer is about one-fifth of that expressed with A-type fl-lactamase. In fact, further analysis shows that this response is due to expected antiserum binding to a fl-laetamase molecule with about onefifth the turnover number of A-type fl-lactamase. The fourth type of response (type D) gives a lower maximum degree of stimulation (about 1.5-fold at most) with a lower apparent titer. 13 In this case, the turnover number of the fl-lactamase variant is the same as that of A-type enzyme, and therefore the serum interacts with the enzyme with lower affinity. Subsequent experiments have shown that types A, B, and C fl-lactamase from S. a u r e u s (and probably type D as well) are minor variants of a common structure probably with one or a few amino differences in their primary sequence. Similar experiments have been done with fl-lactamases from other bacterial species, and in general these experiments have led to the view that these enzymes tend to be species specific, but that within a given species a relatively large number of minor variants are to be found. 6''4 The cross reaction found between the minor variants within a given species, but the absence of cross reaction between the fl-lactamases from different species suggests that perhaps interaction between enzyme and antiserum is particularly sensitive to changes in the primary sequence of the enzyme protein, and that a change in only one or two residues can be tolerated before the binding of the serum becomes suSstantially impaired. Nor is such a view incompatible with the normal high specificity of antibody molecules. ~ N. Cirri and M. R. Pollock, Advan. Enzymol. 28, 237 (1966).
96
METHODS FOR THE STUDY OF ANTIBIOTICS
[5]
2. The Use o] Antisera to Study Laboratory-Induced Mutations The main objective here, since one starts with the information that any changes in reaction are due to changes in amino acid sequence, is to try to discover whether the mutation has affected only the turnover number of the enzyme, or whether there has also been some alteration in affinity for antibodies. A typical example is the examination of the pen P2 mutant of staphylococcal fl-lactamase. 1~ Bacterial cultures synthesizing this mutant enzyme express about 5% of the normal wild-type level of activity. Comparison of the serum interaction of this mutant enzyme with the wild type shows that the total stimulation obtained is the same (i.e., about 4.5-fold), but that the antiserum titer with the mutant enzyme is about ½oth of that found with the wild type. These results suggest strongly, therefore, that the mutational change in P2 has resulted in a protein with about 5% the turnover number of the wild-type enzyme but with no alteration in the affinity of interaction between serum and enzyme. This result is only an indication and must be confirmed by purification of the enzyme; and indeed in the case of mutant P2 the turnover of the purified mutant enzyme is found to be about 5% of the unmutated parental fl-lactamase. As yet this approach to the study of fl-lactamases is largely unexploited since no systematic analysis of the effect of primary sequence changes on enzyme activity and reaction with antiserum has yet been made. Once this approach is developed, however, the use of sera in an appropriate way will give much useful preliminary information about the properties of mutant enzymes, even if the final situation can be confirmed only by the painstaking isolation and purification of each mutant.
3. Use o] Antisera to Analyze One Component in a fl-Lactamase Mixture The one group of strains where the general rule that fl-lactamases tend to be species specific breaks down in that group of gram-negative bacteria susceptible to R factor infection. In this group it is possible to find bacterial isolates that express two distinct types of fl-lactamase,8 and it can therefore be valuable to have methods for determining the relative amounts of the two enzymes expressed by such strains. One typical example is the use of anti-type I I I a fl-lactamase serum to analyze the enzyme produced by a strain of E. coli which can be shown by electrophoresis of a crude enzyme preparation to make two enzymes, one a cephalosporinase of type Ib and the other an R factor-mediated enzyme (type IIIa). The type Ib enzyme does not react with the anti-type IIIa serum ~ See footnote 4, and also unpublished experiments b y M. It. Richmond.
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IMMUNOLOGICAL TECHNIQUES FOR STUDYING ~-LACTAMASES
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whereas the t y p e I I I a e n z y m e - - b e i n g the enzyme against which the serum was raised in the first p l a c e - - g i v e s a neutralization reaction with a residual activity of about 35% (see Fig. 1). About 100 units of the enzyme mixture was titrated with anti-type I I I a serum as a constant antigen titration. The serum partially neutralized the activity of the mixture, and the residual activity was about 67% of the initial activity (Fig. 6). Since (a) only the type I I I a enzyme reacts with the antiserum, and (b) the presence of type I b enzyme does not interfere with the reaction, it is possible to calculate the relative amounts of the two enzymes in the original mixture using the fact that the residual activity obtained with type I I I a enzyme and this antiserum is 35% of the initial activity. I n this particular example, in fact, the calculation shows that the original mixture was made up of about 50 units of type Ib enzyme and 50 units of t y p e I I I a . This approach to analyzing the composition of mixtures of fl-lactamases is very useful, but it is subject to two limitations. First, the antiserum must be specific to one of the two component enzymes; and second, the amount of the two components must be approximately equal. Clearly the total activity of small amounts of type I I I a enzyme in the presence of large amounts of a nonreactive fl-lactamase will show relatively little decrease of activity in the presence of antiserum, and any subsequent calculations must be subject to large errors. 120
\ 8( .>
E
'esidual activity
40
0.1 ml
0.2
I
of a n t i s e r u m
FIG. 6. Neutralization of a mixture of type Ib and type IIIa fl-lactamase with anti-type IIIa serum. E, equivalence point. Type Ib enzyme does not react with anti-type IIIa serum. Moreover this serum neutralizes type IIIa fl-lactamase to 35% residual activity. The actual neutralization observed in Fig. 6 is 35 units out of a total of 103 present; and this corresponds to the neutralization of 54 units of type IIIa fl-lactamase. The type Ib component therefore amounts to 49 units, by difference.
98
METHODS FOR THE STUDY OF ANTIBIOTICS
[6]
B. Precipitation Analysis ~6
Even if antisera to fl-lactamases do not affect enzyme activity, they will precipitate enzyme protein provided the concentration of the reactants is high enough. However, since most enzymes have such high specific enzyme activities it is unusual to have sufficient enzyme protein in a test system to produce an effective precipitate. It follows, therefore, that quantitative precipitation analysis of fl-lactamases is usually confined to rather special circumstances; and it is normally necessary to label the enzyme protein with radioactivity in order to follow the process accurately. Quantitative precipitation analysis has been used effectively to discover whether fl-lactamase I induction in Bacillus cereus was due to the biosynthesis of enzyme de novo, or whether conversion of an inactive precursor was involved.16 In these experiments a culture of Bacillus cereus growing exponentially was induced by adding 6 gg of benzyl penicillin per milliliter, and at the same time a source of 14C-labeled amino acids was added to the culture. Incubation was continued, and samples of the culture were withdrawn at intervals for precipitation analysis. The amounts of fl-lactamase I present in the growth medium in this experiment (the enzyme is an extracellular protein in B. cereus) were far too low for precipitation to occur without the addition of carrier fllactamase. Accordingly, each sample from the culture supernatant was treated with enough purified nonradioactive B. cereus fl-lactamase I to bring the total activity of the sample to 9000 units, followed by a 50% excess of specific antiserum. The precise experimental sequence was carried out as follows.16 Samples of whole bacterial culture (4.0 or 5.0 ml) were pipetted directly into a centrifuge tube containing a few crystals of oxine (8-hydroxyquinoline) and sufficient solid sodium chloride to bring the final concentration to molar strength. After shaking to dissolve the solids, the tubes were cooled rapidly and the bacteria centrifuged down. The penicillinase activity in the supernatant was assayed and enough carried fl-lactamase added to 3.0 ml of the total sample to bring the total penicillinase activity to 9000 units. This amount of enzyme is equivalent to about 3 mg of pure enzyme. The carrier supplemented samples were then dialyzed overnight at 2 ° against 1 mM KH2P04/K~HPO~ buffer, pH 7.0. After overnight dialysis, the carrier-supplemented enzyme preparations were transferred to centrifuge tubes and 0.15 ml of a suspension of "fine-mesh" glass powder (100 mg of glass per milliliter) added to each. The preparation was left at room temperature for 3 min, then the glass powder was centrifuged off and washed once with 5 ml of dis~6M. R. Pollock and M. Kramer, Biochem. J. 70, 665 (1958).
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IMMUNOLOGICAL TECHNIQUES FOR STUDYING ~-LACTAMASES
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tilled water. The enzyme was then eluted from the glass with two successive washes (2.5 ml and 1.0 ml, respectively) of alkaline 1 M NaC1 at pH 8.5. These two washings were combined and filtered through an Oxoid membrane filter to sterilize them and made up to 5.0 ml with distilled water; their fl-lactamase content was assayed. Enough carrier fl-lactamase to return the total activity of the preparation to 9000 units was then added to 4.0 ml of the eluate. Anti-exo-penicillinase serum was then added to exceed the equivalence point by 50%, and the mixture was incubated overnight at 35% The next morning the precipitate was centrifuged down after cooling to 2 °, resuspended in 2 ml of distilled water, and centrifuged down once more. This precipitate was then prepared for radioactivity measurements in the conventional way. If this procedure is followed meticulously, at least 90% of the fl-lactamase activity is precipitated as an antigen/antibody complex; and the application of these precipitation techniques allowed Pollock and Kramer TM to show conclusively that B-lactamase induction was not due to the activation of an inactive precursor, but was the result of de novo fl-lactamase synthesis. Gel Precipitation and Other Qualitative Procedures The main objective in this article has been to describe the preparation of specific anti-fl-lactamase sera and to show how they may be used for the quantitative study of penicillinases and cephalosporinases. Once the sera have been obtained, however, they may be used for the full range of gel precipitation and immune-electrophoresis procedures available for other enzymes and proteins. These have frequently been reviewed, and the reader is referred to them. 17 One special point must be borne in mind when applying these techniques to fl-lactamases. Several of the enzymes, but notably all the B-lactamase variations synthesized by plasmid carrying strains of Staphylococcus aureus are relatively basic proteins and adsorb strongly to acidic polymers such as agar. Gel diffusion experiments involving these enzymes should therefore be carried out in the presence of 1 M NaC1. Without this no diffusion of enzyme occurs and precipitation, if it occurs at all, is either on the edge of the well or even in the enzyme well itself. Unfortunately, it is impossible to add such high salt concentrates to electrophoretic systems, and so far immune electrophoresis of staphylococcal fl-lactamase has been unsuccessful. These enzymes do not adsorb to starch gels, but the precipitation bands are difficult to locate in this medium unless they are visualized by staining the gels for enzyme activity. The best method of doing this is to treat starch gels after electro1, C. A. Williams and M. W. Chase, Eds., "Methods in Immunology and Immunochemistry," Vol. 3, pp. 118 and 234. Academic Press, New York, 1971.
100
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
phoresis with the reagent used to detect fl-lactamase by colonies growing on starch agar. 1 This method is less effective than might be expected, however, since the reaction spreads rapidly and the precipitation bands appear clearly only for a short time.
[7] Paper Chromatography of Antibiotics By VLADIMIRBETINA I. II. III. IV.
V.
VI.
VII.
VIII.
IX.
X. XI. XII.
Introduction . . . . . . . . . . . . . . . . . . General T e c h n i q u e s . . . . . . . . . . . . . . . . Bioautography . . . . . . . . . . . . . . . . . Paper C h r o m a t o g r a p h y of fl-Lactam Antibiotics . . . . . . . A. Penicillins a n d 6-Aminopenicillanic Acid . . . . . . . . . B. Cephalosporin C F a m i l y , 7-ACA, a n d C e p h a m y c i n s . . . . . C a r b o h y d r a t e Antibiotics . . . . . . . . . . . . . . A. Aminoglycosidic Antibiotics . . . . . . . . . . . . B. Streptothricin Group . . . . . . . . . . . . . . C. Oligosaccharides w i t h C h r o m o p h o r e . . . . . . . . . . D. E v e r n i n o m i c i n a n d L i n c o m y c i n Group . . . . . . . . . E. Other Sugar D e r i v a t i v e s . . . . . . . . . . . . . Macrocyclic Lactone Antibiotics . . . . . . . . . . . . A. N o n p o l y e n e Glycosidic Macrolides . . . . . . . . . . B. Polyene Macrolides . . . . . . . . . . . . . . . C. Other Macrocyclic Antibiotics . . . . . . . . . . . . Quinone Antibiotics . . . . . . . . . . . . . . . A. Tetracyclines . . . . . . . . . . . . . . . B. A n t h r a c y c l i n e s a n d A n t h r a c y c l i n o n e s . . . . . . . . . Amino Acid a n d Peptide Antibiotics . . . . . . . . . . A. Diketopiperazine D e r i v a t i v e s . . . . . . . . . . . B. H o m o p e p t i d e s . . . . . . . . . . . . . . . C. H e t e r o m e r Peptides . . . . . . . . . . . . . . D. Sideromycins . . . . . . . . . . . . . . . . E. Bleomycin Group . . . . . . . . . . . . . . F. A c t i n o m y c i n s . . . . . . . . . . . . . . . G. E c h i n o m y c i n - T y p e Antibiotics . . . . . . . . . . N i t r o g e n - C o n t a i n i n g Heterocyclic Antibiotics . . . . . . . A. M i t o m y c i n Group . . . . . . . . . . . . . . B. P y r i m i d i n e Nucleosides . . . . . . . . . . . . . C. P u r i n e Nucleosides . . . . . . . . . . . . . . O x y g e n - C o n t a i n i n g Heterocyclic Antibiotics . . . . . . . . Alicyclic Antibiotics . . . . . . . . . . . . . . . A r o m a t i c Antibiotics . . . . . . . . . . . . . . A. Chloramphenicol a n d I t s Derivatives . . . . . . . . . B. Novobiocin . . . . . . . . . . . . . . . .
101 102 105 110 110 116 119 119 123 126 128 128 129 129 134 136 137 137 141 144 144 145 146 146 148 150 150 152 152 152 153 154 156 159 159 160
