| dc.description.abstract | The thesis entitled “Chemical Models in Penicillin Allergenesis” is divided into three chapters.
Chapter I – Introduction and Background
The first chapter deals with a résumé of the problem and enumerates the present status relating to:
the known immunochemical mechanisms in drug allergy and the types of allergic reactions,
the role of different determinants in penicillin allergy,
chemical aspects of penicillin immunogenesis,
clinical applications,
the earlier work done in this laboratory on physicochemical interactions on synthetic templates containing the structural elements of penicillin, which led to the isolation of a specific carrier receptor protein (CRP) both from immunised rabbits and a penicillin?sensitive human subject, as well as electrophoretically homogeneous anti?penicillin antibody.
This chapter ends with the aims and objectives of the current investigation:
The origin of the carrier receptor protein (CRP)
The nature of the rabbit anti?penicillin antibody
The stoichiometry of the binding of CRP with penicillin
The stoichiometry of the antigen–antibody reaction
The competitive binding of several analogues of penicillin to CRP in the presence of ¹?C?penicillin
The competition for binding of CRP–analogue conjugates versus CRP–penicillin conjugate at the antibody binding sites
The thermodynamic parameters of CRP–penicillin or CRP–analogue binding processes and of the antibody interaction
The kinetics of CRP–penicillin and CRP–analogue reactions
Chapter II – Methodology
The second chapter describes the detailed methodology used in the work.
Modified methods of synthesis of the polymeric affinity templates HP3, containing the structural elements of benzylpenicillin, and 7DOHP3, containing 7?deoxy benzylpenicillin, have been outlined:
NH–CO–CH2–C6H4–CO–(CH2)2–CO–NH–(CH2)6–N+H2–CO (HP3)
NH–CO–CH2–C6H4–CO–(CH2)4–CO–NH–CH2–O–CO–N+H2–CO (7DOHP3)
Synthesis of several analogues such as o- and p-nitrobenzyl penicillin, o- and p-aminobenzyl penicillin, benzylcepham, 6?phenylacetamidopenicillanyl alcohol, as well as ¹?C?benzylpenicillin and ¹?C?7?deoxypenicillin, has been reported. Other analogues were obtained courtesy of Beecham Laboratories, U.K.
Techniques used for immunisation of rabbits with penicillin alone and with a BPO–penicilloyl–BSA conjugate, as well as immunological assay methods, are described. Procedures for different analytical and physicochemical techniques employed in the investigation are also outlined.
Chapter III – Results and Discussion
Sera from immunised animals were fractionated on HP3 and 7DOHP3 polymers. After removal of non?specifically associated proteins with 0.5 M phosphate buffer, the antibody was isolated as an electrophoretically homogeneous protein by elution with 0.9 M thiourea.
CRP was isolated from the HP3 polymer in the penicilloylated conjugate form as a fully functional antigen; penicillin?free CRP was obtained from 7DOHP3 by elution with 4–8 M urea. The “free” CRP does not show appreciable cross?reaction with the antibody but becomes a reactive antigen upon incubation with penicillin.
Origin of CRP
Sera from rabbits prior to penicillin exposure contained 12–99 µg CRP/mL, and these levels increased with immunisation. Animals with higher CRP content showed a quicker and stronger antibody response. A penicillin?insensitive human subject contained 34 ng/mL, while a penicillin?sensitive subject with a 20?year history showed 70 ng/mL. Thus CRP appears to be a constitutive protein.
The antibody elicited with the BPO–BSA conjugate had identical electrophoretic mobility to that elicited by penicillin alone, indicating that the same antibody arises from determinants of the BPO group, whether present exogenously in penicillin or generated in vivo via the CRP–penicillin conjugate.
The isolation of electrophoretically homogeneous CRP and antibody provided a successful in vitro model for detailed studies of the chemistry and kinetics of CRP (MW 60,000) and benzylpenicillin analogues.
Stoichiometry and Binding
Using radioactive penicillin, CRP–penicillin binding was found to be 1:1. The rabbit IgG antibody (MW 150,000; chains of MW 50,000 and 23,000) was shown to be divalent in binding CRP–¹?C–penicillin conjugate.
Competitive Binding – CRP
Competitive binding ratios showed the following order of reactivity (highest to lowest):
Methicillin > 6?APA > Carbenicillin > o-nitrobenzylpenicillin > Cloxacillin ~ Benzylpenicillin ~ Penicillanyl alcohol ~ Benzylcepham < p-aminobenzylpenicillin ~ p-nitrobenzylpenicillin > Ticarcillin > o-aminobenzylpenicillin > Amoxycillin > 7?deoxybenzylpenicillin > Ampicillin
Competitive Binding – Antibody
Order of competition:
7?Deoxybenzylpenicillin > Benzylpenicillin > o-aminobenzylpenicillin ~ p-aminobenzylpenicillin > Penicillanyl alcohol > Cloxacillin > 6?APA > Benzylcepham > Ampicillin > Carbenicillin > Amoxycillin > p-nitrobenzylpenicillin
Methicillin, ticarcillin, and p-nitrobenzylpenicillin did not compete with CRP–penicillin for antibody binding.
Thermodynamic Parameters (Antigen–Antibody)
K=2.662×107?M?2K = 2.662 \times 10^{7} \, M^{-2}K=2.662×107M?2 at 4°C;
K=2.853×107?M?2K = 2.853 \times 10^{7} \, M^{-2}K=2.853×107M?2 at 37°C
?G=?12.0\Delta G = -12.0?G=?12.0 kcal/mol at 4°C;
?G=?13.5\Delta G = -13.5?G=?13.5 kcal/mol at 37°C
?H=361\Delta H = 361?H=361 cal/mol
?S=30\Delta S = 30?S=30 eu/mol
CRP–7?deoxybenzylpenicillin binding showed K?2.8×105M?1K \approx 2.8 \times 10^{5} M^{-1}K?2.8×105M?1 and ?G??8\Delta G \approx -8?G??8 kcal/mol.
Kinetics
CRP + benzylpenicillin:
Rate constant = 0.812 × 10³ L·mol?¹·min?¹
CRP + 6?APA: faster reaction
Binding Site Topology
Evidence suggests a positive charge near the ??position of the benzyl group within the CRP binding pocket.
Fluorescence Studies
Two conformational changes occur in CRP upon binding benzylpenicillin:
a rapid change from non?covalent binding (similar to 7?deoxybenzylpenicillin)
a slower change accompanying covalent binding
For 6?APA, only the second (covalent?related) change occurs.
Relevance to Penicillin Allergy
The CHR–IgG model system is highly relevant to understanding penicillin allergenesis and provides mechanistic insights into antigen formation and immune recognition. | |
