Bence-Jones proteins and light chains of immunoglobulins. VII. Localization of antigenic sites responsible for immunochemical heterogeneity of kappa chains.

Abstract The evaluation of numerous anti-κ chain antisera through immunochemical studies of κ light chains for which complete or partial sequence data were available has resulted in the localization of three distinct regions in the variant half of the light chain which are associated with specific antigenic sites. The residue at position 9 appears to be associated with an antigenic site which, in general, may be termed a group-related antigenic site. The residues at positions 45 and 94 through 96 are associated with two distinct antigenic sites which are recognized specifically by numerous antisera and are responsible for certain intergroup similarities and intragroup differences among κ chains. Many of the antisera readily distinguish among proteins which have been structurally assigned to the same subgroup. The localization of distinct regions which markedly influence the nature of the antigenicity of the light chain molecule provides a molecular basis for the observed heterogeneity among κ chains and contributes to the knowledge of the tertiary structure of the polypeptide chain.

The establishment of the identity between Bence-Jones proteins and light polypeptide chains which are common to all immunoglobulins (1) emphasized the importance of the Bence-Jones protein as a homogeneous source of material for studies directed toward elucidation of the relationship between molecular structure and function of immunoglobulins.
Furthermore, in view of the contributory role of light chains in antibody specificity (2-4), studies on Bence-Jones proteins are especially significant. The basic primary structure of Bence-Jones proteins, i.e. light polypeptide chains, has been established (5-7) ; comparisons of the primary structure of many Bence-Jones proteins (summarized in Reference 8) revealed that light chains of the same type (K or L)  due 108 to 214). The amino acid sequence of the variant half of each light chain is unique, whereas the sequence of the constant half is common to all light chains of the same type.
The ability to cleave specifically the BenceJones protein into its variant half and constant half made possible the preparation and isolation of each half in sufficient quantities for studies on the properties of each half (9). Knowledge of the properties of each half is important in determining whether a property of the intact light chain is an expression of the whole molecule or is a characteristic of only one-half of the molecule.
This report presents the results of x-ray diffraction studies on the crystals of the variant half of a human K Bence-Jones protein.

EXPERIMENTAL PROCEDURE
Isolation and IdentiJication of Variant Half of Bence-Jones Protein LEN-Details of the isolation of Bence-Jones protein LEN' were presented previously (9). The monomeric form of Bence-Jones protein LEN was cleaved enzymatically into its constituent variant half (V,) and constant half (C,) with pepsin, and each half was isolated by zone electrophoresis and purified by gel filtration (9). Antisera against Bence-Jones protein LEN, the isolated VL of protein LEN, and heterologous K type Bence-Jones proteins were prepared according to the method presented previously (9). Crystallization of Variant Half of Bence-Jones Protein LEN-Ten milligrams of isolated V, LEN were dissolved at 37" in 1.4 ml of 0.1 M potassium phosphate buffer, pH 6.0, and maintained at 37" for 30 min. The sample was transferred from the 37" water bath to a freezer. After 1 hour at -2O", the sample was transferred to a 5" refrigerator. After 96 hours at 5", crystals were present.
This method of crystal preparation is referred to as Method A. Crystalline V, could be obtained by the following method which did not require previous isolation of V,: 10 mg of lyophilized Bence-Jones protein LEN were dissolved in 1 ml of 0.05 M glycine-HCl, pH 3.4, and the final pH was 1 Bence-Jones protein LEN has been classified immunochemitally RS a KII protein (10). The primary amino acid sequence of KII light, chain is characterized by an insertion of 5 to 6 residues between positions 29 and 30 (11). Therefore, while the molecular weight of the monomeric form of KI and KIII Bence-Jones proteins is approximately 22,500, the molecular weight of the monomeric form of KII Bence-Jones proteins is approximately 23,000. adjusted to 3.6 by addition of microliter quantities of 1 M HCl. Fifty microliters of pepsin (1 mg per ml) dissolved in the glycine-HCI buffer, pH 3.4, were sdded to the sample at 37", and after 18 min at 37" the pepsin was inactivated by adjusting the pH to neutrality with 1 M NaOH. The sample was transferred from the 37" water bath to a 5" refrigerator, and after 96 to 144 hours at this temperature crystals of Vr, were present. We refer to this procedure of crystal preparation as Method B. X-ray Diffraction-Crystals prepared by Methods A and B were mounted in a cold room (4") and sealed in thin walled quartz capillaries containing a droplet of mother liquor. The transference of a capillary onto a precession camera equipped with a gas stream cooling device which maintained the crystal at a temperature of 2 f 1" was performed wit'h minimal exposure to higher temperature.
Subsequently, it was found that a crystal exposed to x-rays for several days and then allowed to warm to room temperature (~25") gave a diffraction pattern not noticeably inferior to the previous pattern recorded at the lower temperature.
X-ray precession photographs were taken with nickel-filtered CuKar radiation.

Identification and Characterization of Variant
Half of Bence-Jones Protein LEN-The results of immunoelectrophoretic analyses of a preparation of Vn LEN were compared with that of a sample containing intact Bence-Jones protein LEN, the V,, and the CL (Fig. 1) The results from starch gel electrophoretic analyses and amino-terminal amino acid analyses were indicative of the homogeneity of the Vr, LEN preparation.
The amino-terminal amino acid residue of the Vr, LEN was determined by the "dansyl" technique (12). A single dansylated derivative was detected and identified as aspartyl; the amino-terminal residue of the intact Bence-Jones protein LEN was likewise identified as aspartyl.
The integrity of the Vn produced by pepsin cleavage was substantiated through the comparison of the peptide maps of the tryptic digests of the V,, CL, and Bence-Jones protein LEN.
Tryptic digestion of the Vr, and the CL produced 11 peptides from each half. The superimposition of the peptide maps of each half revealed that, with the exception of one peptide, the peptides of the Vr, were distinct from those of the CL (9). Moreover, the peptide map of the tryptic digest of Bence-Jones protein LEN was identical with the map obtained by superimposing the peptide maps of Vr, and C,,.

Properties of Variant
Half of Bence-Jones Protein LEN-Crystals of Vr, LEN with maximum dimensions of approximately 0.75 mm were obtained by Method A. The crystals were colorless in unpolarized light and appeared as shown in Fig. 11 of Reference 9; under polarized light they appeared to consist of alternately colored layers. Crystals of Vr, LEN with maximum dimensions of approximately 0.3 mm were obtained by Method B. The crystals were colorless prisms with varying face development.
One crystal examined with x-rays was found to be extended along the crystallographic c axis (see below).
Several crystals prepared by Method A appeared in precession photographs to exhibit twinning.
However, a crystal prepared by Method B gave normal diffraction patterns (Fig. 2). The crystal was monoclinic, and the unit cell dimensions obtained from 13" precession photographs were a = 65.6 f 0.1 A, b = 37.9 f 0.1 A, c = 43.5 f 0.1 A, p = 90.0 f 0.2". The standard deviations were estimated on the basis of the statistical error,? of measurement and of previous calibrations of precession film measurements by diffractometry.
The calculated unit cell volume was 108,200 A3. Since no systematic absences were observed, the space group was determined to be P2 ( Reference 13). Space groups Pm and P2/m, which are also characterized by a lack of systematic absences, could be excluded on the basis of the optical activity exhibited by the protein. The number of protein molecules per unit cell was 2n, where n, the number of molecules per asymmetric unit, was estimated by comparing the fractional volume of solvent implied by various values of n with the normal range of values 0.27 to 0.65 for crystals of globular proteins (14). The molecular weight used in these calculations was assumed to be within the range 11,500 f l,OOO.l For n = 1, the calculated fractional volume, 0.72 to 0.76, greatly exceeded the normal range. For n = 2, the fractional volume was 0.43 to 0.52; for n = 3, the fractional volume was 0.15 to 0.28. Although two of the 116 crystal forms analyzed by Matthews (14) had a value lower than or equal to 0.28, we elected to conclude that n = 2 because this value of n yielded a fractional volume which was in close agreement with the most frequently occurring range of 0.40 to 0.48. Therefore, on the basis of n = 2, we concluded that there were 2 molecules of ~11,500 molecular weight in the asymmetrical unit, and 4 molecules. related in pairs by 2-fold rotation axes in the unit cell.