Cross-linking formalin-treated RNAse

FORMATION OF INTRA- AND INTERMOLECULAR CROSS-LINKS IN FORMALIN-TREATED RNASE A... 

Figure 15.3 (a) Heat absorption in solutions of native RNase A (trace 1) and RNase A kept in 10% buffered formalin for 2 days (trace 2) and 6 days (trace 3) at pH 7.4 and 23°C. All samples were dialyzed against 75 mM potassium phosphate buffer (pH 7.4) prior to DSC. (b) Dependence of Td of the dialyzed RNase A samples on time of incubation in 10% buffered formalin at pH 7.4 and 23°C. (c) Heat absorption of solutions of formalin-treated RNase A fractions isolated by size-exclusion gel chromatography monomer (trace 1), dimmer (trace 2), and a mixture of oligomers with >5 cross-linked proteins (trace 3). Protein concentrations were 0.5 mg/mL. The thermal denaturation transition temperature (Td) is defined as the temperature of the maximum in the excess heat absorption trace associated with the protein s endothermic denaturation transition. See Rait et al.10 for details. 

Figure 15.4 SDS-PAGE of formalin-treated RNase A fractions taken before (lanes 1-3) and after (lanes 4-6) the DSC scans shown in Figure 15.3c. Lanes 1 and 4, monomer lanes 2 and 5, dimer lanes 3 and 6, mixture of oligomers with >5 cross-linked proteins M, molecular mass markers as in Figure 15.2. See Rait et al.10 for details.

In summary, formalin-treated does not significantly perturb the native structure of RNase A at room temperature. It also serves to stabilize the protein against the denaturing effects of heating as revealed by the increase in the denaturation temperature of the protein. However, formalin-treatment does not stabilize RNase A sufficiently to prevent the thermal denaturation of the protein at temperatures used in heat-induced AR methods as shown by both DSC and CD spectropolarimetry. This denaturation likely arrises from the heat-induced reversal of formaldehyde cross-links and adducts, as shown in Figure 15.4 of Section 15.4. Further, cooling formalin-treated RNase A that had been heated to 95°C for 10 min does not result in the restoration of the native structure of the protein, particularly in regard to protein tertiary structure. 

Figure 15.9 The results of capture ELISA on native RNase A and formalin-treated RNase A. Right panel, native RNase A (curve 1) and unfractionated formalin-treated RNase A (curve 2). Left panel, individual fractions of formalin-treated RNase A monomer (curve 3), dimmer (curve 4), trimer (curve 5), tetramer (curve 6), and a mixture of oligomers with >5 cross-linked proteins (curve 7). The ELISA plate wells were coated with monoclonal antibody against bovine pancreatic RNase A (1 pg/mL) overnight at 4°C and then blocked with bovine serum albumin. The wells were incubated for lh at 37°C in the presence of various concentrations of antigen in lOOpL of PBS. After washing, each plate well received a 1 4000 dilution of horseradish peroxidase conjugated rabbit polyclonal anti-RNase A antibody followed by incubation at ambient temperature for lh. After washing, detection was achieved using a mixture of 2,2,-azino-di-(3-ethylbenzthiazoline-6-sulphonate) and hydrogen peroxide. Absorbance was monitored at 405 nm. See Rait etal.11 for details.

Capture ELISA on selected oligomeric fractions of formalin-treated RNase A (see curves 3-7 in Fig. 15.9) also reveal that the plateau values increase with an increase in the number of cross-linked molecules in the fractions. This is due to an increasing proportion of bound epitopes per binding site or, in other words, epitope density on the surface. Thus, the nearly identical plateau values for the titration of native RNase A and formalin-treated unfractionated RNase A (curves 1 and 2 in Fig. 15.9) are fortuitous, being caused by the particular composition of oligomers present in the formalin-treated RNase A preparations that was analyzed. 

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