D.M. Power

Ion binding of penicillins to proteins

Abstract 1. 1. A new method is described, utilising the reactions of hydrated electrons ( e aq − ), to demonstrate that reversible ion binding occurs between a series of penicillins and the proteins, bovine serum albumin and lysozyme. 2. 2. The structure of the penicillin side chain considerably influences the binding process with bovine serum albumin. The first molecular of benzyl or phenoxymethyl penicillin which binds results in a greater decrease in the reactivity of the protein towards e aq − than subsequent molecules. 6-Aminopenicillanic acid, however, exerts an opposite effect and initially increases the reactivity of bovine serum albumin towards e aq − . Methyl penicillin and ampicillin binding decreases bovine serum albumin reactivity linearly with penicillin concentration. If the free carboxylic group is protected as in methyl and acetoxymethyl esters of benzyl penicillin no such ion binding is observed. The results are discussed in terms of the types of binding possible between these penicillins and bovine serum albumin. 3. 3. Six penicillin molecules only can bind to lysozyme and our results indicate that these are associated with the lysine residues. 4. 4. The protein-penicillin complex can be dissociated with salt. Complete breakdown of the benzyl penicillin complex with bovine serum albumin is achieved at approx. 1 · 10 −3 M KCl, but to dissociate the lysozyme complex approx. 1 · 10 −2 rmM KCl is required.

Interactions of bovine serum albumin with penicillins and cephalosporins studied by pulse radiolysis

Abstract 1. 1. The reactions of hydrated electrons produced during pulse radiolysis have been utilised to investigate the binding of eleven penicillins and four cephalosporins to bovine serum albumin. 2. 2. A primary binding site exists in the serum albumin molecule, which the results indicate to be a hydrophobic cleft in the surface of the molecule separated by a distance >0.5 nm from a cationic amino acid residue, probably lysine. 3. 3. Interaction of benzylpenicillin, phenoxymethylpenicillin, cloxacillin or phenethecillin with this binding site leads to a conformational change in the protein resulting in a decrease in reactivity towards hydrated electrons. 4. 4. Interaction of cephalosporin C and 6-amino penicillanic acid with serum albumin involves another site, which consists of a cationic amino acid residue separated from an anionic residue by a distance >0.7 nm. Drug binding at this site induces a conformational change in serum albumin, leading to greatly increased reactivity towards hydrated electrons. This increase is associated with an increased availability of disulphide bridges. 5. 5. Cephalosporin C also binds hydrophobically to bovine serum albumin, resulting in a decrease in hydrated electrons reactivity; such binding can be inhibited by the presence of palmitic acid. 6. 6. The potential of the pulse radiolysis method for revealing conformational changes in proteins is demonstrated.

Interaction of aminoacridines with nucleic acids. A pulse-radiolysis study.

Pulse radiolysis has been used to study the interaction of aminoacridines with nucleic acids. The data confirm that there are two modes of binding. These are: a weak interaction which has a maximum binding ratio of one site per dye; and a strong binding process effected by both electrostatic and Van der Waals interactions. The limit of this latter, strong binding mode is approximately six sites per dye. The radiation-induced transient absorption spectrum of benzoflavine is characterized by a pronounced bleaching at 440 nm, which is quenched by the addition of nucleic acids. Mechanisms have been proposed for the reactions of both eaq-and .OH with benzoflavine which account both for the observed bleaching of benzoflavine solutions and for the protective effect of nucleic acids. It is proposed that eaq-reacts with benzoflavine to form a stable benzoflavine semiquinone radical and that .OH reacts with subsequent formation of a very stable benzoflavine hydroxycyclohexadienyl radical.

The interaction of cytochrome C with phospholipids. A pulse-radiolysis study.

The reactions of the hydrated electron (eaq-), produced during pulse radiolysis, have been used to study the binding of phosphatidyl choline (PC), phosphatidyl serine (PS), phosphatidyl ethanolamine (PE), and phosphatidyl inositol (PI) vesicles with horse-heart cytochrome C. An interaction could only be detected between cytochrome C and either PS or PI. An apparent equivalence point in the binding was reached for both phospholipids at a molar ratio of phospholipid : protein of 6 : 1. At this point, the reactivity of (eaq-) towards the cytochrome C was very markedly reduced. Indeed, the rate of disappearance of (eaq-) under such conditions was the same as the rate of eaq- disappearance in triply-distilled water. The inclusion of cholesterol at a molar ratio of 1 : 1 within the phospholipid vesicles changed the stoichiometry of the interaction. Evidence that protonated epsilon-amino groups of lysine residues are involved in the interaction is presented. Possible models for the complexes formed are discussed.

Chemical changes following γ-irradiation of benzylpenicillin in aqueous solution

The effects of γ-irradiation on aqueous solutions of sodium benzylpenicillin have been investigated. Pulse radiolysis indicates that both hydrated electrons and hydroxyl radicals are responsible for the degradation between 10–4 and 10–2M solute concentrations. Reaction of hydroxyl radicals with the benzene ring of the penicillin side-chain leads to the formation of o-, m-, and p-hydroxybenzylpenicillins. Hydroxyl radicals also induce cleavage of the β-lactam ring, yielding benzylpenilloic and benzylpenicilloic acid. Reaction of hydrated electrons with penicillins generally lead to molecular rearrangements yielding products such as benzylpenillic acid.

Interaction of Chondroitin-4-sulphate with Cationic Dyes

SummaryPulse radiolysis has been used to estimate the thermodynamic parameters associated with the binding of six cationic dyes to tracheal chondroitin-4-sulphate. Very similar enthalpy and entropy values were obtained for the dyes of markedly different chemical structure. The extent of destruction of the complexes with increased temperature has been compared with known data of their stability in the presence of ethanol and urea.

The interaction between heparin and antithrombin III: a comparison of two different heparin-dye conjugates

The interactions of antithrombin III with two heparin-dye conjugates have been compared using their fluorescence anisotropy. The first, heparin labelled with 5-isothiocyanatofluorescein, where the dye was mostly bound to unsulphated glucosamine residues, exhibited binding which was characteristic of heparin with a low affinity for antithrombin III. The second, heparin labelled with a reactive naphthalene dye (DENMT), showed similar binding character. However, when the heparin was treated with an amino group blocking agent prior to labelling with DENMT, the resultant heparin-dye conjugate showed binding behaviour, the strength of which was consistent with heparin molecules having both high and low affinity for antithrombin III. Heparin molecules with a high affinity for antithrombin III did not possess free amino groups. The implications of these findings are discussed with regard to the reliability of the data obtained using heparin-fluorescein conjugates.

Interactions of Alkyl Sulphates with Bovine-serum Albumin Studied Using eaq− as a Probe

The reactions of hydrated electrons produced during pulse radiolysis habe been used to investigate the binding of a range of alkyl sulphates to bovine-serum albumin. Binding to ten high-affinity sites is detectable for all compounds (methyl, hexyl, octyl, decyl, and dodecyl sulphates) studied. Sodium dodecyl sulphate, in contrast to the other analogues, causes large increases in the reactivity of BSA as a result of further binding. Possible mechanisms for this increase are discussed.

The location of apoprotein in plasma high-density lipoproteins: photochemical labelling studies.

In a convincing model [ 1 ] of human high-density lipoprotein (HDL) structure, it is proposed that the apoproteins have a predominantly surface location. While supporting evidence for such an arrangement exists [2] , models involving a more extensive penetration of the lipid core of HDL have been described [3]. If photochemical labelling experiments from within the lipid core [4] of HDL could be conducted, a minimal degree of labelling of apoprotein would be expected if the model described in [l] proved realistic. We have carried out such experiments using 3H-labelled phenylnitrene photo-generated from phenylazide as a non-specific reagent. The results obtained, taken in conjuction with parallel data obtained with membrane systems where penetration of the lipid region by protein is well documented, point to a predominantly surface location of apoproteins in both human and bovine plasma HDL.