beta-meso-Phenylbiliverdin IX alpha and N-phenylprotoporphyrin IX, products of the reaction of phenylhydrazine with oxyhemoproteins.

Abstract

Oxyhemoglobin and oxymyoglobin were allowed to react aerobically with phenylhydrazine and p-tolylhydrazine. The chloroform extract of each reaction mixture, after treatment with H2SOdmethanol, yielded a blue pigment and a.green pigment, which were identified by electronic absorption, mass, and proton NMR spectroscopy as the dimethyl esters of -mao-arylbiliverdin IXa and N-arylprotoporphyrin IX, respectively. NPhenyiprotoporphyrin IX dimethyl ester formed complexes, with Zn2+, Cd'+, and Hg'+ but not with other cations. The proton NMR spectrum of the zinc complex suggested binding of the phenyl group to one ofthe two pyrrole rings ofprotoporphyrin IX with a propionic acid substitutent. The effectiveness of phenylhydrazine as an inducer of Heinz body formation may be due to destabilization of the hemoglobin. molecule by the replacement of heme with phenyl adducts of biliverdin and protoporphyrin.The green hemoglobin produced in the reaction ofhemoglobin with phenylhydrazine was found by Kiese and Seipelt (1) to differ from verdoglobins produced by other reagents. Lemberg and Legge (2) showed that biliverdin production was increased in the erythrocytes ofrabbits treated with phenylhydrazine, and they concluded that this in vivo result of phenylhydrazine activity corresponded to the in vitro formation ofbiliverdin in the coupled oxidation ofhemoglobin and ascorbic acid. Beaven and White (3), on the other hand, did not obtain biliverdin in the in vitro reaction of phenylhydrazine with oxyhemoglobin; instead they isolated a green pigment with an absorption band in the Soret region suggestive of an intact porphyrin ring.Studies from this laboratory indicated that substituents on the benzene ring affected the hemolytic activity of substituted phenylhydrazine and the binding ofsubstituted phenyldiazenes to methemoglobin in parallel fashion (4). Subsequent studies showed, however, that the hemolytic activity of ring-substituted nitrosobenzenes was not related to their affinities as ligands of deoxyhemoglobin (5).A two-electron transfer to oxyhemoglobin was suggested as the initial step in the degradation ofheme mediated by ascorbic acid (6), and the same transfer was postulated in the reaction of phenylhydrazine with oxyhemoglobin (4). Although the processes may be initiated in the same way, different reported products, biliverdin with ascorbic acid (2) and an intact porphyrin with phenyihydrazine (3), indicate that subsequent steps are different. We therefore undertook to isolate and characterize the products of heme degradation when hemoglobin is treated with arylhydrazines, and we found two types of compounds: one is a biliverdin IXa derivative with an aryl group on a meso carbon, and the other is a protoporphyrin IX derivative with an aryl group on a pyrrole nitrogen.EXPERIMENTAL PROCEDURES Materials and Methods. To a solution of metmyoglobin (5 g, type III from horse heart, Sigma) in potassium phosphate buffer (pH 7.4, 0.01 M, 200 ml), a 50-fold excess of powdered Na2S204 was added under N2. This mixture wa...

Iron release and membrane damage in erythrocytes exposed to oxidizing agents, phenylhydrazine, divicine and isouramil

“…In the case of divicine and isouramil, active oxygen species are formed mainly by redox cycling [30]. In the case of phenylhydrazine, oxidase and peroxidase reactions of oxyhaemoglobin and methaemoglobin have been described [22] which lead to formation of both O-and phenylhydrazine radicals (phenylhydrazine and phenyl radicals) [25][26][27]. These radicals can denature the haemoglobin molecule [20]; iron is probably released from denatured haemoglobin and can induce lipid peroxidation if the cell is depleted of GSH.…”

“…Phenylhydrazine oxidation results in the formation of superoxide and hydrogen peroxide [21][22][23][24]. Several reactive intermediates of phenylhydrazine [25][26][27] are formed as well as complexes of these reactive products with haemoglobin. In the erythrocyte membrane, lipid peroxidation can occur [28] as well as formation of a new antigen which is recognized by autologous IgG [29].…”

Reactions of hemoglobin with phenylhydrazine: a review of selected aspects.

“…The chemical nature of the modified heme, free from globin, was established almost simultaneously by papers from the laboratories of Ortiz de Montellano (19) and Itano (38). Both groups isolated protoporphyrin IXcontaining green pigment as the dimethyl ester by treating the product from reaction of HbO2 with PHZ with acidic methanol under air and used spectroscopic and mass spectrometric techniques to make structural assignments.…”

Reactions of Hemoglobin with Phenylhydrazine: A Review of Selected Aspects

“…We have not discussed, in this section, the formation of modified hemoglobins whose heme group can be extracted in the form of II (38). As little is known about possible intermediates on the pathway to this type of modified hemoglobin, there seems little to be gained by postulating possible mechanisms for its formation.…”