Caroline W. Easley

The heat precipitation of Bence-Jones proteins. I. Optimum conditions.

Abstract The heat precipitation of Bence-Jones proteins has been studied with respect to the type of protein, protein concentration, temperature, and electrolyte concentration and composition. A set of conditions for heating was established which resulted in the heavy precipitation of a wide variety of Bence-Jones proteins but not of normal serum proteins. On this basis, a simplified heat test for the detection of Bence-Jones protein in urine is proposed.

Application of specialized techniques for specific staining of peptide maps on various media

Combinations of specific color reactions useful in peptide mapping on W h a t m a n filter papers have been described in a previous communicat ion 1. This paper introduces a new combinat ion of specific reactions for staining peptide maps on W h a t m a n No. 3MM paper 2, and to a limited extent, on thin-layer plates. Cellulose plates used were E. Merck thin-layer chromatographic cellulose-precoated plates. Silica gel plates were prepared ~ from Merck silica gel G and Koch-Light amylopectin. Staining ofpeptide maps on paper. An optimal combination of several staining reactions on one peptide map is desirable, in order to eliminate variations seen in duplicates and to conserve time and materials. The new staining sequence described here combines 5 reactions on a single 3MM paper. Best results m a y be obtained on a peptide map prepared from an aliquot of digest representing at least o . I / ,mole original protein. A distinct advantage of this staining sequence is the combination of a highly sensitive test for arginine with the Pauly reaction for histidine and tyrosine; the Sakaguchi reagent for arginine and the Pauly reagent are incompatible on the same paper 1. A disadvantage is tha t the tyrosine reaction is weak as applied here ; occasionally it does not work at all. These reactions are performed in the order in which they are described. To avoid confusion, the spots are marked after each reaction, and the results incorporated on a composite tracing. I. The platinic iodide reaction for reducing sulfur is performed as described previously 1. The paper must be thoroughly dried after electrophoresis, since remaining traces of the solvents used, as well as pyridine or collidine in the atmosphere, seem to interfere with this reaction.

Site of cleavage of γ-globulins by papain

Abstract Limited cleavage of γ-globulins of several species by a variety of proteolytic enzymes yields 3.5-S fragments apparently representing adjoining structural moieties of the parent molecules. The locus, specificity, and stoichiometry of the peptide bonds involved have been studied by N-terminal group assay. Sulfhydryl-activated plant proteases such as papain, bromelain, ficin, and chymopapain all cleave similar bonds in human γ-globulin and form similar 3.5-S fragments. The γ-globulins of different species vary in susceptibility to papain; rabbit γ-globulin and pathological human proteins are most readily cleaved. Ficin and papain yield indistinguishable fragments from rabbit γ-globulin, namely Fractions I and II, and Fraction III. The latter can be resolved into three subfractions, IIIA, B, and C. In general, the sensitive bond involves a branched-chain amino acid, usually leucine. However, a transient threonyl N-terminal group is liberated in early stages of the cleavage of normal γ-globulin later being replaced by leucine. Both peptide-bond rupture and disulfide-bond reduction are requisite for the formation of 3.5-S fragments, but the cleavage is apparently initiated by the proteolysis which exposes a critical disulfide bond. However, no structure common to the γ-globulins of the various species can yet be formulated because they differ in the nature and apparent number of N-terminal groups and do not yield the same number or types of 3.5-S fragments on cleavage by papain.

SICKLING PHENOMENA OF DEER

In an apparently random survey of the blood from several species of zoo animals, in 1840, Gulliver noted on microscopic examination that the erythrocytes of various deer would assume a number of bizarre shapes. Sharp angulations and spicules projecting in various directions were the principle features of these cells. It is likely that the observations was entirely fortuitious, based as it was on a propensity for the cells containing oxyhemoglobin to sickle so readily that it is difficult to observe them in room air without their becoming sickled. Complete deoxygenation of blood samples abolished sickling, but at an elevated pH sickling occurs at relatively low oxygen tensions. This relationship is clearly demonstrated in FIGURE 1, in which is depicted the relationship of oxygen saturation to sickling in an environment where pH was fixed at 7.8 by an isotonic sodium phosphate buffer containing potassium at a physiological concentration. At this pH a PO, of only 25-35 mm Hg is required to produce the degree of oxygen saturation at which sickling becomes pronounced. Intermittent observations on the sickle cells of a limited number of various wild-killed or zoo deer were reported over the years. Most notable of these were the studies of Undritz * who reported that the sickling of deer erythrocytes is reversible and related to changes in pH. However, information regarding the sickling phenomena of deer erythrocytes remained scanty. In 1960, with the assistance of the Florida Game and Fresh Water Fish Commission, we established an experimental herd of White-tailed deer (Odocoileus virginianus), which has been under continuous investigation at the University of Florida since that time. The objective was to study various physiological parameters, genetic transmission of the hemoglobin chains, structural characteristics of the hemoglobins, the determinants of the morphological changes of the erythrocytes and possible methods for modification of these alteration^.^^ No pathological effects from the sickling have been noted in the deer, but many other features of the deer hemoglobins have turned out to be as unusual as are their erythrocyte morphologies. These include the presence of duplicate a-chain genes, multiple @-chain variants, marked structural differerences between allelic @ chains rather than single point mutations, and the association of specific @chain types with distinct morphological aberrations and patterns of hemoglobin polymerization. This presentation will review the structural and genetic features that produce the remarkable hemoglobin heterogeneity of the White-tailed deer, and will report new information on the structural differences between the @-chain types, particularly those that are associated with the characteristic morphological changes.

Multiple hemoglobin α-chains in the sika deer (Cervus nippon)

Investigation of the hemoglobin α-chains of an Asiatic deer, the sika (Cervus nippon), was prompted by the heterogenity of α-chain gene loci in the Virginia white-tailed deer (Odocoileus virginianus). Although electrophoresis of hemoglobin chains from 10 sika revealed only a single α-chain band, peptide mapping demonstrated variations in the α-TPIII∗ and α-TPIV peptides. Substitutions at positions 15, 20, and 22 produce a minimum of five α-chains; two possible additional chains could not be proven because of inseparability of the whole α-chains. The most common chain contains Asp-15, Lys-20 and Pro-22 but in other chains glycine is present at position 15, Asx at position 20, and either serine or Asx at position 22. The probable explanation for the large number of α-chains is gene duplication which may have been produced by breedings between subspecies from different geographical areas. Comparison with the α-chain structure of the white-tailed deer suggests that the sika may have evolved from the lineage which produced the white-tailed deer after the α-chain genes of the latter species had duplicated. In addition, these data provide further examples of the unusual variability of this portion of the α-chain.

A comparative study of enzymatic digestion profiles of apolipoprotein B from four human subjects.

A methodological approach for comparative structural study of apolipoprotein B has been developed. Low-density lipoproteins from four human subjects were digested in three separate enzyme systems, utilizing trypsin, chymotrypsin and Staphylococcus aureus protease V8, each in the presence of 1% sodium dodecyl sulfate. The peptides were separated by electrophoresis on polyacrylamide gels in SDS; the stained gels were scanned spectrophotometrically to produce characteristic profiles. Comparison of the profiles revealed good reproducibility and a high degree of similarity among the different subjects. Of the four subjects studied, one subject had one apparent difference in the tryptic digest profile and also in the S. aureus protease V8 digest profile. The structural significance of these variations can be evaluated only after a larger number of subjects, including those presented here, have been examined; this study is now in preparation.

Studies on Fd-fragments of human immunoglobulins: I. A comparative peptide mapping study on l-chains, Fab- and Fd-fragments of G-myeloma proteins

Abstract The Fab-fragments and corresponding L-chains of four G(1)-Gm(f+) myeloma proteins were studied by comparative peptide mapping. Of the 15–20 peptides found to be derived from the Fd-fragment, 7 were considered to be common in regard to position and staining to the Fd-fragments of the myeloma globulins studied. The total number of Fd-peptides found in the composites was too low, as judged from the amino acid analysis and from sequence data from the literature. This was due to an overlap of tryptic peptides from Fd-fragments and L-chains. The Fd-fragment of one monoclonal protein could be isolated and studied by peptide mapping. The number of Fd-peptides thus found (25–28) was in good agreement with the expected number from the amino acid analysis. The homologous peptides of Fd and corresponding L-chains could be traced in the peptide maps. In connection with this aspect the relevance of finding “common” peptides is discussed. The Gm(f+) peptide (free arginine) could be localized in the Fd-maps.