Anil Saha

Mechanism of cyro-precipatation—I characteristics of a human cyroglobulin

Abstract Studies were undertaken to evaluate the physico-chemical and immunochemical characteristics of a particular human cryoprotein obtained from the serum of a patient suffering from idiopathic cryoglobulinemia. Physico-chemical characterization studies showed that this particular protein represents a single species of protein molecules and belongs to human immunoglobulin G class with lambda type light polypeptiode chain and γ1 type heavy chain specificity. The solubility of the cryoprotein depends on temperature, pH and the concentration of ionic and non-ionic solutes. Although the number of basic amino acid residues remained the same as the patients own IgG, the cryoprotein has relatively larger proportion of hydrophobic amino acid residues and lesser number of acidic amino acid residues. Studies on the hydrodynamic properties at pH 5·0 suggest that the cryoprotein has a shape more spherical than that of normal human IgG. Studies on the transition from ‘gel’ to ‘sol’ state under certain conditions of temperature, pH, and ionic and non-ionic solutes indicate that the phenomenon of cryoprecipitation as observed here reflects an inadequacy in water: protein interaction.

Partial characterization of nonhistone nuclear proteins which are decreased in hepatomas of the rat.

1. 1. Nuclear proteins which are soluble in 0.35 M NaCl were studied in Morris hepatomas and livers of tumor bearing rats. 2. 2. Isoelectric focusing revealed an increase in hepatomas of proteins having isoelectric points below pH 6 and a decrease in two proteins with isoelectric points of pH 8.2 and 8.5. 3. 3. The proteins which are decreased in hepatomas were studied with respect to [32P]phosphate incorporation, apparent molecular weights and amino acid composition. 4. 4. The amino acid compositions of these proteins distinguish them from histones and high mobility group proteins.

Studies on the urinary proteins of an IgG myeloma patient—I characteristics of urinary IgG

Abstract Studies on the protein components excreted in the urine of an IgG (G2:λ) myeloma patient (Ga) revealed the presence of both the L and H chain components. The urinary protein components were resolved by column chromatography on DEAE-cellulose followed by gel filtration on Sephadex G-75. The minor urinary protein components were λ type Bence Jones protein and Fc fragment. The major urinary protein component possessed the antigenic determinant sites of λ type Bence Jones protein and G2 type H chain involving both the Fd and Fc fragments. This urinary protein component designated as urinary IgG showed an apparent molecular weight of 117,000 and a carbohydrate content of 9·54 per cent. Ga myeloma IgG had an apparent molecular weight of 150,000 and a carbohydrate content of 3·2 per cent. The ratio of mannose+galactose : fucose : glucosamine : galactosamine : sialic acid residues was 12:3:8:0:2 for Ga myeloma IgG and 44:10:5:1:2 for urinary IgG. Fluorescence emission maxima of Ga myeloma IgG and urinary IgG occurred at 335 mμ and 330 mμ, respectively. Transition pH as determined by pH-dependent fluorescence characteristics was pH 11·6 for myeloma IgG and pH 11·46 for urinary IgG. Spectrophotometric studies on tyrosine ionization showed that the number of tyrosyl groups located in the restricted environment was 36 out of 58 residues for myeloma IgG and 22 out of 42 residues for urinary IgG. Apparent molecular weight of the reduced-alkyklated H chain was 48,000 for myeloma IgG and 36,000 for urinary IgG. Papain digestion of urinary IgG produced two protein components separated by DEAE-cellulose column chromatrography and dialyzable peptides. One of the components of apparent molecular weight 41,000 possessed antigenic determinats of Fc fragment, while the other protein component those of L chain and Fc fragment but without the Fd determinant sites.

Studies on the depolymerization of the heme-proteins. I. Dissociation of chick hemoglobin 1.

Abstract The dissociation of an avian hemoglobin, chick oxyhemoglobin 1, was studied in the presence of ionic type (LiCl, NaCl, KCl and CaCl 2 ) and non-ionic type solutes (formamide, urea, guanidine hydrochloride and 2-chloroethanol), and in high alkaline pH conditions. An increase in the solute concentration and alkalinity of media produced increasing dissociation as measured by sedimentation velocity coefficient values. Sedimentation equilibrium studies employing an ultraviolet monochromator and automatic double-beam photoelectric scanner revealed that an equilibrium state exists between the different subunits, tetramer, dimer, and monomer, depending on the condition of the media. Alkaline dissociation of chick hemoglobin to half-molecule began at pH 10.6 and was completed at pH 11.6. The degree of alkaline dissociation implies that chick hemoglobin is more resistant than the mammalian hemoglobins, adult human and bovine hemoglobins. Localized conformational change as evaluated by the solvent perturbation difference spectra was observed with CaCl 2 , and all non-ionic solutes. Spectrophotometric titration of tyrosine ionization showed that of the 17 tyrosine residues evaluated by amino acid analysis of chick hemoglobin 1, 15 tyrosine residues were ionized at pH 12.5–pH 12.75 and 8 tyrosine residues became available between pH 10.3 to pH 11.3 where tetrameric chick hemoglobin dissociated to half-molecule. Urea was more effective in reducing heme-heme interaction and producing hyperbolar oxygen equilibrium curves than NaCl. The transition from sigmoidic to hyperbolar oxygen equilibrium curve is dependent on the solute concentration.

Studies on cryoprecipitation: covalent structure of a human IgG cryoglobulin and its gelation characteristics.

Abstract Cleavage of a human IgG cryoglobulin (6·5 S, γ 1:λ, Gm factor 4) into its constituent L and H chains could only be accomplished in the presence of mercaptoethanol. Dissociation of Dv cryoglobulin could not be affected by structure-diruptive solutes like urea (8 M ) and guanidineHCl (6 M ). The weight-average apparent mol. wt as determined by sedimentation equilibrium analysis were 50,500 ± 1000 and 26,000 ± 1000 for H and L chains of Dv cryoglobulin, respectively. Sedimentation velocity analysis showed the self-association of the H chains, depending on the solvent environment. The reduced-aminoethylated H chains of Dv cryoglobulin exhibited visible cryo-gel formation and under identical experimental conditions, the L chains remained in solution. These studies indicate that the sites responsible for self-association leading to cryo-gel formation are located on the H chains which show a higher degree of resiliency after being with strong protein denaturants.

Conformational stability of a human cryoglobulin.

Studies on a single component human cryoimmunoglobulin (cryo-IgG) (gamma 1 : lambda, Gm 4) were undertaken to gain a better understanding of the conformational stability of macromolecular interfaces essential for self-association of cryo-IgG leading to the formation of visible gel mass. Changes in the gross and localized conformation of cryo-IgG and a monoclonal IgG (gamma 1 : lambda, Gm 4) isolated from a myeloma patient (Hy) (Hy IgG) (gamma 1 : lambda, Gm 4) in alkaline media were determined by analytical ultracentrifugation, fluorescence characteristics, tyrosine ionization and H+ titration. Ultracentrifugal studies revealed that major transition in gross conformation took place at pH 11.4 for cryo-IgG and pH 11.7 for Hy IgG, whereby the number of charges and tyrosine residues exposed to aqueous environment was 110 and 26 for cryo-IgG, and 111 and 48 for Hy IgG, respectively. Beyond this transition pH fragmentation of both the proteins occurred and cryo-IgG lost its capacity for gel formation. Self-association of cryo-IgG was observed upto pH 11.4 in decreasing order with increase in denaturation pH. Cryo-IgG renatured from exposure to higher alkaline pH upto pH 11.4, showed the capability for forming gel, in spite of the irreversible local conformational changes as established by direct and reverse fluorimetric titration and tyrosine ionization studies. Cryo-IgG could be maintained in the optically clear sol phase at pH 10.5, at which pH 12 out of 62 tyrosine residues became exposed to aqueous media. There are distinct differences in the accessibility of tyrosine residues of cryo-IgG and Hy IgG as reflected in their tyrosine ionization profiles.

Conformational stability of antigenic determinant sites of bovine liver catalase

Abstract Macromolecular conformations which present various active sites associated with specific biological properties of BLC ∗ were affected under the experimental conditions of solvent perturbation. Ab-comb capacity, enzymic activity and monomer-association were destroyed when BLC was subjected to 7 M formamide, 5 M urea and pH 11·0. BLC, renatured from 4 M NaCl, retained 75 per cent ab-comb capacity, 57 per cent enzymic activity and exhibited tetramer-dimer equilibrium (9·0 S). Physico-chemical parameters evaluated provided indices of conformational changes. The fluorerescence emission maxima of BLC showed a red shift from 325 nm to 345 nm in 9 M formamide, 348 nm in 8 M urea, 350 nm in 6 M guadinine-HCI, 336 nm in 1·0 M CaCl 2 , 335 nm at pH 11·0 and at 56·5°C, indicating thereby a change in the microenvironment of trp residues from hydrophobic to polar milieu. Chemical modification of BLC with tetranitromethane and trinitrobenzenesulfonic acid affected biological activities. With 9 tyr residues nitrated per BLC monomer, Ab-comb capacity and enzymic activity was 73 and 82 per cent, respectvely. A similar effect was observed with 5 lys residues trinitrophenylated per monomer, showing 91 per cent Ab-comb capacity and 73 per cent enzymic activity. Capacity of modified monomers for self-associationto form tetramers was lost when 10 tyr residues or 6 lys residues were modified. Monomers produced by chemical modification or thermal denaturation and isolated by SDG ultracentrifugation, did not show biological activities. Chemical modification produced increasing number of tyr residues available for ionization as compared to native BLC, at an identical pH.

Influence of heat on the conformational stability of a human IgG cryoglobulin.

Temperature-induced conformational changes of a human immunoglobulin G cryoglobulin (cryoIgG) (IgG) (gamma 1:lambda, Gm4) was investigated and compared with a human myeloma IgG (gamma 1:lambda, Gm4) employing spectrofluorimetric and immunochemical methods. Fluorescence measurements revealed the major changes in protein conformation of both proteins at a temperature of 62 degrees C and above, the measurements being carried out with excitation wavelengths at 278 and 295 nm, respectively. Studies on both cryoIgG and myeloma IgG, which were heat denatured at high temperatures and subsequently cooled at 25 degress C, indicated that both proteins underwent progressively irreversible conformational changes beyond 65 degress C and cryoIgG appeared to be more temperature sensitive than myeloma IgG. Evaluation of the changes on specific antigenic determinant sites using antigen-antibody interaction revealed that the Fc determinant sites of both the proteins were disorganized to a greater extent at a temperture of 68 degress C than Fd or lambda-chain antigenic determinant sites. The Fd determinant sites of myeloma IgG were, however, found to be more heat labile than those of cryoIgG.