Byeong-Kuk Seon

Antibodies of limited heterogenety: L chains of a single mobility

Abstract The specifically purified anti-p-azobenzoate antibody from two rabbits (out of nine rabbits that had been injected with a heterogensous antigen (azo-p-benzoate-bovine-γ-globulin conjugate)) was found to be relatively homogensous on the basis of several criteria including homogeneity of the hapten-binding constant, and light chains giving only a single band upon disc electrophoresis, a relatively unique amino acid composition, and a relatively simple tryptic peptide map. Such homogeneity would not be expected on the basis of the concept that antibodies produced to a particular hapten by an individual animal represent a continous heterogeneous population composed of antibodies with binding constants following a probability distribution function. It appears rather, that only a few molecular species of antibody make up the bulk of the antihapten antibody produced in each individual animal. Thus, the observed limited antibody heterogeneity in an individual rabbit appears to depend on the stumulation of only a few of the many cells capable of producing antibody againts a given hapten, rather than on a structural identity of the environment around each individual group on the antigen molecule as postulated by others.

Isolation and characterization of A CH2 domain fragment of human IgG

Abstract A fragment corresponding to the C H 2 domain of IgG was isolated and chemically characterized. This C H 2 domain fragment was isolated by gel filtration of the reduced and alkylated IgG fragment which was obtained by the high-temperature peptic digestion of myeloma IgG. An amino-terminal amino acid sequence study revealed that the amino acid sequence of the C H 2 domain fragment corresponds to that of a γ-chain segment which begins with isoleucine 253. The mol. wt of the fragment was estimated by gel filtration in the presence of 6 M guanidine-HCl to be 8800.

Environments of individual tyrosines in protein: Differences in rates of mono- and dhodination

Abstract The relative rates of iodination of tyrosine to monoiodotyrosine and of monoiodotyrosine to diiodotyrosine in proteins: Tyrosine → k 1 monoiodotyrosine → k 2 diiodotyrosine have been a subject of controversy for some time. We have now shown that within a particular protein, a human Bence Jones protein, there are tyrosines for which k1 is greater than k2 and other tyrosines for which k2 is greater than k1. The value of the ratio, k1/k2, is dependent on the particular tyrosine involved.

Reactivity of tyrosine residues in the constant portion of type K Bence Jones protein

Abstract Tyrosine 173 of the constant portion of a human type K Bence Jones protein (Col) appears to be on the surface of the protein since it is the most reactive tyrosine toward iodination. This surface position for tyrosine 173 may be the case in general for type K Bence Jones proteins and L chains since it seems to be highly reactive in another type K Bence Jones protein (Koo) and the κ type L chain from a human myeloma IgG. Tyrosine 192, next to the valine characteristic of InV (b+) allotype, and tyrosine 186 and 140 appear to be less exposed. Tyrosine 140 is much less reactive than any of the other three of the constant portion and thus appears to be least exposed.

Reduction of IgM and characterization of the products.

Abstract We have found that mild reduction of human IgM with 2-mercaptoethylamine (MEA)§ yields 2 major components, monomeric subunits (IgMs) and what appears to be the half molecule of IgMs, H–L. In non-dissociating buffers, 2 molecules of H–L form a non-covalently-associated IgMs. Five monoclonal IgM proteins were mildly reduced with MEA under various conditions. The reduced IgM proteins were analyzed by gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The analysis was carried out both 0.5% agarose-2.5% acrylamide gel and 5% acrylamide gel. The reduced preparations of all 5 IgM proteins showed IgMs and H-L in addition to the small amount of the remaining intact pentamer. One of the IgM proteins, IgM Widr, was further studied in detail. The reduced and carboxymethylated IgM Widr was subjected to ultracentrifugal analysis in 0.32 M NaCl biffered with 10 −3 M sodium borate (pH 8.0). Only two peaks of 6.4S and 16S were observed. The approximate ratio of the amounts of the two peaks was 7.3 (6.4S):1 (16S). This result indicates that 2 molecules of H-L associated non-covalently and this associated material is co-sedimented with IgMs. Supporting evidence was obtained by a gel filtration study of the ultracentrifuged material. The gel filtrate on a Sephadex G-200 column showed a small peak eluting at the void volume and a large peak, the mol. wt of which was determined to be 185,000 daltons. Several fractions of the first and the second peaks were analyzed by SDS gel electrophoresis. The first peak contained covalently bonded pentamer and some oligomers. The second peak contained IgMs and H-L.

Common microenvironments of tyrosyl residues in several bence jones proteins

Abstract A comparison was made of the reactivities toward iodination of particular tyrosyl residues in κ Bence-Jones proteins of subgroups I, II and III. They are Roy (I), Cum (II), Ti (III) and Col (III). For each of these proteins the positions of all the tyrosines in the primary structure are known. In all the proteins Tyr 173 is highly reactive and Tyr 186 is less reactive. Another interesting feature is that in all the proteins, the rate constant for incorporation of the first iodine is greater than that for incorporation of the second iodine in the case of Tyr 173 while for Tyr 186 the opposite situation was found. Thus, all four of the Bence-Jones proteins have one common microenvironment around Tyr 173 and another common environment around Tyr 186, showing that the tertiary structure of each of the proteins is the same in the region of Tyr 173 and is also the same in the region of Tyr 186. Tyr 36 in Ti and Col is of low reactivity and thus appears to be partially buried. It appears that in all κ Bence-Jones proteins the microenvironment of Tyr 173 is hydrophilic and that of Tyr 186 is hydrophobic.

Isolation and characterization of 7S IgG, a γ-fragment, β2-microglobulin and a Bence Jones protein in urine of a patient with plasma cell leukemia☆

Abstract Large quantities of a monoclonal 7S IgGκ, a κ type Bence Jones protein, and β 2 -microglobulin along with a smaller amoung of a γ-fragemnt were found in the urine of a patient, TSCH, with plasma cell leukemia. The serum of this patinet also contained a monoclonal 7S IgGκ. Comparison of the isolated urinary IgG and the serum IgG disclosed no significant differences between the two proteins with respect to amino acid composition, amino-terminal amino acid sequence, amino sugar composition, sodium dodecyl sulfate acrylamide gel elecgtrophoresis, ultracentrifugation and antigenicity. Both IgG proteins were intact 7S molecules with a γ chain of γl subclass and with a κ chain of VκIII subgroup. A double immunodiffusion study showed that teh γ-fragment represents a portion of the Fc fragment. This γ-fragment was previously shown to be noncovalently bound to β 2 -microglobulin (Seon & Pressman, 1977). A relatively simple procedure was successfully used to isolate a large amoung of β 2 -microglobulin from the urine of the patient.

Rabbit antibody with light chain having an N-terminal valyl residue☆

Abstract Amino acid sequence studies were carried out with rabbit light chains from several different sources. Light chain from the specifically purified anti- p -azobenzoate antibody from one rabbit showed a single predominant amino-terminal amino acid sequence Val-Glx-Val-Leu-. This sequence is significantly different from that of rabbit light chains reported by other groups and suggests that this amino-terminal sequence may be that of a new subgroup of rabbit κ light chains. The present amino-terminal sequence is also of particular interest since valine has not been reported as the predominant amino-terminal residues for light chains of any species.

The reactivity of tyrosyl residues in Bence Jones protein: Differences in rates of iodination in covalent and noncovalent dimers

Abstract A comparative study of the reactivity toward iodination of the tyrosyl residues of the covalent and noncovalent dimers of a Bence Jones protein showed that at least one of the eight tyrosine residues in the noncovalent dimer iodinated more rapidly than the corresponding residue in the covalent dimer. For each of the other tyrosines, the rate of iodination was the same in the two dimers. This shows that there are conformational differences between the covalent and noncovalent dimers to provide different environments for at least one tyrosine residue. In the ultracentrifuge, the covalent dimer sedimented more rapidly than the noncovalent one, also indicating a conformational difference. Optical rotatory dispersion and circular dichroism measurements did not show any significant difference between the two dimers.

Amino-terminal sequences of heavy chains of rabbit anti-hapten antibodies of limited heterogeneity.

Abstract We have studied the amino-terminal amino acid sequences of heavy chains of rabbit antibodies which are of limited heterogeneity. These antibodies are the anti- p -azobenzoate antibody from rabbit No. 2717 and two fractions (BE-1 and BE-3) of anti- p -azobenzoate antibodies from rabbit No. 2663. No free amino-terminal residue was found for any of these three heavy chains and only a single blocked amino-terminal hexapeptide was isolated in good yields from each of these heavy chains. Their sequences are Glp-Ser-Leu-Glu-Glu-Ser- for No. 2717 heavy chain and Glp-Ser-Val-Glu-Glu-Ser- for both BE-1 and BE-3 heavy chains. These heavy chain amino-terminal sequences are compared with the light chain amino-terminal sequences of the same antibody molecules, and with the reported heavy chain amino-terminal sequences of normal rabbit immunoglobulins and rabbit anti-carbohydrate antibody. It appears that the amino-terminal amino acid sequences of rabbit heavy chains are relatively similar from antibody to antibody in contrast to the much greater variation in the amino-terminal sequences of rabbit light chains.