A major anti-myoglobin idiotype. Influence of H-2-linked Ir genes on idiotype expression.

A rabbit antiidiotypic antiserum raised against an A.SW IgG1K monoclonal anti-sperm whale myoglobin (Mb) antibody, HAL19, and extensively absorbed with normal mouse immunoglobulin and MOPC 21 (IgG1K), was found to detect a common or major anti-Mb idiotype expressed by some but not all anti-Mb monoclonal antibodies, regardless of immunoglobulin G (IgG) subclass, and by 40-50% of the anti-Mb antibodies in immune serum from five high responder strains of mice representing five different Igh allotypes. It did not inhibit antibodies to three unrelated protein antigens. The fraction of antibodies expressing this idiotype, denoted IdHAL19, was regulated by H-2-linked genes that correlated exactly in four independent haplotypes and an F1 with the known Mb immune response (Ir) genes and may be identical to these. Whereas less than 50% of antibodies from high responder mice were inhibitable by anti-IdHAL19, greater than 80% of antibodies from low responder mice, tested at comparable final antibody concentration, were inhibitable. This result was true for both low responder haplotypes, H-2b (B10) and H-2k (B10.BR). The idiotype was found to be present on antibodies that bound to native Mb but not fragments 1-55 or 132-153 of Mb or a denatured form, S-methyl Mb. This specificity for native Mb paralleled that of the monoclonal idiotype HAL19 itself. Therefore, the production of antibodies specific for native in contrast to denatured Mb was studied in H-2-congenic high and low responder strains. Strikingly, low responders produced antibodies that reacted almost exclusively with the native conformation, whereas a larger proportion of antibodies from high responder mice also reacted with the denatured form, S-methyl Mb. Bypassing of the Ir gene defect by immunization with Mb attached to a carrier, F gamma G, resulted in low responder antisera resembling higher responder sera in both idiotype expression and conformational specificity. The simplest explanation of these results is that H-2-linked Ir genes control antibody fine specificity, which is reflected in the idiotypes of the variable regions expressed. We suggest that low responder mice produce a more limited repertoire of antibodies consisting primarily of IdHAL19-positive antibodies specific for the native conformation of Mb. High responder mice produce a greater diversity of antibodies to Mb, so that the IdHAL19-positive, conformation-specific population represents a smaller proportion of the total. Similarly, the use of carrier-specific helper T cells in low responder mice results in a greater diversity of antibodies, which dilutes out the IdHAL19 subset.(ABSTRACT TRUNCATED AT 400 WORDS)

Complete amino acid sequence of the major component myoglobin from Hubb's beaked whale, Mesoplodon carlhubbsi.

The complete primary structure of the major component myoglobin from Hubb's beaked whale, Mesoplodon carlhubbsi, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. In all experiments a Beckman 890C automatic sequencer was used to degrade the peptides. A cyanogen bromide digest was used to fragment the protein at its two methionine residues into three fragments which were separated by gel filtration. In a similar pattern the protein was citraconylated to protect the lysine residues and the modified protein fragmented at its arginine residues with trypsin. The three peptides obtained here were also purified by gel filtration. Sequencer analysis of the whole apoprotein, cyanogen bromide fragments and the peptides cleaved at the arginine residues with trypsin provided 80% of the completed sequence. The remainder of the sequence was obtained by digesting the middle cyanogen bromide fragment with staphylococcal protease and by total tryptic digestion of the whole apoprotein and isolating the resulting peptides by ion-exchange chromatography. The primary structure of the Mesoplodon carlhubbsi myoglobin represents a sequence which may be closey related to the ancestral sequence of all cetacean myoglobins.

Complete amino acid sequence of the myoglobin from the Pacific sei whale, Balaenoptera borealis.

The complete amino acid sequence of the major component myoglobin from Pacific sei whale, Balaenoptera borealis, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. From the sequence analysis of four of these peptides and the apomyoglobin, over 75% of the covalent structure of the protein was obtained. The remainder of the primary structure was determined by the sequence analysis of peptides that resulted from further digestion of the amino-terminal and central cyanogen bromide fragments. The amino-terminal fragment was specifically cleaved at its two tryptophanyl residues with N-chlorosuccinimide and the central cyanogen bromide fragment was cleaved at its glutamyl residues with staphylococcal protease and at its single tyrosyl residue with N-bromosuccinimide. The primary structure of this myoglobin proved identical with that from the gray whale but differs from that of the finback whale at four positions, from that of the minke whale at three positions and from the myoglobin of the humpback whale at one position. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea.

Complete amino acid sequence of the major component myoglobin from the goose-beaked whale, Ziphius cavirostris.

The complete primary structure of the major component myoglobin from the goose-beaked whale, Ziphius cavirostris, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Over 80% of the amino acid sequence was established from the three peptides resulting from the cleavage of the apomyoglobin at its two methionine residues with cyanogen bromide along with the four peptides resulting from the cleavage with trypsin of the citraconylated apomyoglobin at its three arginine residues. Further digestion of the central cyanogen bromide peptide with S. aureus strain V8 protease and the 1,2-cyclohexanedione-treated central cyanogen bromide peptide with trypsin enabled the determination of the remainder of the covalent structure. This myoglobin differs from the cetacean myoglobins determined to date at 12 to 17 positions. These large sequence differences reflect the distant taxonomic relationships between the goose-beaked whale and the other species of Cetacea the myoglobin sequences of which have previously been determined.

Complete amino acid sequence of the myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani.

The complete amino acid sequence of the major component myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani, was determined by the automated Edman degradation of several large peptides obtained by specific cleavage of the protein. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. By subjecting four of these peptides and the apomyoglobin to automated Edman degradation, over 80% of the primary structure of the protein was obtained. The remainder of the covalent structure was determined by the sequence analysis of peptides that resulted from further digestion of the central cyanogen bromide fragment. This fragment was cleaved at its glutamyl residues with staphylococcal protease and its lysyl residues with trypsin. The action of trypsin was restricted to the lysyl residues by chemical modification of the single arginyl residue of the fragment with 1,2-cyclohexanedione. The primary structure of this myoglobin proved to be identical with that from the Atlantic bottlenosed dolphin and Pacific common dolphin but differs from the myoglobins of the killer whale and pilot whale at two positions. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea.

Use of thiocyanic acid to form 2-thiohydantoins at the carboxyl terminus of proteins.

The chemistry of the formation of 2-thiohydantoins on the carboxyl terminal of peptides or proteins was investigated. It was found that thiocyanic acid was much more reactive for the formation of 2-thiohydantoins than were the thiocyanate salts. The physical reasons for this observation are explained. The kinetics of the reaction of a number of proteins, and some of their fragments, with thiocyanic acid were also determined. Simple and safe procedures for the preparation of anhydrous thiocyanic acid solutions were devised. The prospective application of these procedures to sequencing from the carboxyl terminal of a polypeptide is discussed.

Rotational motions in myoglobin assessed by carbon 13 relaxation measurements at two magnetic field strengths.

Proton-decoupled Fourier transform nuclear magnetic resonance spectroscopy of natural abundance 13C was used to obtain spectra of cyanoferrimyoglobin of sperm whale (Physeter catadon) at 14.1 and 23.5 kG. Comparison of the spin lattice relaxation times at these two field strengths allowed the unambiguous assignment of a rotational correlation time of 22 plus or minus 5 ns for the alpha carbon resonances. The spin lattice relaxation time value for a major band attributable to aromatic carbon atoms also corresponded to a single correlation time, attributable to over-all tumbling of the molecule. Certain narrower resonances reflect other modes of rotational motion in addition to the over-all tumbling. Observations of nuclear Overhauser enhancement and line widths accord with these conslusions.

Electrostatic stabilization in myoglobin. Interactive free energies between individual sites.

The pattern of electrostatic interactions between pairs of charge sites in sperm whale ferrimyoglobin was examined as a function of pH in terms of proton site occupancy, static solvent accessibility, and distance of separation. By grouping all examples of the most stabilizing interactions and all examples of the most destabilizing interactions, we can easily show that at pH 7.50 the former is much stronger; that is, the negative contributions to electrostatic free energy far outweigh the positive contributions. Much of the electrostatic energy of stabilization in native myoglobin is provided by specific charge-pair partners that are very highly conserved among 53 mammalian myoglobin species and is invariant substantially from pH 8.5 to 3.5. Destablizing interactions that become most significant, but not actually dominant, near the acid unfolding pH range can be recognized in emerging clusters of uncompensated positive charges. Binding of azide ion by the heme iron effectively reduces the most prominent destabilizing set of such interactions. In general, thoe charged residues that experience the largest summed stabilizing interactions with other groups are the most conserved between species. The histidine residues, however, show their best correlation of conservation with low values of static accessibility. Although histidine residue 64 has an effective pK corresponding to the midpoint of the unfolding transition near pH 4.2 at an ionic strength of 0.10 M and so might be called a "trigger group", its interactions contribute only a modest fraction of the overall pH-dependent free energy change. An examination of the primary stabilizing interactions represented by the charge-pair partners indicates a probably major role of electrostatic interactions in the nucleation and docking stages of the condensation of the polypeptide chain into the compact native structure.

Reassignment of residue 122 in the myoglobin from the killer whale, Orcinus orca.

The complete amino acid sequence of the major component myoglobin from killer whale, Orcinus orca, was determined by automated Edman degradation. In this study residue 122 was found to be glutamic acid instead of glutamine as was originally reported (Castillo et al. 1977). This reassignment affects the phylogenetic relationship of killer whale myoglobin with the myoglobins from other closely related cetacean species and also affects studies concerned with the physical parameters of the protein.

Electrostatic effects in hemoglobin: electrostatic energy associated with allosteric transition and effector binding.

The pH dependence of the summed electrostatic stabilization for deoxy- and liganded hemoglobin was computed for several ionic strength values. The computed contribution to the stabilization of deoxyhemoglobin by binding of 2,3-diphosphoglycerate in the beta cleft compared well with experimental binding behavior for human hemoglobin A0 and hemoglobin F. The contribution of diphosphoglycerate binding to the alkaline Bohr effect was computed correctly for both hemoglobins A0 and F. The computed effects of simultaneous binding of diphosphoglycerate and formation of Val-1 beta carbamino adducts suggested a competition between these effectors. A direct competition was formulated between these two effectors, with extension to include a simple anion such as chloride or bicarbonate binding in competition with diphosphoglycerate but not with Val-1 beta carbamino formation. This model was found to hold at pH 7.3-7.4 over a range of concentrations of the effectors involved and to predict the pH dependence of Val-1 beta carbamino formation over the pH range 7.0-8.0. The pH dependence of the computed differential stability of liganded vs. unliganded hemoglobin A compared well with observation.

Electrostatic effects in myoglobin. Application of the modified Tanford-Kirkwood theory to myoglobins from horse, California grey whale, harbor seal, and California sea lion.

The modified Tanford-Kirkwood electrostatic theory (Shire et al., 1974a) was applied to ferrimyoglobins from the following animal species: sperm whale (Physeter catodon), horse, California grey whale (Eschrichtius gibbosus), harbor seal (Phoca vitulina), and California sea lion (Zalophus californianus). Computations were made of the overall hydrogen ion titration curves of the proteins, and of pH and ionic strength variations of ionization equilibria for individual groups in the protein, with particular reference to the hemic acid ionization of the iron bound water molecule. Coordinates and static solvent accessibility were estimated in terms of the sperm whale myoglobin structure. Where possible, theoretical results and experimental data are compared. Some comparative features of charge and ionization properties among the various myoglobins are presented.