Variable Regions of Heavy and Light Polypeptide Chains of the Same gamma G-immunoglobulin Molecule (original) (raw)
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Amino acid sequence of the VH region of a human myeloma immunoglobulin (IgG New)
Biochemistry, 1977
The amino acid sequence of the heavy-chain variable region of the human immunoglobulin. New has been determined. Since the amino terminus of the heavy chain was blocked, the sequence of residues 1-69 was established by digesting the appropriate CNBr fragment separately with trypsin, chymotrypsin, and thermolysin and sequencing the resulting peptides. The region from residues 70 to 120 was present in another CNBr fragment which was submitted directly to automatic Edman degradation. The result of this T h e three-dimensional structure of the Fab' fragment of the human immunoglobulin New (IgG New) has been determined to a nominal resolution of 2 8, (Poljak et al., 1974). The light (L, A) chain of IgG New has been sequenced and reported (Chen and Poljak, 1974). The study of the amino acid sequence of the heavy (H) chain was undertaken to obtain the necessary information for a complete interpretation of the 2.0-A Fourier map of Fab' New. The amino acid sequence of IgG immunoglobulin H chains is usually divided into four homology regions, V H , CH 1, C H~, and cH3, consisting of approximately 1 10 to 1 15 amino acids (Gally and Edelman, 1972). With the exception of wellcharacterized allotypic variants, human y chains show sequence identity in their CH regions as well as partial identity and strong homology in their variable V H regions (Capra and Kehoe, 1975). Two approaches were used to obtain peptides from the V H region of New. In one case, Fab' fragments were cleaved with CNBr and separated by gel filtration. This gave a fragment comprising residues 1-69. A combination of enzymatic and
Analytical Biochemistry, 1974
An improved immunochemical procedure for the quantitative isolation of labeled minor proteins from tissue homogenates is worked out and is applied to the isolation of glucose&-phosphate dehydrogenase from mouse liver. Goat anti-enzyme serum is used as primary reagent, followed by rabbit anti-goat IgG, and not by carrier enzyme as in currently used methods. The resulting immunoprecipitates are analyzed by acrylamide gel electrophoresis, so that only counts in enzyme bands are registered. An equivalent precipitate formed with serum from nonimmunized goat serves as an efficient control for coprecipitation. ' G6PDH-Glucosed-phosphate dehydrogenase. 386
Analytical Biochemistry, 1975
Polyacrylamide gel electrophoresis of Fluorescamine-modified peptides is a rapid (90 mitt), sensitive (< 1 nmole), and reproducible method which may be used to resolve peptides without the limitations with respect to molecular size or water solubility found in other analytical systems. Fluorescamine-modified peptides, ranging in size from 3 to 215 amino acids, have been examined in a variety of buffer systems between pH 2.3 and 9.8 and gel concentrations varying between 4% and 16% acrylamide. Similar peptides were examined by high-voltage paper electrophoresis and detected by Fluorescamine. Peptides of low solubility have been examined in the presence of urea. The method reported here was used to resolve small peptides differing in size by a single amino acid, as well as peptides of the same size differing only by a single charge. It was successfully employed as a criterion for homogeneity of peptides in the course of purification for amino acid sequence analysis.
The objective of this experiment was to isolate proteins from egg yolk, then to analyze the yield via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV-Vis spectroscopy. The isolation was performed through a series of centrifugations and filtrations, then subsequently purifying the protein via size-exclusion chromatography. The obtained samples were then analyzed for the proteins and their concentrations via SDS-PAGE and UV-Vis spectroscopy using standard bovine serum albumin for the calibration curve. Results of the analysis proved that proteins were successfully isolated (concentrations?). It is recommended in future isolations to devise a way to make the series of incubations and sample preparations in the analysis via UV-Vis spectroscopy more efficient and, if possible, to find a way to improve visualization of the bands in SDS-PAGE. Proteins are known to be an essential component of the daily diet of all living creatures. They serve a myriad of functions within the body owing to their variety in structural features which determine the role they play. They are thus described as the " workhorses " of the cell in that they provide structural stability, motor functions, play a role in harvesting free energy, facilitate reparation and growth of tissues, and mediate communication between the cell and the extracellular environment and much more [2, 6]. The structure of proteins can be classified into four different levels. These comprise the primary, secondary, tertiary and quaternary structure. The primary structure pertains to the sequence of amino acid residues, the building blocks of proteins, throughout a segment of a polypeptide chain. The secondary structure, on the other hand, describes the local conformation of the polypeptide backbone. For example, two common conformations are the alpha helix, wherein the backbone twists in a right-handed fashion, and the beta sheet, wherein the backbones are extended and interact non-covalently with nearby backbones. The tertiary structure pertains
Proceedings of the National Academy of Sciences, 1976
Analyses of amino-acid sequences of the total cell-free products programmed by the mRNA of MOPC-104E gamma light (L)-chain show that over 95% of the products have sequences of a distinct protein that correspond to the L-chain precursor. In this precursor an extra piece is coupled to the NH2-terminus of the mature L-chain. Analyses of products labeled with [3H]alanine, [3H]leucine, and [3H]proline demonstrate that the extra piece is composed of at least 18 residues. Analyses of [35S]methione-labeled product indicate that the extra piece may contain an additional NH2-terminal methionine, which is detected in about 10% of the molecules. Partial recovery of the NJ2-terminal methionine (alanine, leucine, and proline are recovered in yields close to theoretical, greater than 95%) suggests that it is the initiator methionine, which is known to be short lived in eukaryotes due to rapid hydrolysis. Thus, the extra piece seems to be 19 residues in length, and it contains one methionine at the...
Biochemistry, 1970
Six human yG1 myeloma proteins having a carbohydrate moiety on the Fab fragment were analyzed. The carbohydrate was located on the light chain in four of the proteins (three of X and one of K type), on the Fd fragment in one, and one protein had carbohydrate on both the Fd fragment and light chain (K type). The amino acid composition and sequence analysis of the glycopeptides indicated that the point of attachment of the carbohydrate varied from protein to protein. However, in all six proteins studied, the localization of the carbohydrate appeared to be restricted to two regions within the variable portion of the light chains and Fd fragments. One was near the N terminus between positions 22 and 32 and the other near the C-terminal end of the variable I t is well established that human yG-immunoglobulins contain a carbohydrate moiety on the Fc fragment (Franklin, 1960;. Whereas it was previously believed that the carbohydrate was restricted to only the Fc fragment, in a recent study of 76 human yG myeloma proteins, it was demonstrated that approximately 25 % contained a carbohydrate moiety on the Fab fragment in addition to the one on the Fc fragment . The carbohydrate was linked to either the light chains or the Fd fragments, and in a few of the proteins, to both. There was no correlation between the presence of carbohydrate on the Fab fragment with the light-chain type nor with the heavy-chain subclass of the myeloma proteins. Two human X-and two K-type Bence Jones proteins having a carbohydrate moiety have also recently been reported by Edmunson et a/. (1968) and by Hood et a/. (1969), respectively. The purpose of the present investigation was to localize the point of attachment of the carbohydrate on either the light chains or Fd fragment and to determine whether a single point of attachment was involved as is the case with the Fc fragment, or if the carbohydrate was at-* Publication No. 415 from the