G. Blauer

Optical properties of bilirubin-serum albumin complexes in aqueous solution: I. Dependence on pH

Abstract Circular dichroism (CD) measurements in the wavelength range 300 to 550 nm of bilirubin (2.5 · 10 −5 M)-human serum albumin (5.0 · 10 −5 to 12.5 · 10 −5 M) complexes in aqueous solution (27°C) were carried out with differently treated serum albumins and at various pH values in the pH range 3.5 to 10. In all cases, two large and proximate bands of opposite sign were recorded between 400 and 500 nm and, in some cases, one or two smaller additional bands were observed either in the range of 300 to 400 nm or near 500 nm. Large differences in the magnitude of the various bands were measured for complexes involving either charcoal-treated or untreated serum albumins. Bilirubin complexes of all human serum albumin preparations showed a reversible inversion with pH in the sign of the Cotton effects of the two main bands occurring around pH 5, with some concomitant shifts in the position of band extrema. In contrast to CD, the light-absorption spectra did not differ largely under various conditions. The main Cotton effects observed in the visible region are interpreted as coupling between electric transition dipole moments of bound bilirubin leading to exciton splitting. This explanation is substantiated by computer analysis into Gaussian curves of observed light-absorption and CD spectra. From these data, and on the basis of some assumptions, both distances and relative positions in space of the transition dipoles are estimated for various cases. Other possible contributions to the optical activity are also considered. Apart from their theoretical significance, the large and diverse effects of optical activity observed under different conditions are also considered to serve as very sensitive probes for changes in the protein environment at the binding site. Moreover, the CD spectra of the complex bilirubin-serum albumin may be of analytical value for the differentiation between preparations of serum albumin and also between serum albumins from different species.

A comparison between poly-α,l-ornithine and poly-α,l-lysine in solution: The effect of a CH2 group in the side chain on the conformation of poly-α-amino acids

1. 1. The helix content of samples of poly-α,l-ornithine was compared with that of poly-α,l-lysine at 25° in dilute solution (up to 1.2 g/100 ml). Both water and 2-chloroethanol-water mixtures (3:1, v/v) were used as solvents. At high degrees of protonation of the side-chain amino groups, the helix content was effectively zero in all cases, as estimated by the optical rotatory dispersion parameter bo of Moffitts equation in the range 300–600 mμ. At low degrees of protonation, in aqueous alkali, polyornithine of DP = 100 was only about 20% helical while low molecular weight polylysine of DP = 20–25 had about 40% helix content under the same conditions, a judged by the parameter bo. In alkaline 2-chloroethanol-water both poly-amino acids had a high helix content. Hydrophobic interactions between adjacent side chains on the helix due to the additional CH2 group in polylysine are considered to stabilize polylysine relative to polyornithine in aqueous medium. 2. 2. Complexes between poly-α,l-ornithine (6–10−3 monoM; DP = 100) and ferriprotoporphyrin IX (6·10−5–7·10−5 M) in aqueous solution at pH 11 and at 25° were identified by light-absorption spectra measured in the range 370–630 mμ. Differences from previously investigated polylysine complexes in their light absorption, rate of formation and pH-stability can be explained by the differences in initial helix content of the respective poly-amino acids.

Circular dichroism of bilirubin-human serum albumin complexes in aqueous solution.

The physiologically important complex bilirubinserum albumin has been investigated by various chemical and physical methods (for a compilation of literature, see [ 11). ORD* spectra of the system bilirubin-BSA measured under various conditions, have recently been reported [2,3] . At pH 5, a negative Cotton effect curve was observed in the visible region for the bilirubin-BSA complex. Calculated on a bilirubin basis, the molar amplitude of this complex Cotton effect was about 1.5 X 106 deg. cd per decimole. The amplitude decreased markedly at both lower and higher pH values without a change in the sign of the Cotton effect curve. Preliminary CD measurements in the visible region showed both negative (longer wavelengths) and positive bands at pH 5. At pH 7.3 to 7.4, the negative band was smaller and the positive band diminished to a fraction of its magnitude at pH 5. In the absence of protein, dissolved bilirubin did not show measurable optical rotation at either pH 5 or 7.5, in the range of 220 to 600 nm and under the conditions used [3] . To the best of our knowledge, no similar Cotton effects have been characterized for the system bilirubin-HSA, although some measurements of optical rotation at various wavelengths have been reported [4] . A preliminary report of CD data obtained for the

Optical properties of bilirubin-serum albumin complexes in aqueous solution: II. Effects of electrolytes and of concentration

Abstract 1. 1.|The dependence of the circular dichroism (CD) data (300–550 nm) of bilirubin-human serum albumin complexes in aqueous solution (27°C) on the concentrations of added electrolytes and complex components was investigated at different pH values in the range pH 4 to 10. 2. 2.|Inclusion of up to 0.1 M NaCl in the system bilirubin (2.5 · 10 −5 M)-charcoal-treated human serum albumin (5·10 −5 –8·10 −5 M) at pH 4 caused an increase in both visible-range CD bands observed which remained of opposite sign without changes. At pH 7–10 there were no significant changes by increasing the chloride concentration between about 0.002 and 0.1 M. Near pH 5, however, addition of NaCl in that concentration range caused an inversion in the sign of both main Cotton effects and some shifts of band positions, with the main transition occurring in the range 0.02–0.03 M under the conditions used. At similar ionic strength, Na 2 SO 4 also caused an inversion of sign of the Cotton effects which, however, was kinetically much slower. In contrast to CD, the light-absorption spectra did not differ largely in all these cases. 3. 3.|Significant effects of added oleic acid on the CD spectra of the bilirubin-human serum albumin complex were measured, in particular near pH 4.8, while similar experiments carried out with bovine serum albumin indicated lesser effects of the oleic acid. 4. 4.|Analytical ultracentrifugation experiments on both human serum albumin and bovine serum albumin and their bilirubin complexes at various molar ratios, carried out under different conditions, indicated that no significant association between serum albumin molecules is induced by the presence of bilirubin, which appears to sediment mainly with the monomer fraction. 5. 5.|The results obtained by electrolytes at pH 5 are interpreted on the basis of previously suggested electric dipole coupling between transition moments of dipyrromethene chromophores. The binding of electrolytes to the serum albumin is considered to change the mode of binding of bilirubin with concomitant changes in the relative orientation of the transition dipoles leading to inversion in sign of the visible-range Cotton effects. These effects may constitute very sensitive probes for changes at the binding site of the protein.

Further evidence for the interaction of the antimalarial drug amodiaquine with ferriprotoporphyrin IX

Evidence for complex formation of the antimalarial drug amodiaquine (AD) with ferriprotoporphyrin IX (FP) in aqueous medium is presented, in addition to previous preliminary data. A mole ratio of one between the complex components is determined for the insoluble complex at pH 6.7-6.8. Mössbauer data obtained at pH 7-8 and at higher concentrations in the millimolar range confirm the interactions existing between the complex components. These data are considered to aid in removing previous objections to a mechanism of antimalarial action involving complexes of FP with AD and related drugs.

Synthesis and optical properties of the chloroquine enantiomers and their complexes with ferriprotoporphyrin IX in aqueous solution

Chloroquine (CQ) enantiomers were prepared by a novel synthesis starting from either (S)- or (R)-pyroglutamic acid. Light-absorption spectra of CQ and of complexes of ferriprotoporphyrin IX (FP) with CQ were measured in dilute aqueous solutions at pH 7.3 and 11.3. Spectrophotometric titrations at these pH values indicated a mole ratio of FP:CQ of 2:1 for the FP-CQ aggregated complexes. Aqueous solutions of each of the CQ enantiomers (pH 7.3) and of their complexes with FP (pH 11.3) were investigated by circular dichroism (CD). At pH 11.3, the complexes of the two enantiomers showed CD-band extrema of opposite sign at 409–410 nm. CD-titrations at pH 11.3 confirmed a predominant mole ratio of FP:CQ of 2:1 in the complex. The possible origin of the optical activity of the FP-CQ complexes is discussed. Chirality 10:556–563, 1998.

Complexes of bilirubin with proteins

Complexes of bilirubin with chymotrypsin, lysozyme and apomyoglobin in neutral aqueous solution are characterized by circular dichroism spectra in the visible region. These are analogous to previously investigated bilirubin-serum albumin complexes. Ferriprotoporphyrin IX displaces bilirubin from its apomyoglobin complex.

Ferriporphyrins in aqueous alkaline medium: The effects of salt and “ageing”

The effects of NaCl and air on ferrideuteroporphyrin IX and ferrimesoporphyrin IX (1 · 10−3–1 · 10−4 M) in aqueous medium at pH 11–12 and at room temperature are compared with analogous systems containing ferriprotoporphyrin IX by using light absorption spectrophotometry in the 640–320 mμ range and analytical ultracentrifugation. At high NaCl concentrations, both ferrideuteroporphyrin IX and ferrinesoporphyrin IX show a shoulder near 600 mμ, a maximum at 565 mμ and higher intensity (Soret) bands around 380 and 340 mμ, respectively. Similar spectra have previously been obtained with ferriprotoporphyrin IX under analogous conditions, except for shifts to shorter wavelengths of most bands in the case of ferrideuteroporphyrin IX and ferrimesoporphyrin IX. In order to obtain similar spectral effects, progressively high concentrations of either the metalloporphyrin or salt are required in the series ferriprotoporphyrin IX < ferrimesoporphyrin IX < ferrideuteroporphyrin IX, indicating the stabilization of the complex by the porphyrin side chains. From the increase of the area under the refractive index gradient curve with increasing NaCl concentrations in sedimentation velocity experiments, aggregate formation by NaCl can be demonstrated for both ferrideuteroporphyrin IX and ferrimesoporphyrin IX, as has been shown previously for ferriprotoporphyrin IX. In contrast to solutions of ferriprotoporphyrin IX in air, no slow spectral changes (“ageing”) are recorded for ferrideuteroporphyrin IX and ferrimesoporphyrin IX in air, or for ferriprotoporphyrin IX in the absence of air. The fast process, due to the effect of salt, can be observed in all cases.

Mössbauer studies of different types of hematin

Abstract Three types of hematin, β-(synthetic malaria pigment), B- and R-(regular) hematin, which differ in part of their infrared spectra and in their solubility in different solvents, have been investigated by Mossbauer spectroscopy at both 4.1 and 90 K. At the latter temperature, all hematins show an asymmetric shape which indicates slow spin–spin relaxation. The R-hematin shows, in addition, a second component which is considered to be due to antiferromagnetic coupling between dimeric units of ferriprotoporphyrin IX. The quadrupole splitting is smaller in β-hematin compared with B- and R-hematin, indicating a more symmetrical charge distribution around the iron in β-hematin. The larger relaxation times observed for β-hematin indicate larger iron–iron distances than in B- or R-hematin.

Optical activity of human serum in the visible region compared with that of the complex bilirubin—serum albumin

On the basis of recent investigations on the optical activity of the complex formed between bilirubin and serum albumin in aqueous solution* [l--4], it was anticipated that blood serum would also show optical activity in the visible region. It is known that human adult blood normally contains between about 0.2 to 1.4 mg/lOO ml of total bilirubin and 4.2 to 5.4 g/ 100 ml of serum albumin [ 5,6]. Bilirubin has been shown to be bound to albumin in blood serum [7, 51. The present results indicate that the optical activity in the visible region of human serum is indeed due to a large extent to that of the bilirubin bound to serum albumin.