Melisenda J. McDonald

Nonenzymatic glycosylation of human hemoglobin at multiple sites

The most abundant minor hemoglobin component of human hemolysate is Hb A1c, which has glucose bound to the N-terminus of the beta chain by a ketoamine linkage. Hb A1c is formed slowly and continuously throughout the 120 day lifespan of the red cell. It can be synthesized in vitro by incubating purified hemoglobin with 14C-glucose. Other minor components, Hb A1a1 and Hb A1a2 are adducts of sugar phosphates at the N-terminus of the beta chain. Hb A1b contains an unidentified nonphosphorylated sugar at the beta N-terminus. In addition, a significant portion of the major hemoglobin component (Hb Ao) is also glycosylated by a glucose ketoamine linkage at other sites on the molecule, including the N-terminus of the alpha chain and the epsilon-amino group of several lysine residues on both the alpha and the beta chains. The results indicate that the interaction of glucose and hemoglobin is rather nonspecific and suggests that other proteins are modified in a similar fashion.

Antibody orientation enhanced by selective polymer–protein noncovalent interactions

A unique interaction has been found between protein G’ (a truncated recombinant bacterial “alphabet” protein which aligns by noncovalent attachment to the antibody stem) and poly(methyl methacrylate), a thermoplastic polymer substrate, which can be easily fabricated using high-rate processes. Significantly improved orientation efficiency with traditional passive adsorption for this system (termed ALYGNSA) has been achieved as compared to the same assay performed on a polystyrene substrate with protein G’. Results were consistent with an average alignment of 80% of the human immunoglobulin G capture antibody which translated into a 30% to 50% improved alignment over an array of industry standards tested. Laser scanning confocal microscopy confirmed the immunological results. Studies of additional poly(methyl methacrylate) polymer derivatives and protein biolinker (A and AG) combinations have been conducted and have revealed different degrees of antibody alignment. These findings may lead to additional novel noncovalent methods of antibody orientation and greater sensitivity in immunological assays.

The Origin of the Adams-Schuster difference spectrum of oxyhemoglobin☆

Abstract The characteristic difference spectrum reported by Adams and Schuster (Biochem. Biophys. Res. Commun. 1974, 58 , 525) on the addition of inositol hexaphosphate to oxyhemoglobin is similar to the difference spectrum between (i) isolated α- and β-chains, (ii) α- and β-semihemoglobins, (iii) addition of inorganic phosphate to oxyhemoglobin, (v) change in temperature of a solution of oxyhemoglobin, (v) change in pH of carp carboxyhemoglobin and (vi) addition of inositol hexaphosphate to α-semihemoglobin. The spectrum may also be generated by differentiation of the spectra of oxyhemoglobin and carboxyhemoglobin, implying that the common feature of the results reported above is a shift in the position of the absorption bands. This shift may arise from several causes and so its interpretation is uncertain.

Ordered heme binding ensures the assembly of fully functional hemoglobin: a hypothesis.

The exact mechanism by which four Fe-Protoporphyrin-IX (heme) moieties and four nascent globin chains combine to form human hemoglobin (alpha(2)beta(2)) remains a mystery. Recent Soret spectral static and kinetic studies of the incorporation of CN-Hemin derivatives into an array of human globin species have provided in vitro evidence of an ordered assembly pathway, through an alphaheme-betaglobin intermediate, that ensures correct formation of active hemoglobin tetramers.

Wavelength-Dependent Spectral Changes Accompany CN-Hemin Binding to Human Apohemoglobin

The interaction of apohemoglobin with two heme derivatives, CN-protohemin and CN-deuterohemin, was monitored at multiple Soret wavelengths (417–423 and 406–412 nm, respectively) in 0.05 M potassium phosphate buffer, pH 7.0, at 10°C and revealed, as previously reported, a multiphasic kinetic reaction. Wavelength-dependent reactions were observed for both CN-protohemin and CN-deuterohemin derivatives with the a chain (bathochromic entity) displaying faster (4- to 7-fold) rates throughout the courses of both heme-binding reactions. The basis of this spectrally heterogeneous kinetic phenomenon could be deduced from molecular modeling studies of α- and β-chain structures. Key differences in the number of stabilizing contacts of the two chains with the peripheral a propionyl 45(CE3); 58(E7); 61(E10) as well as the b vinyl 38(C4); 71(E15); 106(G8) groups were found. Furthermore, RMS plots comparing apo- and heme-containing subunits reveal substantial structural disparities in the C-CD-F-FG helical regions of the αβ dimer interface.

A comparison of the functional properties of two lepore hemoglobins with those of hemoglobin A1

Abstract Lepore hemoglobins result from crossovers between normal beta and delta chain genes. Structural investigation of two newly discovered examples of Lepore hemoglobins revealed one of them to be structurally identical to hemoglobin Lepore Hollandia α 2 A δ 22 -x- β 50 , a rarely occurring Lepore variant, while the second had the structure of hemoglobin Lepore Boston α 2 A δ 87 -x- β 116 . Studies of the equilibrium and kinetic properties of the liganding reactions of these two Lepore hemoglobins, which differ only in three amino acid residues, and comparison of these with the known properties of hemoglobin A 1 ( α 2 β 2 ) and hemoglobin A 2 ( α 2 δ 2 ) have been carried out. A high value of n , the Hill coefficient, indicating normal heme-heme interaction, was observed in each hemoglobin along with a normal Bohr effect. However, a slight but definite increase in oxygen affinity was observed for each Lepore hemoglobin. Furthermore, kinetic studies indicated a slight but consistently increased rate of ligand combination and a somewhat decreased rate of oxygen dissociation for hemoglobins Lepore Hollandia and Lepore Boston at pH 7 and 20 °C. Apparently, the higher oxygen affinity of these Lepore hemoglobins over those of the normal hemoglobins A 1 and A 2 reflects changes of sequence that are common to both types of hemoglobin Lepore.

AFM imaging of ALYGNSA polymer–protein surfaces: evidence of antibody orientation

Previous investigations found the combination of recombinant bacterial protein G (rProG) and poly(methyl methacrylate) (PMMA) to produce a greater proportion of oriented antibodies. PMMA–rProG yielded a sixfold greater availability of antibody Fab regions compared with other bacterial affinity linker protein and polymer pairings, including commercially available polystyrene (PS) high-binding 96-well microplates. Given the name ALYGNSA, the PMMA–rProG combination was developed into a fluorescence assay and evaluated in conjunction with commercially available cancer biomarker enzyme-linked immunosorbent assays (ELISAs). In each study, a lower limit of detection was seen with the ALYGNSA assay. The purpose of this investigation was to examine the ALYGNSA substrate in contrast with a commonly used ELISA substrate and analyze the affinity-immobilized antibodies for additional evidence of orientation. Non-contact atomic force microscopy is a logical method as it operates in ambient conditions, can be used directly on biological samples without modification, and offers the resolution necessary to identify the position of the antibody on the surface. Dynamic contact angle studies were employed to examine untreated PMMA and PS samples and revealed important differences in their surface characters. Comparative height threshold grain analysis of the prepared ALYGNSA surface, a similarly treated mica surface, and a gold colloid sizing standard evaluated and confirmed the antibody orientation of the ALYGNSA system.

Monitoring the effect of subunit assembly on the structural flexibility of human alpha apohemoglobin by steady-state fluorescence

A single energy transfer distance, between the sole intrinsic tryptophanyl donor [14 (A12)] and a nonfluorescent sulfhydryl acceptor probe (4-phenylazophenylmaleimide, PAPM) attached to the only cysteine [104 (G11)], has been employed to examine the effect of subunit assembly on the structure of the heme-free humanα-hemoglobin. Efficiencies of energy transfer were measured in 0.05 M potassium phosphate buffer,pH 7.0, at 5°C, and the structural flexibility ofα-apohemoglobin, in the absence and presence of humanβ-heme-containing chains, was examined by a steady-state solute quenching technique. The quenched efficiencies (EO) and Förster distances (R0O) were analyzed by least-squares to determine the goodness of fit (χR2) for the assumed distribution parameters: average distance ¯r and half-widthhw. Data forα-apohemoglobin in the absence and presence ofβh chains yielded values for ¯r of 18 and 22 Å andhw of 20 and 8.5 Å, respectively. Although the increase in ¯r forα-apohemoglobin in the presence ofβh chains was presumably a consequence of additional quenching from the heme moiety, the change in the half-width strongly indicated a decrease in the flexibility of theα-apohemoglobin chain within the assembled protein. A transition in structural flexibility similar to that demonstrated here may be an important aspect of human hemoglobin assembly.

Fluorescence studies of normal and sickle beta apohemoglobin self-association.

The acrylamide quenching of the intrinsic tryptophanyl fluorescence of normal and sickleβ apohemoglobins has been studied in 0.05 M potassium phosphate buffer,pH 7.5, at 5°C over a protein concentration range from 1 to 50μM. Analysis of quenching dynamics revealed a strong dependence on acrylamide concentration for the intrinsic fluorescence of both normal and sickleβ apohemoglobins, suggesting that one tryptophanyl residue [presumably that at position 37(C3)], was more accessible to collisional quencher than the otherβ tryptophanyl residue [15(A12)]. Additional studies, which altered viscosity and subunit assembly experimental parameters, supported the assignment of residue 37 as the more dynamically accessible residue. Finally, the quenching data were also found to be dependent on protein concentration, implying that this difference in the mobility between the two residues is a sensitive probe of self-aggregation. Extrapolated dynamic quenching constants at low concentration of acrylamide were used to estimate the dimer-monomer equilibrium dissociation constants of normal and sickleβ apohemoglobins, and were found to be 5.6 and 2.4μM, respectively, thus demonstrating distinct self-association properties ofβA andβS apohemoglobins.

HB Rancho Mirage [β143(H21)HIS→ASP]; A Variant in the 2,3-DPG Binding Site Showing Normal Oxygen Affinity at Pewsioidgical pH

Hb Rancho Mirage was detected in a 17-year-old male in association with a mild anemia. Hemoglobin electrophoresis revealed the variant had a mobility between Hbs A and J on cellulose acetate (pH 8.6) and a mobility like Hb F on citrate agar (pH 6.4). A substitution of His→Asp was found at position 143 in the s chain, a residue that contributes to the anionic 2,3-DPG binding site in Hb. This variant exhibited normal oxygen affinity at physiologic pH and reduced affinity at alkaline pH. This suggested a subtle shift in the allosteric equilibrium due most likely to the introduction of a negative charge that stabilized the 2,3-DPG pocket. Both homotrophic (heme-heme) and heterotropic (2,3-DPG and protons) effects were reduced; this might be a consequence of an alteration in the carboxyl terminal region of the s-subunits. Although a His→Asp substitution would be considered to cause reasonable disruption of the 2,3-DPG and C-terminal conformation of the s- subunits, the properties of Hb Rancho Mirage suggest th...