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The digestion of a human IgG1 K myeloma protein with pepsin in the presence of 8M-urea was observed to produce a fragment, designated Fb′2, which differed from the products of aqueous peptic digestion and from other characteristic immunoglobulin digestion products. 2. Fragment Fb′s was also found when two other IgG1/K proteins were treated similarly. 3. Sedimentation-equilibrium studies showed the mol.wt. of fragment Fb′2 to be 56800. 4. On reduction, two equivalents of each of three peptides were released from fragment Fb′s; these were characterized by N- and C-terminal determinations and by amino acid sequencing. 5. Fragment Fb′2 was shown to consist of the constant regions of both light chains, from residue Ile-117 to the C-terminus, and the CH1 domains and hinge region of the heavy chains, from residue Val-113 to residue Met-252, with a gap of five residues within the intrachain disulphide loop, between residues Leu-174 and Tyr-180.

A wheat germ protein (Ec), the dominant site of cysteine incorporation during early (E) germination of isolated wheat embryos, has been partially sequenced by automated Edman degradation. The sequence of residues 1–59 is not significantly similar to the amino acid sequence known for any other protein, including wheat germ agglutinin which, like Ec, is very rich in half-cystine and glycine. The partial sequence for Ec contains an almost identical pattern of half-cystine residues in segments 6–20 and 35–48, a duplication which includes 10 of the 12 half-cystine residues in the sequence. The partial sequence of Ec may include a large part of the complete sequence, but this remains uncertain because it has not been possible to arrive at a definitve estimate of molecular weight using different physical techniques. Protein Ec can be prepared from a reticulocyte lysate in which cell-free synthesis is programmed by bulk wheat germ mRNA. Determination of the distribution of half-cystine moieties between residues 1 and 20 by Edman degradation of the [35S]cysteine-labeled product of cell-free synthesis shows that it is devoid of an N-terminal extension. Unlike wheat germ agglutinin, Ec does not seem to arise by processing of a conspicuously larger precursor protein. Unlike Ec, another wheat germ protein, Em, the most conspicuous methionine-labeled protein when cell-free protein synthesis is directed by wheat germ mRNA, is refractory to direct sequence analysis by Edman degradation. However, again unlike Ec, uncertainty about the molecular weight of Em, based on its mobility in different sodium dodecyl sulphate – polyacrylamide gel systems, has been resolved by virtue of Em being ideally suited to study by the Yphantis high-speed sedimentation equilibrium method.

Goblet-cell mucin (mucin 1) was isolated and purified from human small-intestinal scrapings. After application of mucin 1 to DEAE-Bio-Gel (A) columns, most of the glycoprotein (76–94% of hexoses) was eluted in the first peak (designated mucin 2). Minor amounts of acidic glycoproteins were eluted with 0.2m- and 0.4m-NaCl in later peaks. Analyses of mucin 1 and mucin 2 revealed mucin 2 to be a monodisperse highly glycosylated glycoprotein containing 6.3% by wt. of protein, N-acetylgalactosamine, N-acetylglucosamine, galactose and fucose. Mucin 1 was similar in composition, but was polydisperse and contained more protein (12.3% by wt.) as well as N-acetylneuraminic acid. Analytical CsCl-gradient ultracentrifugation showed both mucin 1 and mucin 2 to have a major component with an average buoyant density of 1.47000g/ml. Mucin 1 also contained a slightly less-dense minor glycoprotein component. After exhaustive reduction and alkylation mucin 1 retained its major component, but partly dissociated into two lighter glycoprotein components. Mucin 2, in contrast, did not change its density distribution after reduction. Band ultracentrifugation in 2H2O-containing iso-osmotic buffers showed that mucin 1 contained a major fast-sedimenting component (so=37±2S), and a minor amount of a slower-sedimenting component. After reduction there was an increased quantity of the latter component, for which an so value of 14.5S was calculated. In contrast, mucin 2 was unaltered by reduction (so=33±2S). These findings indicate that the major component of goblet-cell mucin (mucin 2) does not dissociate after S–S-bond reduction, and thus does not apparently rely for its polymeric structure on the association of subunits through covalent disulphide bonds. However, the effects of reduction on mucin 1 suggest that in the native mucin intramolecular disulphide bonds in the minor glycoproteins may stabilize their structure, permitting secondary non-covalent interactions to develop with the major dense mucin (mucin 2) protein.

Inactivation of C3 by enzymatic cleavage, nucleophilic addition, or slow freezing and thawing resulted in the acquisition of similar end-state conformations as judged by near-UV circular dichroism. Although inactivation by the two nonenzymatic processes involves no peptide bond scission, the inactivated C3 resembled C3b in that it possessed a free sulfhydryl group not present in the native protein and an increased surface hydrophobicity as evidenced by enhanced binding of the fluorophore 8-anilino-1-naphthalensulfonate (ANS). The C3b-like functional properties of modified C3 [Pangburn, M. K., & Muller-Eberhard, H. J. (1980) J. Exp. Med. 152, 1102-1114] may thus be understood in terms of the similarity of its conformation to that of C3b. The rate of the conformational change following proteolytic cleavage was fast and appeared to be limited by the rate of the enzymatic reaction. In contrast, the rate of conformational change following addition of methylamine was slow and rate limited by the conformational rearrangement itself, not by the chemical modification. A kinetic analysis of the changes in circular dichroism and ANS fluorescence enhancement suggested that the nucleophilic addition was spectroscopically undetectable and was followed by a minimally biphasic, spectroscopically demonstrable conformational rearrangement. The appearance of C3b-like functional activity in nucleophile-modified C3 largely parallels the time course of the spectroscopically detectable conformational change but is distinctly slower than the rate at which hemolytic activity is lost. While fully transconformed methylamine-inactivated C3 can bind factor B and is susceptible to cleavage by C3b inactivator and its cofactor beta 1H, this cleavage occurs at a substantially slower rate than the equivalent process in C3b. The implications of these findings in terms of the mechanism through which the alterative pathway of complement is initiated are discussed.

An iterative program, Gaushaus, has been adapted to analyze complex DNA reassociation kinetics. With valid experimental data, rapid convergence to a solution is always achieved by this program even when the starting parameters are grossly in error. The output of the program also includes useful statistical information.

A numerical method is described for the elimination of Rayleigh scattering from protein fluorescence emission spectra. The method is based upon the observation that Rayleigh scattering is symmetrical about a wavelength at or near the wavelength of excitation. It works best when an automated, computer-based approach can be applied to spectra collected on a data-logging device, although manual correction of chart-recorded spectra is also possible.

The fluorescent probe, 5-iodoacetamidofluorescein (5-IAF), was specifically attached to the COOH-terminal cysteine residue of the L chains of two monoclonal human IgKκ proteins. The induced circular dichroism and fluorescence properties of the bound 5-IAF probe were used to study changes in its microenvironment upon reassociation of autologous or heterologous L chains and their domains with Fd' fragments. Recombination of 5-IAF-L with Fd' at pH 5.4 was accompanied by a red-shifted visible difference spectrum, an increase in the magnitude of the induced optical activity and an enhancement of fluorescence. These results were compatible with the transfer of the 5-IAF moeity to a less polar and more asymmetric environment. Equilibrium and kinetic data obtained from difference spectroscopy and fluorescence studies indicated that 5-IAF-L bound with high affinity to Fd' in a 1:1 ratio with a second-order rate constant of 1618 M −1 sec −1 at 25°C. Using the same approach, it was shown that the labelled constant region fragment (5-IAF-C κ ) bound to Fd' with an association constant of 5 × 10 6 M −1 and a forward rate constant of 30 M −1 sec −1 . When 5-IAF-C κ was recombined with a preformed Fd'V κ complex, however, the intensity of the visible difference spectrum, the fluorescence emission, the binding affinity and the forward rate constant of the reaction, were significantly increased. In contrast, no spectroscopic changes were observed when V κ was recombined with a preformed Fd'-5-IAF-C κ complex. These data suggest that: (1) the high affinity interaction between Fd' and L results from the summation of relatively weak interactions between paired domains; (2) the binding of Vκ to Fd' modulates the reactivity of Cγl toward Cκ, probably through conformation changes transmitted via V H -Cγl contacts; and (3) conversely, once 5-IAF-Cκ is bound to Fd', the addition of the complementary Vκ domains does not induce any detectable change in the vicinity of the fluorescent probe.

Circular dichroism and ultraviolet absorption difference spectroscopy have been used to study the binding of a series of divalent and trivalent cations to porcine intestinal calcium-binding protein (CaBP). When calcium is bound to the single high-affinity site on CaBP, the aromatic optical activity is greatly increased. Analysis of the circular dichroic spectra, obtained in the presence and absence of calcium, suggested that although changes in the optical activity of the single tyrosyl residue accounted for much of the overall change observed upon binding calcium, one or more of the five phenylalanyl residues was also perturbed. All the cations tested, with the exception of lead which gave rise to unique spectral effects, caused the same changes in optical activity between 300 and 250 nm. In the peptide absorption region, CaBP exhibited optical activity typical of an α-helical protein and no significant changes were observed in the presence of any of the cations tested. Cation-binding curves obtained from the circular dichroic data for the cations bound with high affinity (i.e., calcium, strontium, and the trivalent lanthanide ions) showed that the apparent number of binding sites was inversely related to the protein concentration. This phenomenon was accounted for by the concentration-dependent aggregation of CaBP observed in earlier studies. The binding data, obtained using circular dichroism, clearly indicated that the affinity of CaBP for the various cations was related to their ionic radius. Absorption difference spectra were observed when calcium was bound to CaBP. The features of these spectra confirmed that phenylalanyl as well as tyrosyl transitions were perturbed upon calcium binding. The extent to which the tyrosyl side chain was exposed to solvent was determined by solvent perturbation difference spectroscopy using perturbing agents of differing molecular radius. The apparent degree of exposure increased as the perturbant size decreased suggesting that the side chain was located in a cleft. Bound calcium did not change the degree of exposure. These data, together with complementary data obtained with bovine CaBP, were discussed in terms of the geometry of the cation-binding site.

Spectral properties of pig intestinal Ca2+-binding protein (CaBP) and its apoprotein have been examined by fluorescence, absorption and c.d. Direct fluorescence from some of the five phenylalanine residues is observed and excitation spectra show that there is also energy transfer from some phenylalanine residues to the tyrosine. Absorption and c.d. spectra show that the tyrosine hydroxy group does not ionize significantly below pH 12. Tyrosine fluorescence is reversibly quenched by a lysine residue with a pK of 10.05 in the Ca2+ form. At low pH the tyrosine fluorescence is enhanced with transitions with pK values of approx. 4.2. The c.d. spectrum of the Ca2+ form shows a decrease of the ellipticity band at 276nm with a transition similar to that of the fluorescence titration. The apoprotein, however, shows an additional transition with a pK of about 6. The results are interpreted in terms of the recently published structure of the cow intestinal CaBP [Szebenyi, Obendorf & Moffat (1981) Nature (London) 294, 327-332]. The single tyrosine has a very high pK, although it apparently lies on the surface of the protein molecule.

A lambda light chain, isolated from an immunoglobulin G molecule, was found to reversibly precipitate at low temperatures. This cryoprecipitation was a function of pH, ionic strength, protein concentration, and time as well as temperature. The lambda chain underwent a cooperative conformational change as the temperature was lowered from 26 to 0 degrees C as judged by ultraviolet difference spectroscopy and circular dichroism. Normal lambda chains showed no conformational change. By difference spectroscopy it was possible to calculate the equilibrium constant governing the conformational change. The change was strongly exothermic (delta H approximately -80 kcal mol-1) and accompanied by a large decrease in entropy (delta S approximately -280 eu). The midpoint of the transition was dependent on the initial protein concentration, suggesting that only the noncovalent dimer of the lambda chain exhibited the conformational change. The existence of a monomer-dimer eqiulibrium (KA approximately 4 X 10(5) M-1) was confirmed by sedimentation velocity. No conformational change was observed by circular dichroism at concentrations where greater than 95% of lambda chain was in the form of a monomer. Although high ionic strength inhibited cryoprecipitation, it had no effect on the conformational change. Stabilization of the dimer by forming an interchain disulfide bond between two monomers abolished both the conformational change and cryoprecipitation. A fragment corresponding to the constant region was isolated from both peptic and tryptic digests of the lambda chain. This fragment neither cryoprecipitated nor showed temperature dependence conformational changes. It proved impossible to isolate a fragment corresponding to the variable region. Both qualitative and quantitative models are presented to account for the behavior of the lambda chain at low temperatures.

Previous studies have shown that the forward rate constants governing the non-covalent association of heavy and light chains of different IgG myeloma proteins vary considerably. In the present study rate constants, absorption difference spectra enthalpies associated with recombination have been determined using kappa chain of known variable-region subgroup specificity. The basic experimental approach was to allow a single species by γ chain to react reparately with several kappa chains belonging either to the κIII subgroup. The rate constant varied by two orders of magnitude but there was no evidence that this variability was related to subgroup specificity. There was as much variation within a subgroup as between subgroups. A correlation was noted, however, between the rate constants and the species of heavy chain used in the recombination reactions. The shape and magnitude of the difference spectra generated upon subunit association were also variable but again this could not be correlated with subgroup specificity. Variations in enthalpies of association were less striking but there were some indications that values for δH depended on the heavy chain used. Only for one of the heavy chains used was there evidence to indicate that the preferential recombination of autologous subunits observed in equilibrium systems arises because autologous subunits associate more rapidly than heterologous chains. The data presented indicate that sequence variations in either the hypervariable regions, or the framework within a subgroup, play an important role in subunit association.

A procedure is outlined by which sedimentation analyses of small quantities of mucin glycoproteins can be performed. Rat intestinal goblet cell mucin was stained with periodic acid-Schiff reagent to permit detection by light absorption at 555 nm. PAS treatment resulted in chemical modification of sialic acid and 55% of fucose residues in the mucin. No other chemical or physical alterations were detected. The stained mucus was subjected to band ultracentrifugation using D2O-containing solvents. Sedimentation was monitored by scanning at 555 nm. Results compared favorably with those reported earlier for conventional boundary ultracentrifugation of intact goblet cell mucin. Because of the low concentrations of mucin used in band ultracentrifugation (0.2–1.5 μg protein/ml), S20,w values are comparable to sedimentation coefficients at zero concentration (So values), determined by conventional means.

Single component guinea pig insulin was found by sedimentation equilibrium centrifugation to exist in neutral solution as a monomeric species only, even in the presence of zinc ion. This insulin, in contrast to bovine insulin, did not bind zinc ion at pH 7.4. The biological activity of guinea pig insulin, estimated by mouse convulsion assay, was 2.14 I. U. per mg.