Elizabeth A. Robinson

Protein-protein interaction among hemoglobin subunits: A comparison of adult, fetal, and bovine carboxyhemoglobin

Abstract Alkali resistance, sedimentation velocity, and molecular weights of human adult, fetal and of bovine carboxyhemoglobin were compared. Sedimentation coefficients and molecular weights were determined at a number of pH values between pH 5.7 and 11.9. Bovine hemoglobin, the subunits of which do not recombine, was considerably more alkali resistant than fetal hemoglobin. Hemoglobin A was least resistant. It appears that there are no appreciable differences in the mode of dissociation of these three species of hemoglobin, except at pH 11.9. The differences seen at this pH presumably result from the alkali stability of the symmetric dimer subunit. Monomer formation is a necessary step for the occurrence of recombination after symmetric dissociation. From the close similarities in sedimentation and molecular weight data obtained with the three species of hemoglobin studied, it was concluded that appreciable amounts of monomer are not present in the equilibrium when dissociation is produced by an elevation of pH. The number of titratable groups essential for the integrity of the hemoglobin tetramer between pH 9 and 11.6 ( n ), together with dissociation constants for the three hemoglobins, were calculated. The average value for n obtained was 1.33. The biochemical and crystallographic data available indicate that either of the two tyrosine residues at position α42 or β (γ) 35 were the groups titrated.

Characterization of a chemically homogeneous tumor antigen from a methylcholanthrene-induced sarcoma, Meth A

A tumor antigen isolated from the cytosol of a methylcholanthrene-induced sarcoma (Meth A) has been purified to homogeneity by the criteria of two-dimensional gel analysis and NH2- and COOH-terminal sequencing. The purified antigen has a mol. wt of 82,000 by SDS gel electrophoresis. However, the apparent mol. mass of the antigen was found to be 71,600 and 67,700 by gel filtration chromatography and sedimentation analysis, respectively. It is not a glycoprotein, possesses an acidic isoelectric point (6.0) and exists as dimeric and monomeric species. The dimer is not held together by disulfide bonds. The purified protein retains its ability to induce transplantation immunity in syngeneic hosts when challenged with Meth A sarcomas. Chemical analyses of the NH2- and COOH-termini gave the following sequences: NH2-PKPINVRVTTMDAELEFAIQPN and IDE(F,A)EM-COOH, respectively.

Purification and characterization of mouse beta-2 microglobulin: allelic variants from two different strains.

Beta-2 microglobulin (beta 2M) is a 12,000 dalton protein associated with membrane-bound cell surface antigens. Variants of beta 2M, beta 2MA and beta 2MB, were first detected by Michaelson et al. (Immunogenetics 11, 93-95, 1980). An improved method was used to purify beta 2MA and beta 2MB from BALB/c and C57BL/6 mouse livers, respectively. Reproducible yields of 10% were obtained. The purifications were accomplished by a 3 M sodium thiocyanate (NaSCN) extraction of a crude membrane fraction, an acid precipitation step, gel filtration on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose and CM-cellulose in that order. The elution profile of beta 2MA and beta 2MB on the ion-exchange columns was found to be different, indicating the presence of structural changes. beta 2MA was found to be more acidic (pI = 7.35) than beta 2MB (pI = 7.68) by isoelectric focusing in gels. Complete sequence analysis of beta 2MA and partial sequence analysis of beta 2MB (61 of 99 residues) were performed by automated Edman degradation of the intact chain and of the overlapping peptides obtained by: (a) tryptic cleavage at arginines after acetimidation of lysine side chains, (b) BNPS-skatole cleavage at tryptophan residues and (c) hydroxylamine cleavage at asparagine-glycine linkages. A comparison of the primary structure of beta 2MA to the partial amino acid sequence obtained for beta 2MB revealed a single amino acid substitution (aspartic acid for alanine at position 85) that accounts for the differences in biochemical properties observed.

Biochemical analysis of a 40,000 mol. wt mouse serum protein which binds β2-microglobulin and has serological cross-reactivity with H-2 antigens

Abstract A 40,000 (40 K) mol. wt mouse serum protein which binds β 2 -microglobulin (β 2 M) has been purified to homogeneity by a modification of the procedure of Kvist & Peterson (1978). Biochemistry 17, 4794–4801. After gel filtration, sodium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography and affinity chromatography on an anti-H-2 a immunoabsorbent column, four proteins with apparent mol. wts of 12, 40, 55 and 75 K were specifically immunoprecipitated with rabbit anti-H-2 a . These individual proteins were subsequently purified to homogeneity by preparative SDS-PAGE. The identity of the 12 K mol. wt protein was unequivocally established to be mouse β 2 M based on amino acid composition, N -terminal analysis and reactivity with rabbit anti-β 2 M. The 40 K mol. wt protein was the only protein that bound β 2 M since radioimmunoprecipitation analysis of the high mol. wt fraction from fresh serum (with rabbit anti-β 2 M) yielded only the 40 K mol. wt protein and β 2 M. This result also precluded the possibility of a pre-existing complex of the 55 and 75 K mol. wt proteins with the 40 K mol. wt protein and β 2 M. The copurification of the 55 and 75 K mol. wt proteins with the 40 K mol. wt protein and β 2 M, was explained on the basis of their immunological cross-reactivity. The 55 and 75 K mol. wt proteins were found to be structurally distinct from the 40 K mol. wt protein as judged by amino acid analysis and peptide mapping. The similarity in amino acid composition of the 40 K mol. wt protein and H-2, in addition pto its cross-reactivity with H-2 xenoantisera, suggested the possibility of their structural relationship. However, the 40 K mol. wt protein differed from H-2 in the following respects: (1) H-2 alloantigenic determinants were not present on the 40 K. mol. wt protein, (2) the 40 K mol. wt proteins from two strains of mice had similar peptide maps indicating the lack of structural polymorphism in these strains, (3) the 40 K mol. wt protein had a more acidic and less restricted p I value than H-2, and (4) no common peptides were found in proteolytic digests of H-2 and the 40 K mol. wt proteins as analysed by two different methods. These results demonstrate that the β 2 M-binding murine serum protein is not specifically associated with other proteins, is not generated from a precursor present in the serum and bears no apparent homology to H-2 molecules despite serological cross-reactivity.