Frederick Aladjem

Subunit structure of the apoprotein of human serum low density lipoproteins

Abstract Human serum low density lipoproteins (d 1.027–1.043 g/cm 3 ) were prepared by preparative ultracentrifugation and delipidated with sodium deoxycholate. By electrophoresis in sodium dodecyl sulfate polyacrylamide gel, the apoprotein was fractionated into major components with apparent molecular weights of 77,000, 66,000, 47,000, 33,500, 21,500, 13,000, and 9,500, respectively; and minor components of higher molecular weight. The data indicate the existence of at least two fundamental subunits of molecular weights of approximately 9,500 and 13,000 daltons.

Isoelectric heterogeneity of the major polypeptide of human serum high density lipoproteins

Abstract The major polypeptide of human serum high density lipoproteins was subfractionated by isoelectric focusing in narrow pH gradient. Five isoelectrically distinct subfractions were isolated with isoelectric points of 5.62, 5.54, 5.46, 5.39, and 5.32, respectively. The relative protein distribution of these five fractions was 12:32:34:17:5. These isoelectric distinct subfractions had identical amino acid compositions.

A method for the determination of heterogeneity of antibodies.

Abstract The average number of univalent homogeneous hapten molecules bound per n-valent antibody molecule, ν ( H ) , is given by the expression ν ( H ) = ∫, 0 ∞ ( kH ) ( i + kH ) −1 n P(k)dk, where H is the concentration of free hapten, and P(k)dk is the probability that reaction between hapten and a particular antibody site will have equilibrium constant between k and k + dk. The integral equation is solved to give P(k) in terms of ν ( H ) ; no assumption about the form of the probability density function is required. Since ν ( H ) is experimentally measurable, the probability density function which describes the heterogeneity of antibodies can be computed directly from experimental data. Experimental considerations pertinent to the use of the solution are discussed. Binding measurements are required over a wide range of free hapten concentrations. The method of analysis should be useful not only for the study of hapten-antibody reactions but also, whenever the above relation is valid, for the analysis of heterogeneity of binding in other systems such as ion or dye binding by proteins or nucleic acids, hormone-protein binding, and binding of small molecules to cells.

Immunochemical heterogeneity of human high densiy serum lipoproteins

Abstract High density lipoproteins, HDL2 (d. 1·065–1.124 g/ml) and HDL3 (d. 1·25–1·210 g/ml) were isolated from pooled human serum by preparative ultracentrifugation. The major peptides were isolated from the apoproteins by DEAE-cellulose column chromatography in 8M urea, and characterized by analytical ultracentrifugation, acrylamide gel electrophoresis, amino acid analysis, and by UV absorbance. Antisera were prepared against native lipoproteins, apoproteins and peptides. The reactions between native HDL, apo-HDL and the major peptides with the various antisera were studied by qualitative precipitin analysis, immunodiffusion and immunoelectrophoresis. The results lead to the following conclusions: (1) The antigenic heterogeneity of HDL is due to the presence, within HDL, of subunits of varying peptide composition. (2) The three peptides R-X, R-gln, and R-thr of HDL2 are immunochemically identical to the three major peptides of HDL3. These three peptides are each immunochemically distinct and do not cross-react with each other. (3) Apo-HDL contains some apoprotein subunits which consists of R-gln without R-thr, some which consists of R-thr without R-gln, and some which contain both R-gln and R-thr. (4) Native HDL contains some molecules which are composed only of R-thr. (5) The peptides R-gln and T-thr are predominantly on the surface of the native HDL2 and HDL3, whereas the peptides R-X is not on the surface. (6) The αLpB antigen of HDL3 contains only one of the three major peptides, R-thr. It is suggested that αLpB is generated from HDL2 molecules rich in R-thr.

The antigen-antibody reaction: V. A quantitative theory of antigen-antibody reactions which allows for heterogeneity of antibodies☆

Abstract A quantitative theory to describe the reactions between multivalent antigen and bivalent antibody is developed. Unlike the previous theory (Palmiter & Aladjem, 1963) the present theory allows for heterogeneity of antibodies. Heterogeneity is described in terms of a multivariate probability density function (PDF). A method for finding the PDF from experimental measurements of the amount of antigen-antibody precipitate and of soluble complexes is given; briefly, the method consists of (a) making an initial estimate of the PDF and computing by means of the theory the distribution of complexes, (b) comparing the computed distribution of complexes with the experimental data, and (c) using the criterion of least squares and iterative procedures for consecutively better approximations, finding that distribution of complexes and hence that PDF which best fits the experimental data. The theory suggests experiments and a new method of interpretation of the results.

The antigen-antibody reaction. IV. A quantitative theory of antigen-antibody reactions

Abstract A theory is developed which describes the distribution of antigen-antibody complexes resulting from the reactions between bivalent antibody and f -valent antigen. It is assumed, as in Goldbergs theory (1952) , that antigen and antibody are immunochemically homogeneous and that no intra-aggregate reactions occur which yield cyclical complexes. Unlike Goldbergs theory, in which all antigen-antibody bonds are assumed to be equivalent, the present treatment allows independent rate or equilibrium constants for the reactions between each of the f -antigen sites with each of the two antibody sites, i.e. it is developed in terms of 2 f equilibrium constants or 4 f rate constants. In outline, the procedure involves the calculation of the number of antibody molecules and antigen molecules with any particular number of reacted sites, and the determination of the most probable distribution of complexes by means of a mathematical procedure similar to that used by Stockmayer (1943) . As a special case, if the existence of one intrinsic equilibrium constant is assumed, Goldbergs expression for the most probable distribution of complexes is obtained from the expressions derived here. Subject to the stated assumptions, the theory provides a numerical procedure for the computation of equilibrium and rate constants of antigen-antibody reactions, and should be useful for the calculation of the behaviour of model systems.

The heterogeneity of antibodies with respect to equilibrium constants: Calculation by a new method using delta functions, and analysis of the results☆

Abstract A method of data analysis has been developed which approximates the probability density of association constants of the reaction of hapten with antibody by a minimum number of delta functions, and alternatively, by a large number of delta functions. The method has been computerized and found able to approximate any physically possible affinity distribution. We have calculated the delta function distributions which describe known distributions of association constants and analyzed experimental data,. From 2 to 4 delta funticons we3re required to describe the probability density of Sips distributions. Not more than three delta functions were needed to describe the probability density functions of each of three antibody populations obtained from pooled antisera. These delta function distributions were found in one case to fit the data significantly more accurately than a Sips distribution. In the other two cases the fit was about the same as the Sips distribution. Affinity heterogeneity analysis using a large number of delta functions gave roughly equivalent results in terms of closeness of fit to the data to the results using a minimum number of delta functions. It is shown that binding measurements do not contain sufficient information to prove homogeneity of antibody. An analysis of factors affecting the resolution of delta functions is given.

Further studies on the subunit structure of human serum low density lipoproteins

Abstract The apoprotein moiety of human serum low density lipoprotein was separated by SDS-gel electrophoresis into several components ranging in molecular weight between approximately 80,000 and 10,000 daltons. Amino acid analyses and N-terminal determination were done on each fraction. Every fraction was found to have essentially identical amino acid composition. We interpret the data to indicate that apoLDL is an aggregate composed of a small number of fundamental subunits of similar amino acid composition.

Isoelectric heterogeneity of human serum high-density of lipoproteins

Abstract Human serum high-density lipoproteins isolated in the density range 1.063–1.125 gm/ml (HDL2) has normally been considered to be homogeneous, though immunochemical heterogeneity of HDL2 was indicated by Aladjem et al. (1) on the basis of simple immunochemical analysis. Immunochemical heterogeneity has also been reported by Scanu (2) and Levy and Frederickson (3). Heterogeneity of HDL has recently been confirmed by two different approaches. We have observed that subpopulations of HDL2 exist which contain both the polypeptides R-Thr and R-Gln (classified by their C-terminal amino acid 2 and others which contain only R-Thr (4, 5). Kostner et al. (6) have observed that upon analytical isoelectric focusing of HDL2 four distinct bands occur. In the present report we show that HDL2 is composed of at least seven different isoelectric subpopulations and that each differs with respect to polypeptide composition.

Allergy to chymotrypsin

HIS report presents a case of allergic sensitivity to chymotrypsin (bovine) in a laboratory worker. The sensitivity apparently was induced by inhalation of the powdered material. With the increasing use of purified enzyme preparations, the hazard of sensitization should be recognized. A larger number of scientific workers are being exposed to inhalation of the dried materials. Injectible chymotrypsin is being used to reduce tissue swelling following trauma and infection.’ The other pancreatic proteolytic enzyme, trypsin, is used medically, both by injection in inflammatory disorders”, 3 and in the form of an aerosol to liquefy mucus in cases of asthma, bronchitis, and bronchiectasis.4, 5 Chymotrypsin is produced by the action of trypsin on chymotrypsinogen. It acts on the peptide link in the carboxylic side of aromatic amino acids. Because of the proteolytic action, the enzymes have been used to dissolve mucus or fibrin. During tests for specific skin sensitivity, the proteolytic activity of trypsin and chymotrypsin, with its resulting irritative wheal and tla,re, introduces the factor of nonspecific reactions, and this must be evaluated in each individual test. To reduce this source of error, we used the nonproteolytic precursor of chymotrypsin (chymotrypsinogen) in the passive transfer tests reported below. Chymotrypsinogen and chymotrypsin have been reported to be distinguishable by means of the Schultz-Dale reaction in the guinea pig