Irving M. Klotz

PHYSICOCHEMICAL ASPECTS OF DRUG‐PROTEIN INTERACTIONS: A GENERAL PERSPECTIVE

Protein-drug binding is, in essence, an interaction between a biological macromolecule and a small molecule. This type of complexation is only one of many different types of protein interaction, all of which are molecularly intertwined in a cellular or organ environment. In a general perspective, it is convenient to view such interactions as falling into two broad categories (FIGURE 1 ) , intramolecular and intermolecular. Intramolecular interactions are manifested in the different conformations that may coexist for a particular species of protein. Intermolecular complexes may be further subdivided into two classes (FIGURE 1 ), those with small molecules and those with other macromolecules. Included among the latter are the quaternary structures constituted of identical subunits, in addition to heteromeric ensembles of various types, for example, protein-protein, protein-nucleic acid. These interactions are the foundation of an interlocked set of macromolecular dynamic rearrangements that provide the fine modulation within and between parts of an organism. Small-molecule binding plays a crucial role in this manifold because it usually provides the molecular coupling device for interlinking the macromolecular states and for adjusting their relative concentrations. A general overview of protein-small-molecule binding should cover the following aspects of such interactions: 1. stoichiometry; 2. energetics; 3. molecular nature; 4. dynamics; and 5 . biological function. This brief review will describe a few representative studies from these topics to illustrate some of the general principles that govern macromolecule-minimolecule interactions.

Near‐Infrared Spectra of H2O—D2O Solutions

Near‐infrared spectra have been obtained for HOD in D2O solutions containing a variety of solutes. A sharp band occurs at 1.416 μ (7062 cm−1) and other bands at 1.525 (6557 cm−1), 1.556 (6427 cm−1), and 1.666 μ (6002 cm−1). The absorbances at 1.416 and 1.556 μ increase and decrease, respectively, with increasing temperature. From this variation, a ΔH° of formation of −2.4 kcal/mole has been estimated for the O–H···O bond in these solutions.Nineteen electrolytes and one polymer, polyvinylpyrrolidone, have been examined for their effects on the spectra of water in this overtone region. No frequency shifts have been observed with added solute, only changes in absorbance. Sodium perchlorate shows the strongest structure‐breaking effect. Tetrabutylammonium bromide and polyvinylpyrrolidone exhibit structure‐making character. A definite correlation has been found to exist between the effects of salts on the aqueous solvent and their ability to affect the conformation of a macromolecule.

Diaspirins that cross-link beta chains of hemoglobin: bis(3,5-dibromosalicyl) succinate and bis(3,5-dibromosalicyl) fumarate.

Two double-headed aspirins, bis(3,5-dibromosalicyl) succinate and bis(3,5-dibromosalicyl) fumarate, have been found to be potent acylating agents of intracellular hemoglobin (A or S) in vitro. Furthermore, each of these reagents cross-links beta chains of hemoglobin, probably at the beta cleft. The modified hemoglobins show increased oxygen affinities and reduced gelation or sickling tendencies.

Protein affinities for small molecules: Conceptions and misconceptions

Abstract There is much confusion and error in published treatments of data for multiple binding of ligands (e.g., substrates) by proteins (e.g., enzymes). There is a widespread impression that if the equilibrium binding, r, of ligand, A, by a protein with n sites can be fitted to an equation with two hyperbolic terms, i.e., r= n α k α (A) 1+k α (A) + n β k β (A) 1+k β (A) (n α +n β =n) then kβ and kβ are always the intrinsic binding constants for two sets of sites. Such a conclusion is often incorrect. For example, in many cases, the protein is constituted of identical protomers with initially identical sites for binding ligands, and yet graphical representations of the binding data appear to behave as if the sites are partitioned between two classes. Although the use of a linear combination of hyperbolic terms to represent binding of ligands by macromolecules always yields empirical parameters kα, kβ … kλ, they cannot correspond to site binding constants when there are interactions between sites. In some circumstances their values may even be imaginary, complex numbers. On the other hand, stoichiometric binding constants can be assigned unambiguously without making any assumption regarding the nature of the interactions among binding sites. These principles are illustrated concretely by analyses of binding measurements for several different proteins containing two to six sites.

The nature of the active site of hemerythrin

Abstract The oxygen-carrying pigment, hemerythrin, contains 0.81% Fe and has 2.4 Fe atoms per O2 molecule bound. It has been demonstrated that 2 of the 2.4 iron atoms are bound directly to the protein through sulfhydryl side chains, and that these two are close neighbors. It is these two iron atoms which participate directly in holding the O2 molecule. The near-ultraviolet spectrum of oxyhemerythrin resembles closely that of methemerythrin [Fe(III)], not at all that of deoxyhemerythrin [Fe(II)]. These similarities, added to the observed valence changes in oxygenation and the positive tests for peroxide ion in oxyhemerythrin, indicate that the Fe(II) in deoxyhemerythrin is converted, reversibly, to Fe(III) during the oxygenation process.

Introduction of sulfhydryl groups into proteins using acetylmercaptosuccinic anhydride

Protected mercaptan groups may be introduced into a variety of proteins under very mild conditions with S-acetylmercaptosuccinic anhydride as reagent. The effects of pH, temperature, type of protein, and other variables on the extent of reaction are described. The protecting acetyl group may be removed and the free-SH generated with the aid of a nucleophilic reagent, hydroxylamine being particularly effective in this respect.

Spectroscopy and structure of hemerythrin.

Abstract The absorption and circular dichroic spectra of hemerythrin in its deoxy, oxy, and several met states have been interpreted by comparison with those of several model systems and correlated with Mossbauer and magnetic data. The properties of the methemerythrin complexes are consistent with the presence of a dimeric pair of high spin iron (III) atoms antiferromagnetically coupled via an oxo bridge. It is proposed that oxyhemerythrin also has the same structure as methemerythrin, the oxygen being present as a second bridging peroxo group. In deoxyhemerythrin it is proposed that the two iron atoms do not interact, the oxo bridge being absent, and that they are in a high spin iron (II) state. The proposed structure predicts that considerable conformational change occurs on oxygenation.

Interactions of calcium with serum albumin

Abstract Binding of calcium ions by bovine serum albumin has been found to be essentially independent of temperature over the range 0 to 25 °C. On the other hand, pH has a very pronounced effect. No complexes with calcium are detectable until the protein is appreciably above its isoelectric point. Thereafter, uptake increases rapidly from pH 5.5 to about 8 and then seems to approach a plateau. At pH 8 and 0 °C. ΔF1∘ is equal to −3870 cal./mole, ΔH1∘ equals 0, and ΔS1∘ is equal to 14 cal./ mole/deg. Serum albumin with three salicylate groups introduced by a diazonium salt coupling showed no appreciable change in its affinity for calcium.

Dissociation of β-lactoglobulin near neutral pH☆

Abstract β-Lactoglobulin, which is constituted of two identical subunits, undergoes a conformational change with a midpoint near pH 7.5. Paralleling this change is an increase in reactivity of the single sulfhydryl group per monomer and in the binding of the organic anion methyl orange. It seemed of interest, therefore, to compare the degree of dissociation of β-lactoglobulin at pH values above and below 7.5, to examine the effects of sulfhydryl reagents and small anions, and to compare the behavior of genetic variants. Sedimentation coefficients dropped from about 3S to 2S at protein concentrations below about 1 mg/ml, as expected for dissociation of dimers into monomers, but the concentration-dependence of s20,w was similar at pH 8.2 as at 6.9. Binding of methyl orange anions did not affect the dissociation pattern. On the other hand if the sulfhydryl group (of β-lactoglobulin A) is treated with 4-(p-dimethyl-aminobenzeneazo)-phenylmercuric acetate, the protein is converted fully to the monomer. An association constant of 4.88 × 104, m −1 fits the monomer-dimer equilibrium of β-lactoglobulin A: 14.2 × 104 is the corresponding value for β-lactoglobulin B.

Properties of erythrocruorin from Cirraformia grandis.

Abstract The respiratory protein erythrocruorin from the annelid worm Cirraformia grandis has been subjected to a detailed physicochemical characterization. The amino acid composition and heme content for this protein have been determined. Molecular weight measurements indicate a value of 3.0 × 106 for the native species and 18,500 for the subunit. The number of subunits in the intact molecule is estimated as 162 ± 24. Oxygen binding experiments revealed a high affinity for oxygen, an unusually large Bohr effect, and little or no interaction among the heme groups in the pH range of 6.5–7.2.