Single Point Attachment Thermal Unfolding

3.1.1. Activation of Silica Beads

1. Wash 10 g (dry weight) of CPC-silica beads with washing buffer.

2. Add 20 mL of an aqueous glutaraldehyde solution.

3. Shake vigorously for 45 min at about 0°C (ice-water bath).

4. Wash the activated beads extensively with washing buffer.

3.1.2. Protein Immobilization

1. Dissolve 30 mg of RNase in 8 mL of phosphate buffer.

2. Add 1 g of glutaraldehyde activated glass beads.

3. Shake gently for 5-7 h at room temperature, then wash thoroughly with the appropriate buffer solution.

4. During the immobilization reaction, check the decrease of the protein concentration as a function of time by measuring the absorbance at 278 nm (e = 9800 M/ cm) of the supernatant solution. Usually between 80 and 90% of the initial protein can be immobilized on the glass beads.

3.1.3. Characterization of the Immobilized Protein

The concentration of the immobilized protein can be as high as 20-25 mg/g of beads. The concentration of the immobilized RNase can be determined in three ways:

1. From the absorbance change during immobilization (see Subheading 3.1.2., step 4)

2. By amino acid analysis, after acid hydrolysis at 110°C. Control experiments showed no interference from the glass.

3. By Lowry-Folin method (14). The method can be applied after dissolving the samples (the glass beads) at pH 12.0 at 100°C

The uncertainty is ±5% for methods 1 and 2 and ±15% and for method 3.

3.1.4. Number of Linkages Between Protein and Support

The number of linkages between protein and support may be indirectly estimated by measuring the amount of protein-free amino groups still reactive after treatment of the protein with valeraldehyde, a monofunctional aldehyde. The conditions of the reaction between valeraldehyde and the free protein have to be the same as those used in the reaction between glutaraldehyde-activated glass beads and protein.

1. Dissolve 40 mg of RNase in 8 mL of phosphate buffer.

2. Add valeraldehyde, 10% (v/v) and mix thoroughly for 5 to 7 h at 25°C.

3. Titrate the remaining free amino groups with trinitrobenzenesulfonic acid, according to ref. 15.

4. Compare the results with an appropriate reference sample of native RNase.

In these conditions it has been found that the amount of reacted amino groups were 1.2 ± 0.4 mole/mole of protein.

3.1.5. Calorimetry (see Notes 2-5)

Highly sensitive commercial microcalorimeters are currently available. Instruments such as DAMS-4 (Pushchino Scientific Center,, MC-2 (Microcal Inc., Northampton, MA), and Micro DSC (SETARAM, Caluire, France) are suitable instruments for protein thermal stability studies. They all employ relatively small sized cells, which can be filled with heterogeneous material, although with some practical limitations. Modern instruments such as VP-DSC (Microcal) and Multi Cell MC-DSC 4100 (Calorimetry Science Corp., Spanish Fork, UT) are more sensitive than earlier versions (1970-1990) and therefore the sample size can be considerably reduced. They are all equipped with a solid sample device. Older instruments needed larger samples because they operated with larger volume cells, more easily filled and more practical to handle.

Analysis of the thermograms according to two-state approximation and the underlying theory of protein unfolding in solution are well documented (16,17). This can be applied to immobilized proteins as well. Data processing can be also conveniently performed by commercial standard software programs such as Origin Software (Microcal). Thermal analysis and deconvolution (single component) programs are usually standard software accessories of the commercial calorimeters.

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