Variable regions of heavy and light polypeptide chains of the same gammaG-immunoglobulin molecule.

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Each yG-immunoglobulin molecule contains two identical light chains and two identical heavy chains.'Light chains (designated x or X) consist of an amino terminal portion of variable amino acid sequence followed by a region of approxi- mately the same length having a constant sequence.The variable regions of x chains fall into three subgroups based on the degree of homology of their amino acid sequences.2It is currently believed that the diversity of sequence in anti- body molecules is related to the variety of antigenic determinants to which they can bind.Heavy and light chains both appear to contribute to the antigen- binding site,' and thus heavy chains should also contain a variable region.The location, extent, and type of variability in heavy chains is being actively investigated in a number of laboratories.3-8In the present communication, we report the amino acid sequence of the first 87 residues of the heavy chain of a human yG-immunoglobulin, Eu.Our major aims are: (1) to compare the heavy and light chains of the same yG-immuno- globulin molecule, (2) to locate the variable region of heavy chains by comparing the sequence of the Eu heavy chains with that reported3 for another heavy chain (Daw) of the same 'yG1 subclass, and (3) to compare the nature of the sequence variation in heavy chains with that already known for light chains.Our results suggest that heavy chains follow rules of variation similar to those governing the structure of light chains.Materials and Methods.-FragmentsHi, H2, H3, and H4 (Fig. 1) were prepared by treat- ing the myeloma protein Eu with cyanogen bromide (CNBr) as described previously.5Fragment H, was further purified by chromatography on carboxymethyl cellulose (What- man CM 52, W. and R. Balston, England) in 0.02 M sodium acetate, pH 4.8, with the use of an exponential gradient from 0 to 0.1 M KCl.Fragment H3 was separated from fragment H7 by gel filtration on Sephadex G-50 in n-propanol: acetic acid: water (3: 1: 8, v/v).Digestion with enzymes: CNBr fragments HI and H3 were treated with trypsin (Calbiochem, TPCK-treated, Lot 65345) at pH 8.0 and 370 for 1 hr with an enzyme/protein ratio of 1% by weight.Digestion with a-chymotrypsin (Worthington, 3 X crystallized, CD1- 6150-1) was performed at pH 8.0 for different periods of time at an enzyme/protein ratio of 1-5% by weight.Pronase digestion (Calbiochem, B Grade, Lot 63807) was performed at pH 8.2 and 370 at an enzyme/protein ratio of 4% by weight.All digests were done in water maintained at the desired pH by the automatic addition of the appropriate concen- tration of NH40H in the cell of a Radiometer TTT-lb titrator.Peptides were treated with leucine aminopeptidase at 300 for various times in 0.01 M tris(hydroxymethyl)aminomethane buffer, pH 8.0, which was 0.2 M in MgCl2.Peptide concentration was 2 mg/ml, and the enzyme/peptide ratio was 1% by weight.Digestion with carboxypeptidases A and B was carried out in 0.2 M1 N-ethylmorpholine buffered at pH 8.6 with acetic acid.Partial acid hydrolysis: Partial acid hydrolysis was carried out in vacuo in 0.03 N HCl for 12 hr at 105' at a peptide concentration of 1 mg/ml as described by Tsung and Fraen- kel-Conrat.9