Mossbauer studies of anhydrous hemoglobin and its subunits

The similarities in the Mossbauer spectra of Hb and its α and β subunits, both in the oxygenated and deoxygenated states, were observed. The relative intensities of the absorption dips in the Mossbauer spectra of ⁵⁷Fe in anhydrohemoglobin (AHb) and anhydrous separated alpha (Aα) and beta (Aβ) chain samples were found to be very sensitive to the conditions of samples preparation. This indicates the formation of hemochromogen as an impurity in the absorbers during the process of preparation. When contributions due to the impurity in the samples were subtracted, Mossbauer spectra of ⁵⁷Fe in AHb, Aα, and Aβ remained similar. Therefore, the electronic structure of the iron cation is the same for Hb and its subunits in the anhydrous state also.     

Gelling properties of apohemoglobin S alone and in mixtures with hemoglobin S

Apohemoglobin S formed a gel in the cold (5 degrees C) with a protein concentration in the supernatants after centrifugation of the gels (Csat) near 27 g/dl, in 0.02 M phosphate buffer at pH 7.2. Under the same experimental conditions in mixtures of apohemoglobin S and deoxyhemoglobin S the solubility of hemoglobin S in the cold was decreased from Csat greater than 40 g/dl in the absence to about 18 g/dl in the presence of apohemoglobin S. Conversely, in the same mixture, Csat of apohemoglobin S was decreased to about 5 g/dl. Also, gelling occurred in mixtures of oxyhemoglobin S and its apoderivative. Apohemoglobin A alone did not form gels; however, it induced fiber formation in deoxyhemoglobin S in the cold; unlike apohemoglobin S, it was not included in the precipitate. Gels of apohemoglobin S were not birefringent, and inspection at the electron microscope failed to show the presence of organized structures. Excluded volume effects were probably at the origin of the decreased solubility of hemoglobin S and apohemoglobin S in the presence of each other.     

Geminate recombination in carboxy hemoglobin A and its relation to overall carbon monoxide reactivity

The geminate recombination of CO with carboxy hemoglobin (Hb₄(CO)₃) following a ten nanosecond laser pulse and the overall combination of the fourth CO with Hb₄(CO)₃ has been studied as a function of pH in the presence and absence of inositol hexaphosphate. The results indicate that the kinetics of both reactions are independent of pH and phosphate concentration. The results are discussed in terms of a two-step mechanism: a pre-equilibrium step followed by heme—ligand bond formation. The latter is also known as the geminate recombination reaction (Hb + CO α Hb · CO α HbCO).     

Heterogeneity of the isolated subunits of the fetal and adult human hemoglobin in solution, detected by XANES spectroscopy

Differences in the local structure of the heme in the isolated alpha-, beta- and gamma-chains of the adult and fetal human hemoglobin are detected by XANES (X-ray absorption near-edge structure) spectroscopy. The ligand bonding angle to the iron ion in the ligated forms and the displacement of the Fe respect to the porphyrin plane in the deoxy forms are found to be different for each chain.     

Location of normal and abnormal tyrosyl residues in ribonuclease

Iodination at pH 9.5 has converted three of the six tyrosyl residues of ribonuclease to 3,5-diiodotyrosyl residues. The iodinated ribonuclease is still enzymatically active toward ribonucleic acid. The diiodotyrosyl residues have approximately normal pK's, while the tyrosyl residues remaining have abnormally high pK's. Hence, iodination has taken place on the normal tyrosyl residues.Two of the iodinated tyrosyl residues have been shown to be No. 115 and No.92; hence, these appear to be normal tyrosyl residues. One of the non-iodinated tyrosyl residues is No. 97, and hence is one of the abnormal tyrosyl residues. The location of the other normal tyrosyl residue (or the other two abnormal ones) remains uncertain.     

Fluorescence anisotropy decay studies upon hemoglobin A and its subunits

Fluorescent conjugates of hemoglobin A, its isolated β-chain, and the apo-derivative of the β-chain have been prepared in which the β-93 sulfhydryl was conjugated with 1,5-AEDANS. Radiationless enery transfer to the heme group results in a major decrease in fluorescence intensity and decay time. Measurements of the time decay of fluorescence anisotropy, employing single-photon counting, indicate that the apparent rotational correlation time is, in each case, substantially reduced from the value expected for a rigid molecule of the same molecular weight. This observation raises the possibility that internal degrees of rotational freedom exist.     

Radiolytic and enzymatic dimerization of tyrosyl residues in insulin, ribonuclease, papain and collagen

Insulin, ribonuclease, papain and collagen solutions saturated with nitrogen, N2O or air were irradiated with doses of 10 to 640 Gy of gamma rays. Protein solutions were also oxidized enzymatically in a system of horse-radish peroxidase: hydrogen peroxide. Column chromatography (Sephadex G-75 or Sephacryl S-200) of treated protein solutions revealed that they contain protein molecular aggregates. Nitrogen saturation of solution before irradiation was most favourable for radiation-induced aggregation of proteins. Fluorescence analysis of protein solutions resulted in detection of dityrosyl structures in irradiated as well as in enzymatically oxidized proteins. Concentration of dityrosine in proteins studied was determined fluorimetrically in their hydrolysates separated on BioGel P-2 column. In irradiated proteins, dityrosine was present almost exclusively in their aggregated forms. In proteins oxidized enzymatically, dityrosine was also present in fractions containing apparently unchanged protein. Mechanisms which could account for differences in the yield of dityrosine formation in radiolysis and in enzymatic oxidation of proteins are suggested.     

The reversible titration of tyrosyl residues in human deoxyhemoglobin

The reaction of hemoglobin with N-acetyl imidazole at neutral pH indicated that in carboxyhemoglobin 1.80 residues per heme were acetylated while in deoxyhemoglobin only 1.15 residues were available to the reagent. The reversible titration of these residues in alkali was followed by difference spectrophotometry at 245 nm. Hill plots of the titration data, assuming 2 residues titrable per heme an3 Δε = 10500 per tyrosyi residue upon ionization, showed a slope of 1.5 and a pH near 11. The average pK of these groups in carboxyhemoglobin was previously found to be near 10.5. Also. by difference spectrophotometry it was shown that exposure of deoxyhemoglobin to alkaline pH was accompanied by a modification of the Soret region of the absorption spectrum, which might indicate the appearance of liganded conformation in the deoxyhemoglobin system. The sedimentation velocity of deoxyhemoglobin demonstrated that at alkaline pH dissociation into duners occurred at pH's lower than 10, where no ionization of tyrosines was detectable. The titration of tyrosines was independent from protein concentration.The low availability of tyrosyl residues to acetylation in deoxyhemoglobin, the cooperativity of proton binling of these residues and the change in conformation of hemoglobin concomitant with their titration are all consistent with results of Simon et al., Moffat, and Moffat et al., and with the model proposed by Perutz for explaining the heme-heme interaction. The free energy of the pK shift of the tyrosyl residues in carboxy and deoxyhemoglobin can be included in the free energy of the heme-heme interaction.     

Oxygen-linked CO2 binding to isolated beta subunits of human hemoglobin.

It is known that most of the oxygen-linked carbamate which is formed in normal adult human hemoglobin (Hb A) is confined to the beta subunits rather than to the alpha subunits. In order to find out if similar differences exist in the isolated protomers of Hb A we have measured the effect of various pressures of carbon dioxide (pCO2) on the oxygen affinity in the following heme pigments: isolated alpha and beta subunits with free --SH groups (alphaSH, betaSH), mercurated beta subunits (betaPMB), myoglobin (Mb), and betaSH/PLP in which the terminal alpha-amino group of betaSH was irreversibly blocked with pyridoxal phosphate (PLP). Similar measurements were done on Hb A and the fraction of oxygen-linked carbamate calculated from the effect of pCO2 (at constant pH) on the oxygen half-saturation pressure (p50). A distinct influence of CO2 on p50 was observed in betaSH which was absent in betaSH/PLP and thus indicates that the terminal alpha-amino group mediates the oxygen-linked binding of CO2 in betaSH as it does in the beta subunits of Hb A. However, the fraction of oxygen-linked carbamate was much less dependent on pH and pCO2 in betaSH than in Hb A. Neither alphaSH, betaPMB, or Mb, all of which are known to exist largely or wholly as monomers but have free terminal alpha-amino groups, showed a shift of p50 upon addition of CO2. As both betaSH and betaSH/PLP were shown to be tetrameric molecules, we conclude from this study that homotetramers composed of isolated beta subunits do exhibit a reciprocal interaction between the binding of O2 and CO2.