The complete amino-acid sequence of the constant (C) region of the a2 heavy chain of a human IgA2 protein of the A2m(1) allotype has been determined. Excluding the hinge region and the carbohydrate content, this a2 allotype differs from the al chain in only 14 amino-acid positions; all of these are identical to the A2m(2) allotype of the a2 chain and confer subclass (or isotypic) character on the a2 chains. However, the A2m(2) allotype differs in six positions where A2m(1) and al are identical; the first two are just before the hinge and the other four are in the last (CH3) domain. The A2m allotypic character of a2 chains is attributed to several conformational factors in the sequence at positions 211-221, just before the hinge. The isoallotypic determinant shared by al chains and the A2m(1) allotype of a2 resides in the identity of their CH3 domains. Thus, the A2m(1) allotype appears to be a hybrid chain that is identical with al in the CH3 domain and identical with the A2m(2) a2 chain in the CHI and CH2 domains and in the hinge, except for the allotypic determinants arising from four structural differences from residues 211-221. The genetic origin of isotypes, allotypes, and isoallotypes of the a chain has involved several events of homologous crossing over and neutral point mutations accumulated late in the evolutionary development of IgA immunoglobulins. Since the crossing over appears to occur between CH2 and CH3, heavy chain domains may be coded for by independent units in embryonic DNA that are analogous to the variable (V) and C segments of light-chain genes.Correlation of the structure, function, evolution, and immunogenetics of antibodies is a primary goal of our studies of the amino-acid sequence of human immunoglobulins of different classes, subclasses, and allotypes. We have recently reported the complete amino-acid sequence of a human immunoglobulin A of the IgAl subclass (1) and also of the a2 heavy chain of the A2m(2) allotype of the IgA2 subclass (2). We report here the complete sequence of the constant (C) region of the a2 heavy chain of an IgA2 immunoglobulin of the A2m(1) allotype. This work has permitted the location and identification of the amino-acid substitutions and the differences in kind, number, and location of oligosaccharides that determine the structural characteristics of all the known subclasses and allotypes of human IgA. The recently discovered (3) isoallotype (or nonmarker) nA2m(2) is shown to be due to the identity in sequence of the last domain (CH3) of the a l chain and the A2m(1) allotype of the a2 chain. Like the Lepore phenomenon in human hemoglobin, the existence of hybrid a heavy chains of IgA poses an interesting question about the evolution and genetics of IgA and suggests an event of homologous crossing over early in human evolution. This may have involved reassortment of independent genetic units that code for individual domains but that are separated by untranslated sequences in embryonic DNA.

Amino acid sequence analysis of the 67,000-dalton fragment that is the amino-terminal half of human ceruloplasmin has revealed internal triplication in the primary structure of the entire molecule. This is illustrated by comparison of 620 residues representing homologous domains of the 67-kDal fragment and of the 50-kDal and 19-kDal fragments that together comprise the carboxyl-terminal half of the molecule. The polypeptide chain is divided into three covalently linked homologous segments, each of about 340 residues. All three homology units have about 30% identity in sequence, and each pair exhibits at least 40% identity. The statistical significance of the 3-fold internal duplication was established by computerized analysis of the sequence. These results and studies of the sites of limited proteolytic cleavage support a model for the ceruloplasmin molecule consisting of an alternating structure of six domains of two different kinds (or possibly nine domains of three kinds). The 3-fold internal homology suggests that the ceruloplasmin molecule evolved by tandem triplication of ancestral genes.Ceruloplasmin [Cp; ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1] is a blue a2-glycoprotein that binds 90-95% of blood plasma copper and has multiple functions (1). Although subject to spontaneous proteolysis, it consists of a single polypeptide chain (MAr 135,000) that exhibits internal duplication in amino acid sequence (2). Three types ofobservations suggest that the ceruloplasmin molecule may be divided into a series of homologous domains: (i) the internal homology in primary structure (2); (ii) the existence of sites of limited proteolytic cleavage resulting in a series of fragments, the principal ones having approximate molecular weights of 67,000, 50,000, and 19,000 (3-8); and (iii) the presence of six copper atoms of three different kinds, as distinguished by spectroscopic and other physical properties (9, 10).We have reported the complete amino acid sequence of the 19,000-dalton (19-kDal) (5, 6) and the 50,000-dalton (50-kDal) (3) fragments that together account approximately for the carboxyl-terminal half of the ceruloplasmin molecule. By comparison of these, we have shown the existence of two homology regions, each containing 224 residues (2). We have now almost completed determining the amino acid sequence ofthe 67,000-dalton (67-kDal) fragment that is the amino-terminal halfof the molecule. The polypeptide chain consists of three covalently linked homologous segments (homology units), each of about 340 amino acid residues. The entire molecule exhibits a 3-fold internal homology in amino acid sequence, all three homology units having about 30% identity in sequence. This is illustrated here by comparing the sequences of some 620 residues from sections of the 67-kDal and 50-kDal fragments and the entire 19-kDal fragment. On the basis ofthese results and from studies of the sites of limited proteolytic cleavage and the location of the four oligosaccharides, we propose a tentative model for the...

The complete amino acid sequence of the major component myoglobin from Pacific common dolphin, Delphinus delphis, was determined by the automatic Edman degradation of several large peptides obtained by specific cleavages of the protein. More than 80% of the covalent structure was established by the degradation of the apomyoglobin and five peptides from: (1) cyanogen bromide cleavage at the two methionine residues, (2) trypsin cleavage of the acetimidated apomyoglobin at the three arginine residues, and (3) 2-p-nitrophenylsulfenyl-3-methyl-3'-bromoindolenine cleavage at the two tryptophan residues. The rest of the sequence was determined by use of the peptides prepared from further digestion of the central cyanogen bromide peptide with staphylococcal protease and trypsin. The primary structure of this myoglobin proved identical with that from the Atlantic bottlenosed dolphin, Tursiops truncatus, but showed four substitutions with respect to the sequence reported for the Black Sea dolphin which has also been given the designation Delphinus delphis.

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Reconstruction of the sperm whale myoglobin structure was accomplished by a series of aqueous condensations of suitably protected synthetic myoglobin fragments to a large fragment prepared from the native protein. Reaction of NANT9-acetimidomyoglobin with 3-bromo-2-(2-nitrophenylsulfenyl)skatole (BNPS-skatole) yielded the fragment corresponding to residues 15-153. The covalent structure was reformed by sequential coupling of the N-hydroxysuccinimide esters of o-nitrophenylsulfenyl-L-tryptophan (residue 14) and selectively protected peptides corresponding to residues 1-5 and 6-13, which were synthesized by the solid-phase method and removed from the resin by methoxide-catalyzed methanolysis. A mixed aqueous solvent system containing methanol and N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine/trifluoroacetic acid buffer (Quadrol) solubilized the peptide and protein fragments during the condensations. Replacement of the heme moiety and immunoaffinity chromatography made possible the isolation and purification of the reconstructed native molecule. The development of this nondestructive synthetic procedure allows investigation of the structural and functional significance of individual residues by isotopic enrichment or selective amino acid substitutions.Studies using selective, consecutive removal and substitution ofthe NH2-terminal residues ofMb by specific degradation and resynthesis (1, 2) have indicated the subtle contribution of the NH2-terminal charge and the uncharged side chains ofresidues 1 and 2 to the conformation and stability of the native molecule (3). To examine further the effects of synthetic sequence alteration with respect to stability, conformation, and electrostatic and hydrophobic interactions, a method for creating internal sequence variations must be developed. A promising approach is that of semisynthesis, a technique whereby the natural product is selectively cleaved and a fragment is isolated that is suitable for rebuilding the native structure or an analog.Previous attempts at partial synthesis with Mb have utilized tryptic or CNBr fragments in which citraconyl or maleyl protecting groups were used on the e-NH2 groups (4, 5). The current strategy exploits the oxidative lability of the tryptophans at positions 7 and 14 ofthe Mb sequence by the use of3-bromo-2-(nitrophenylsulfenyl)skatole (BNPS-skatole; skatole = 3-methylindole) to effect cleavage, yielding the fragment corresponding to residues 15-153 (designated fragment 15-153) in which the E-NH2 groups have been protected previously by reaction with methyl acetimidate (1). Sequential coupling of the N-hydroxysuccinimide (HOSu) esters of o-nitrophenylsulfenyl-L-tryptophan (NPS-Trp; residue 14) and the selectively protected peptides corresponding to residues 1-5 and 6-13 (designated peptides 1-5 and 6-13) regenerated the native sequence. Isolation of reconstructed Mb capable of correctly positioning the heme moiety was accomplished by passage through successive affinity columns on which the native semisynthetic protein was se...