BSA and RNAse

Typical results for "Type 1 turbidimetric titrations are shown in Figure 1, for BSA and RNAse, in the presence of 0.04 wt. % PDMDAAC at ionic strength 0.2 M. 

As shown in Figure 1, the critical pH values for BSA and RNAse in 0.2 M NaCl are separated by about one pH unit. When a solution containing BSA and RNAse, each at 0.5 g/L, is titrated in like fashion, a single turbidity curve is observed at an intermediate pH. At this point, it is not clear whether both proteins are participating jointly and simultaneously in complex formation, and therefore not being separated. If the titration is stopped, say at 100-96T = 20, the coacervate may be removed and the titration of the supernatant continued. Analysis of these successive coacervate fractions is currently underway, employing size exclusion chromatography to determine the concentrations of the two proteins. 

Molecular Area and Film Pressure. A smaller area/molecule and a greater film pressure caused an increase of surface viscosity (Figures 3-8). However, at the same area/molecule, ceramide was more viscous than ganglioside. A more striking contrast is seen in Figures 9 and 10. When modest quantities of DPL or cholesterol are added to saturated films of BSA and RNase, the pressure increases markedly (e.g., from 21 to 45 dynes/cm with BSA) whereas the viscosity dropped from large, immeasurable values to practically zero. 

Figure 1. "Type 1" turbidimetric titrations for BSA (O) and RNAse (-f-), both 1 g/L, in the presence of 0.4 g/L PDMDAAC and 0.2 M NaCl. Broken line shows evaluation of critical pH.

In most of our SEC-LS/UV/RI studies, only three protein standards (BSA monomer, ovalbumin, and RNase) are used to calibrate the light-scattering instrument (2). Therefore, we can use these same three standards to obtain the (UV)/(RI) calibration constant of Eq. [4] without extra effort. To estimate the error of this approach, we derived a new calibration constant using only these three standards, and then calculated the e of other proteins, which were compared with literature values... 

Protein and Lipid-Protein Systems. The high surface viscosity of BSA and the low surface viscosity of RNase had been observed by a torsion rotational method that used an extremely large torque (2, 6). In the present experiments, the protein is dispersed in the aqueous subphase to a final concentration of 10 /xg/ml. Both film pressure and surface viscosity are measured as a function of time (to 40 min). The film pressure of BSA was 21 dynes/cm at mixing and remained constant for 40 min. Also, surface viscosity reached a high value instantaneously (although the first measurement was made at 5 min) and remained constant for 40 min. In contrast, RNase built up pressure slowly, from 3 dynes/cm... 

Does heat-induced aggregation involve in all cases unfolded protein molecules In Sec. II we saw that BSA and ovalbumin have a definite tendency to aggregate at room temperature this could be also the case for HSA [141] and RNase [161], since the heat stability of these proteins depends on concentration. Does this tendency increase with temperature below the temperature of incipient unfolding How could this aggregation process involving native species affect heat-induced unfolding ... 

One modifier that was commercially available, inexpensive, and reasonably stable, hexamethylphosphoramide, worked very well with other proteins as well as RNase. Table 2 shows the results of modifying BSA, casein, egg albumin, and RNase with HMPA. Without modifier, no fluoro-hydrolase activity was detected for any of these proteins. 

Fig. 6. Plateau-values, I"P1 /mg m 2, of adsorption isotherms of lysozyme (LSZ), ribonuclease (RNase), a -lactalbumin (aLA), calcium-depleted (X -lactalbumin (aLA(-Ca )) and bovine serum albumin (BSA) on hydrophobic polystyrene (PS) and hydrophilic hematite (a — Fe203) and silica (Si02) surfaces. An indication of the charge density of the surface is given by the zeta-potential, C, and of the proteins by + and signs. Ionic strength 0.05 M T = 25°C. (Derived from Currie et al. 2003).

We regularly use bovine albumin (BSA), chicken ovalbumin, and ribonuclease (RNase) to calibrate the light-scattering instrument (2). These protein standards can also be used to obtain the calibration constant for Eq. [4]. 

BSA monomer, ovalbumin (chicken), P-lactoglobulin (bovine milk), serum albumin (human), carbonic anhydrase (bovine), L-glutamic dehydrogenase (bovine liver), a-chymotrypsin (bovine), a-chymotrypsinogen A (bovine), immunoglobulin (bovine milk), pepsin, trypsin (bovine), and heparin were from Sigma. RNase and lysozyme (egg white) were from Calbiochem. The recombinant human basic fibroblast... 

The Kirkwood-Buff integrals and the excess (or deficit) number of molecules of water and PEG around a protein molecule were calculated for 3-lactoglobulin (p-LG), bovine serum albumin (BSA), lysozyme, chymotrypsinogen and ribo-nuclease A (RNase A). Experimental data regarding and V2 for these systems are available in the literature [11,12,14]. The partial molar volumes V and E3 in the aqueous solutions of PEGs were calculated using the experimental data and the correlations suggested in Ref [51]. 

Fig. 3. Dependence of A i2 [mol/mol] on the volume of exclusion (Fg) for various proteins a) BSA (pH=3.0), b) BSA (pH=7.0), c) chymotiypsinogen (pH=3.0), d) lysozyme (pH=7.0), e) RNase A (pH=2.0). The dashed lines are shown for comparison. Experimental data regarding and V2 for these systems were taken from Ref [14].

BSA was a product of Pentex, Kankakee, 111., and salt-free RNase was obtained from Sigma, St. Louis, Mo. 

Nonspherical, surface-imprinted magnetic PMMA (see Fig. 28) nanoparticles could be prepared by Tan et al. [177, 178]. A miniemulsion process was used to prepare magnetite/PMMA nanoparticles on which proteins were either immobilized by adsorption (RNAse A) [178] or covalently (bovine serum albumin, BSA) [177]. After creating a shell of PMMA, the proteins were removed, leaving cavities on the particles surface. The BSA-imprinted nanoparticles showed superparamagnetic properties and exhibited a high rebinding capacity for BSA. 

Add z pL template (RNA analyte or PI primed DNA) to bring the total volume to 25 pL. Template volume will vary depending on concentration and desired number of input molecules, usually lO molecules see Note 10). For negative control reactions template volume will equal 0. (Summary of standard reaction conditions 40 mAf Tris-HCl, pH 8.5, 50 mAf KCl, 12 mAf MgClj, 2 mAf NTPs (each), 1 mAf dNTPs (each), 10 mAf DTT, 0.2 pAf PI, 0.2 pAf P2,15% DMSO, 100 pg/mL BSA, 20 U T7 RNA Polymerase, 8 U AMV Reverse Transcriptase, 0.2 U RNase H, 12.5 U RNAguard , 10 molecules of template). 

Dubin and Park [13] have utilized selective complexation of poly(l-alkyl-4-vinylpyridiniums) with mixtures of proteins to develop a convenient new method for isolation of proteins of interest. For example, they were able to cleanly separate bovine serum albumin (BSA) from ribonuclease (RNase) by a selective coacervation technique. The net negatively charged BSA forms a strong ionic complex with 2 and precipitates to leave only the RNase in solution. 

---