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The 57Fe hyperfine interactions are the useful source of information about the finest structural peculiarities in various iron-containing proteins which can be essential in the natural variety of structure and function relationship in normal biomolecules as well as in its changes in the case of molecular pathology. Mossbauer spectroscopy with a high velocity resolution as the most sensitive probe for the 57Fe hyperfine interactions demonstrates some advances in the study of various hemoglobins, ferritins and pharmaceutical products that can bring new information for the life sciences.

A comparative study of tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a in the oxy- and deoxy-forms was carried out using 57Fe Mossbauer spectroscopy with a high velocity resolution in order to analyze the heme iron electronic structure and stereochemistry in relation to the Mossbauer hyperfine parameters. The Mossbauer spectra of tetrameric rabbit hemoglobin in both forms were fitted using two quadrupole doublets related to the 57Fe in ɑ- and β-subunits. In contrast, the Mossbauer spectra of monomeric soybean leghemoglobin a were fitted using: (i) two quadrupole doublets for the oxy-form related to two conformational states of the distal His E7 imidazole ring and different hydrogen bonding of oxygen molecule in the oxy-form and (ii) using three quadrupole doublets for deoxy-form related to three conformational states of the proximal His F8 imidazole ring. Small variations of Mossbauer hyperfine parameters related to small differences in the heme iron electronic structure and stereochemistry in tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a are discussed.

Normal human adult, rabbit and pig oxyhemoglobins in frozen red blood cell solutions were studied using Mossbauer spectroscopy with a high velocity resolution. Spectra were analyzed using two models with and without accounting for the heme iron electronic structure non-equivalence in α- and β-subunits of tetrameric hemoglobins. The observed differences of Mossbauer hyperfine parameters were related to structural variations in human, rabbit and pig oxyhemoglobins as well as to the differences in ligand binding affinities of these proteins.

A comparative study of normal human, rabbit and pig oxyhemoglobins and oxyhemoglobin from patients with chronic myeloleukemia and multiple myeloma using Mossbauer spectroscopy with a high velocity resolution demonstrated small variations of the 57Fe quadrupole splitting and isomer shift. These variations may be a result of small structural differences in the heme iron stereochemistry of various hemoglobins.

Placental protein 12 was isolated from amniotic fluid. Albumin was removed by means of ion-exchange chromatography on DEAE-Sepharose and chromatography on Blue-Sepharose. Complete purification was obtained by metal-chelate affinity chromatography and hydrophobic interaction chromatography under mild conditions for desorption. Using this procedure large quantities of a highly purified preparation can be obtained in one run.

A comparative study of oxyhemoglobins from pig, rabbit, normal human and patients with blood system malignant diseases was performed using Mossbauer spectroscopy with a high velocity resolution at 90 K. Mossbauer spectra were fitted with the help of two models: using one quadrupole doublet (model of equivalent iron electronic structure in α- and β-subunits of hemoglobins) and superposition of two quadrupole doublets (model of non-equivalent iron electronic structure in α- and β-subunits of hemoglobins). The results obtained using both models demonstrated small variations of hyperfine parameters that were related to the heme iron state variation in different hemoglobins. These results were compared with structural and functional differences of the hemoglobins investigated.

The heme iron in human adult hemoglobin modified by both pyridoxal-5′-phosphate and glutaraldehyde was characterized by Mossbauer spectroscopy and compared with non-modified hemoglobin. Mossbauer spectra of the samples were measured at 87 and 295 K (1yophilized form) and at 87 K (frozen solution). The values of quadrupole splitting for the oxy-form of modified hemoglobin were found to be lower than those of the oxy-form of hemoglobin without modifications in lyophilized form and frozen solution, respectively. On the other hand, the values of quadrupole splitting for the deoxy-form of modified and non-modified hemoglobins in frozen solution were the same. The Mossbauer spectra of the oxy-form of modified hemoglobin were also analyzed in terms of the heme iron non-equivalence in α- and β-subunits of tetramer. The differences of the tendencies of temperature dependencies of quadrupole splitting for the oxy-form of modified and non-modified hemoglobins in lyophilized form were shown. These results indicated that the heme iron electronic structure and stereochemistry were changed in the oxy-form of pyridoxylated hemoglobin cross-linked by glutaraldehyde.

Human adult hemoglobin modified by both pyridoxal-5′-phosphate and glutaraldehyde in the oxy-form was studied by Mössbauer spectroscopy. Mössbauer spectra were measured at 87 and 295 K (hemoglobin in lyophilized form) and at 87 K (hemoglobin in frozen solution). The values of the quadrupole splitting for modified oxyhemoglobin were found to be lower then those of oxyhemoglobin without modifications in lyophilized form and frozen solution, respectively. The Mössbauer spectra of modified oxyhemoglobin were also analyzed in terms of the heme iron inequivalence in α- and β-subunits of the tetramer. Differences of the tendencies of temperature dependencies of quadrupole splitting for modified and non-modified oxyhemoglobin in lyophilized form were shown. Key words: Hemoglobin; Mössbauer Spectroscopy; Quadrupole Spitting. This work was supported in part by the Russian Foundation for Basic Research (grant # 97-04-49482).

The relationship of the heme iron electronic structure and hemoglobin functions is of interest in order to understand the molecular nature of biological function. Previous Mossbauer study of various human hemoglobins demonstrated small variations of the Mossbauer hyperfine parameters (quadrupole splitting △EQ and isomer shift δ) for normal adult and fetal as well as for normal and leukemic oxyhemoglobins [1]. This fact indicates small variations of the heme iron electronic structure and stereochemistry in these hemoglobins. Moreover, it was shown that some features of the Mossbauer spectra related with the iron electronic structure nonequivalence in nonidentical subunits of tetrameric hemoglobins. Further study of human oxyhemoglobin modified by lyophilization demonstrated some changes of the heme iron electronic structure and stereochemistry [2]. On the other hand, some modified hemoglobins appeared useful as blood substitutes. Mossbauer study of hemoglobin modified by glutaraldehyde showed that cross-linking of hemoglobin by this reagent modified the heme iron stereochemistry [3]. In the present work we discuss the results of comparative study of human oxyhemoglobin modified by both pyridoxal-5 ′-phosphate which binds to the NH3-terminal groups of the β-chains and glutaraldehyde which mainly (70 %) cross-linked α- and β-subunits in each hemoglobin molecule.

Placental protein 14 was isolated from the biological material of patients undergoing legal abortions. The major part of ballast protein was removed by ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose. Albumin was separated by chromatography on Blue-Sepharose. Complete purification was obtained by metal-chelate affinity chromatography on Nickel-Chelate Sepharose and hydrophobic interaction chromatography on Phenyl-Sepharose and Octyl-Sepharose. The protein was not exposed to denaturing agents or extreme pH.