Biochimica et Biophysica Acta, 393 (1975) 483-495

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 37054 COMPLEXES BETWEEN S Y N T H E T I C P O L Y M E R L I G A N D S A N D FERRIAND F E R R O - P R O T O P O R P H Y R I N IX

EISHUN TSUCHIDA, KENJI HONDA and ETSUO HASEGAWA Department of Polymer Chemistry, Waseda University, Shinjuku-ku, Tokyo 160 (Japan)

(Received November 29th, 1974)

SUMMARY The complexes of synthetic polymer ligands, i.e. poly-L-lysine, poly-4-vinylpy~'idine, poly-N-vinyl-2-methylimidazole and the higher branched polyethyleneimine, with ferri- or ferro-protoporphyrin IX were studied from the standpoint of polymer ligand effects by comparison with those of their monomeric model ligand complexes and poly-y-benzyl-L-glutamate containing an imidazole nucleus at the chain end. The coordination numbers and formation constants were determined optically and their structures were also estimated. The coordination number of a poly-L-lysine complex was two, but those of other polymer ligand complexes were one. One of the polymer effects, which was indicated by the large formation constants of the polymer complexes, was caused by the increment of the local ligand concentration around the polymer chain. Another was caused by the conformational effect of an a-helical structure in the poly-L-lysine complexes. The interaction of a poly-L-lysine-heme complex with molecular oxygen was also studied. An observed pseudo-allosteric phenomenon may be due to the specific structure of a poly-L-lysine complex which is different from those of other polymer-ligand complexes.

INTRODUCTION Attention has been paid to the metallo-porphyrin complexes from the standpoint of synthetic oxygen carriers in relation to the role of hemoglobin and systematic investigations have been carried out on these complexes with low molecular weight ligands [1, 2]. But few quantitative studies have been reported on the complexes formed with synthetic polymer ligands although a few primitive but qualitative investigations have been made in the poly-L-lysine system [3-7] and the poly-N-vinyl2-methylimidazole system [8]. Even in these systems, the coordination numbers, formation constants and structures have not been elucidated although a paramagnetic susceptibility was measured on a poly-L-lysine complex [5]. In this paper, the complex formation reactions of various polymer ligands with ferri- and ferro-protoporphyrin IX (ferriheme and ferroheme, respectively) are studied quantitatively and the coordination numbers and formation constants are determined. Then the structures of these polymer complexes are elucidated. On the basis of these results, the polymer-ligand effect and the effect of an a-helical confor-

484 mation in the complex formation with hemin or heme will be discussed. The interaction of these complexes with molecular oxygen is also studied and a pseudoallosteric phenomenon observed will be discussed in relation to the structure of the complexes. EXPERIMENTAL Polymer ligands. Poly-L-lysine(poly(Lys))HBr was prepared by e,N-carboIzenzoxy-L-lysine-N-carboxy anhydride polymerization followed by the decarbobenzoxylation with an HBr/acetic acid solution (Mw = 7.34. 104). Poly-y-benzyl-Lglutamate containing an imidazole nucleus at the chain end was prepared by polymerizing ~-benzyl-L-glutamate-N-carboxy-anhydride as described before [9] (average degree of polymerisation = 26). The degree of chain branching, i.e. the fraction of a primary amine per nitrogen, in a branched polyethyleneimine was 0.42 as determined by the Van Slike method (M, = 8.2- 104). Polyethyleneglycol (Mn -- 2.51" 104). Low molecular weight ligands. Reagent grade samples were purified by recrystallization or distillation by the usual methods. Ferri-protoporphyrin I X chloride. It was isolated from human blood by the method of Willstaetter as described before [10]. Freshly prepared solution was used for each experiment. Ferri-ehlorophyllin a and b (sodium salts). Commercial reagent (Nippon Yohryokuso Co. Ltd.) was purified by reprecipitation with acetic acid after extraction with an ether/water mixture and then washed with cooled methanol twice and dried in vacuo. Reduced agent. Reagent grade sodium hydrosulfite was used as the solution. The initial molar ratio of the reduced agent to ferriheme was selected from 50-100. Solvents. N,N-Dimethylformamide was distilled under reduced pressure after dehydration with magnesium sulfate. The pH of the aqueous solution was adjusted to 12.0 by the use of a buffer solution (0.05 M Na2HPO4/NaOH) or by the addition of 1 M N a O H solution. Method of measurement of the absorption of O2. The absorption of molecular oxygen by ferroheme complexes was followed by the use of a Warburg's apparatus. Apparatus. Absorption and circular dichroism spectra were measured by using a Shimadzu MPS-50L spectrophotometer and a JASCO J-20 spectropolarimeter, respectively. The half-wave potential was determined by Shimadzu RP-2 polarography. Viscosities were measured using an Ubbelohde viscometer. A Hitachi-Horiba F-7 pH meter was used. Method of determination of coordination number and formation constant. They were determined according to the method of Miller and Dorough [11].

PM+ h B ~ PMB,; K = [P~B;]/[PM] [B]; =

(1) R/[BI ~

(2)

where PM, B and K are ferri(ferro)protoporphyrin, a base and a formation constant, respectively, h represents a coordination number. R = X/(A -- X), A = D¢ -- D~, X = Dm -- D~

485 Du is the absorbance of ferri(ferro)heme without an axial base. Om indicates the absorbance in mixing ferriheme with an axial base arbitrarily. De is the absorbance at which the absorbance due to PMB; reached the maximum value. If [B]o is much larger than [PM]othe following equations were obtained from Eqn. 2:

(3)

log R = log K ÷ filog [B]0 1

X

1 --

KA

1

[B]~ ÷

1 --

(4)

A

Coordination numbers and formation constants were determined by the use of Eqns 3 and 4. The changes in optical densities were measured at 425, 433, 408 and 408 nm for poly-L-lysine-ferriheme, poly-L-lysine-ferroheme, imidazole-ferriheme and poly-7-benzyl-L-glutamate, respectively with a pendant imidazole-ferriheme complex. RESULTS AND DISCUSSION

Axial coordination number and formation constant of poly-L-lysine-ferriheme (ferroheme) complex. Figs. 1 and 2 show the determination of the coordination numbers and formation constants of poly-L-lysine-ferriheme and poly-L-lysine-ferroheine complexes in pH 12.0 aqueous solution. Although Blauer [3-5] reported the formation of a complex with a low-spin property in a poly-L-lysine-ferriheme system and recently Hatano et al. [7] studied the electronic structure using magnetic circular dichroism spectra, they have not as yet been definitely determined. From Fig. 1, the coordination numbers of the poly-L-lysine-ferriheme and poly-L-lysine-ferroheme complexes were determined to be equal to 2 from the slopes of the straight lines

I

0

0

-1

Q:

~g

cc ,-4

-I

-2 I

--14

I

-3

I

-2

I -

i

log[poly-I.-lysine]o (unit tool/I)

Fig. 1. Determination of the coordination number of the poly-L-lysine-ferri- and ferroheme complexes in pH 12.0 aqueous solution at 20 °C. Poly-L-lysine-ferriheme(©), [ferriheme]0= 2.00.10 -5 tool/l; poly-L-lysine-ferroheme(~), [ferroheme]0= 1.16.10-5 mol/l.

486 10-5 x ]/[poly-L-lysine]~ (12/unit mole2)

2o

b

Qs

i

I

0

I

I

I

2

10-9 X ]/[poly-L-lysine]2o (l 2/unit mole 21

Fig. 2. Determination of the formation constants of poly-L-lysine-ferriheme and ferroheme complexes in pH 12.0 aqueous solution. The experimental conditions were the same as shown in Fig. 1. obtained. Therefore, only one complex species should be formed preferentially in each complex formation reaction.

Axial coordination number and formation constant of a complex of ferriheme with poly-7-benzyl-L-glutamate containing a pendant imidazole. From Fig. 3, the coordination numbers of the ferriheme complexes in N,N-dimethylformamide are both equal to one. The formation constants are also determined from Fig. 4. The characteristic visible absorption maxima are consistent with each other in both complexes. Therefore, these results confirm that an imidazole nucleus of the polymer chain end acts as a ligand preferentially. From the results described above and our previous results [10], the results of the complex formations of various ligands including polymeric and monomeric ligands are summarized in Tables 1 and 1I.

Comparison of the complex formation reactions of polymer ligands with ferrior ferroheme with those of low molecular weight ligands. As shown in Table I, the absorption maxima are the same in both a polymer ligand complex and its monomeric analogue complex, which indicates that the ability zs ligand unit is unchanged in the polymeric or monomeric system. The coordination numbers of the complexes cf the low molecular weight ligands, i.e. n-butylamine, imidazole and pyridine, with ferriheme are all equal to one, which is in agreement with the values of the other polymer ligand complexes except poly-L-lysine. The formation of the complexes with coordination number one can be explained by the solvent effect, the trans effect and steric hindrance of the polymer chain. For example, in a non-coordinative solvent such as chloroform, two ligands can coordinate to a metalloporphyrin compound [12], but in a coordinative solvent such as N,N-dimethylformamide or water, once one strong ligand coordinates at the

487

~ o

0

!

-1 -4

-3 log [B] o

-2 (tool/ 1 )

Fig. 3. Determination of the coordination numbers of the ferriheme complex of poly-~-benzyl-Lglutamate with a pendant imidazole at 25 °C (A) and the imidazole-ferriheme complex at 35 °C ((3) in N,N-dimethylformamide. [ferriheme]e = 1.00.10-5 mol/l.

fifth position, the ability of coordination at the sixth position is weakened by a trans effect, so that only a weak ligand such as N,N-dimethylformamide can coordinate at the sixth position of ferri- or ferroheme. Therefore, the formation of the complexes with coordination number two in poly-L-lysine systems may be due to an inherent factor for poly-L-lysine different from other polymer ligands. Fig. 5 shows the complex formation dependence on the ligand concentration followed by the changes in the optical densities at each characteristic absorption maxima. The ligand concentration which promotes the complex formation is in the order poly-L-lysine