Arun K. Attri

pH-dependent self-association of zinc-free Insulin characterized by concentration-gradient static light scattering

Insulin self-association at pH 1.85, 1.95, 3.0, 7.2, 8.0 and 10 was studied via composition gradient light scattering (CG-SLS). At pH 1.95 in acetic acid, insulin was found to exist as a monomer, and in pH 1.85 HCl as a dimer. At pH values of 3.0-8.0, the dependence of scattering intensity upon total insulin concentration at concentrations of up to 1.5mg/mL may be quantitatively accounted for by a simple isodesmic association equilibrium scheme requiring only a single association constant for addition of monomer to monomer or any oligomer. At pH 10, the association constant for addition of monomer to monomer was found to be smaller than the association constant for addition of monomer to all higher oligomers by a factor of approximately five. The isodesmic association scheme was also found to quantitatively account for the concentration dependence of the weight-average molecular weight derived from previously published sedimentation equilibrium measurements made at pH 7.0, and the best-fit value of the stepwise equilibrium constant obtained therefrom was in excellent agreement with that obtained from analysis of the light scattering data obtained at pH 7.2.

An automated method for determination of the molecular weight of macromolecules via sedimentation equilibrium in a preparative ultracentrifuge

An automated method for quantitating the concentration gradient of a macromolecule brought to sedimentation equilibrium in a preparative ultracentrifuge is described. Between 30 and 80 microliter of macromolecular solution are spun in a combination centrifuge tube and optical cell fabricated from quartz. Immediately following the conclusion of centrifugation the tube is optically scanned along its length using a spectrophotometer sample cell modified for this purpose. Successive scans of the same tube containing first solution and then solvent permit the differential absorbance due to macromolecule to be measured precisely as a function of position within the centrifuge tube, and hence the radial position within the rotor. Molecular weights calculated from data so obtained from 32 centrifugations of eight proteins ranging in Mr from 12K to 340K using either swinging-bucket or fixed-angle rotors agree with previously published values with a standard deviation of 5%.

Technique and apparatus for automated fractionation of the contents of small centrifuge tubes: Application to analytical ultracentrifugation

An automated method is described for dividing the contents of small cylindrical centrifuge tubes into fractions deriving from laminae of solution as thin as 0.1 mm in the direction of the cylindrical axis. Experimental data are presented to demonstrate that fractions as small as 1 microliter may be prepared with a standard deviation of less than 3% in volume delivery and that negligible mixing occurs between the contents of adjacent fractions during the fractionation procedure. The method has been used to quantitate the gradients of a variety of radiolabeled proteins formed in sedimentation velocity and sedimentation equilibrium experiments. Sedimentation coefficients and molecular weights calculated from the gradients obtained agree well with literature values and with values obtained by optically scanning the centrifuge tubes (A.K. Attri and A.P. Minton, 1983, Anal. Biochem. 133, 142-152; 1984, Anal. Biochem. 136, 407-415). The present technique combines a spatial resolution equal to that of optical methods of gradient measurement with a sensitivity which may be several orders of magnitude greater, depending upon the specific activity of labeled solute.

Self-association of Zn-insulin at neutral pH: Investigation by concentration gradient-static and dynamic light scattering

Equilibrium self-association of Zn-insulin at pH 7.0 was characterized over the range 0.3-5mg/mL by simultaneous measurement of static and dynamic light scattering. Analysis of static light scattering yielded a concentration-dependent weight-average molecular weight, and analysis of dynamic light scattering yielded a concentration-dependent intensity-average diffusion coefficient. The concentration dependence of both quantities may be accounted for to within experimental precision by a simple model, according to which the basic structural unit of Zn-insulin at concentrations exceeding 0.3mg/mL is a hexamer H. With increasing total protein concentration, hexameric protomers may self-associate in accordance with an isodesmic scheme in which a protomer may add to any prexisting oligomer H(n) to form H(n+1) with an invariant stepwise equilibrium association constant.

Simultaneous determination of the individual concentration gradients of two solute species in a centrifuged mixture: application to analytical ultracentrifugation

A procedure for determining the absolute activity of 14C-labeled and 3H-labeled solutes in a mixture from the measured counts per minute in two scintillation energy windows is described. It is shown that the method described here provides a substantially more accurate determination of 3H activity in the presence of a larger 14C activity, and a more accurate determination of 14C activity in the presence of a larger 3H activity, than does the standard dual label analysis implemented in a Beckman LS 3801 scintillation counter. The new dual label procedure is combined with the automated fractionation procedure of Attri and Minton [(1986) Anal. Biochem. 152, 319-328] to permit the gradients of each of two differently radiolabeled solute species in a mixture to be individually determined following centrifugation. It is shown that the sedimentation coefficients of each of two differently labeled noninteracting proteins in a mixture may be readily determined in a sedimentation velocity experiment, and that the molecular weights of each of two such proteins in a mixture may be readily determined in a sedimentation equilibrium experiment.

An automated method for determination of the sedimentation coefficient of macromolecules using a preparative centrifuge.

Following centrifugation in a preparative ultracentrifuge at relatively high speeds for 1-3 h, quartz tubes containing approximately 150 microliter of macromolecular solution are optically scanned using an apparatus and method previously described (Attri, A. K., and Minton A. P. (1983) Anal. Biochem. 133, 142-152). The resulting data are processed by microcomputer immediately upon completion of scanning to yield the sedimentation coefficient of the solute. Calculated values agree to within a few percent with those found in the literature and with the results of control experiments carried out using an analytical ultracentrifuge.