Two types of lambda polypeptide chains in human immunoglobulins based on an amino acid substitution at position 190.

Studies in this and in other laboratories have been concerned with the chemical composition of immunoglobulin light polypeptide chains and with the genetic significance of the chemical evidence. Many recent investigations have been devoted to the comparison of amino acid sequences of Bence-Jones proteins of manl-3 and mouse.4-6 These polypeptide chains are about 214 residues in length and consist of variable and invariable halves of about equal length. In man, as in mouse, the invariant portion comprises the 107 amino acids of the COOH-terminal region. There are two classes of light polypeptide chains of man, designated kappa and lambda. Human lambda Bence-Jones proteins share no antigenic determinants with kappa polypeptide chains and there are no tryptic peptides in common.7 Amino acid sequence analysis, however, has shown a large degree of homology (about 44%)8 between kappa and lambda chains. Lambda Bence-Jones proteins share ten common peptides. To date, these ten peptides appeared identical in all lambda BenceJones proteins. Two antigenic forms of lambda polypeptide chains, tentatively designated Oz (+) and Oz (-), have been distinguished by a rabbit antiserum produced to a lambda-type Bence-Jones protein.9 The present investigations were undertaken to define a structural basis for the antigenic specificity. This communication reports the existence among lambda-type Bence-Jones proteins of a single amino acid substitution in the carboxyl terminal half of the molecule at position 190. A good correlation exists between the immunological subtype and the structural variation as demonstrated by peptide analysis. Materials and Methods.-Bence-Jones proteins were initially isolated from urine by 80% ammonium sulphate precipitation. Proteins Ru and Ni were reprecipitated twice with 50% ammonium sulphate. Other proteins were further purified by DEAE-chromatography (phosphate buffer pH 8.0, gradient elution), Sephadex G-100 gel filtration (0.2 M Tris-HCl buffer pH 8.0 plus 0.2 M NaCl), Geon-Pevikon block electrophoresis (veronal buffer pH 8.6 A 0.6), or any combination of these as was necessary. Immunoelectrophoresis and Ouchterlony analysis at concentrations exceeding 15 mg/ml showed that all preparations were homogeneous by these criteria. Polyacrylamide gel electrophoresis (pH 8.8)10 of the Ru and Ni Bence-Jones proteins showed a single component in each case. Reduction of the proteins was accomplished by incubation in 10 M urea and 0.01 M Dithiothreitol under N2 for 3 or 4 hr at 37°C. After reduction, iodoacetic acid was slowly added to a final concentration of 0.022 M maintaining a pH of 8.2 by addition of 0.2 N NaOH. The reaction was terminated after 15 min by the addition of sufficient 0-mercaptoethanol to bring the final concentration to 0.025 M. The carboxymethylated product was dialyzed against water and lyophilized. Performic-acid oxidation of the enzyme was performed according to the method of Moore" but without the use of HBr as a reducing agent. Trypsin (2 X crystallized, lot 6241, Worthington) was treated with -(1-tosylamido-2 phenyl)