Proteins freezing methods

Figure 3.1 shows the influence of different freezing methods on the activity of a recombinant DNA protein. However, in the case of other macromolecules, for example a monoclonal antibody, there may be exceptions from the rule and different factors (e. g. pH-value) play an important role. 

Emulsion Capacity and Stability. A 0.5 g sample of the freeze-dried protein fraction was redissolved in a minimum of 0.3 M citrate-phosphate buffer at pH 7.0 and mixed thoroughly with 50 ml of 1 M NaCl for 1 min in a Sorvall Omnimixer at 1000 rpm in a one pint Mason jar set in a water bath (20°C). Crisco oil (50 ml) was added to the jar and an emulsion formed by mixing at 500 rpm with simultaneous addition of oil at the rate of 1 ml/min until the emulsion broke. The endpoint was determined by monitoring electrical resistance with an ohmeter. As the emulsion broke a sharp increase (l KS2 to 35- 0 KSi) was noted. Emulsion capacity was expressed as the total volume of oil required to reach the inversion point per mg protein. This method is similar to that used by Carpenter and Saffle (8) for sausage emulsions. To establish emulsion stability the same procedure was used except that 100 ml of oil was added and a stable emulsion formed by blending at 1000 rpm for 1 min. A 100 ml aliquot was transferred to a graduate cylinder and allowed to stand at room temperature. Observations were made of the volume of the oil, emulsion and water phases at 30, 60, 90 and 180 min. 

Schultz, C. J., R. N. Dalton, C. Turner, H. A. W. NeU, and D. B. Dunger. 2000. Freezing method affects the concentration and variability or urine proteins and the interpretation of data on microalbuminuria. Diabetic Medicine 17 7-14. 

Patapoff and Overcashier tested freezing methods and annealing upon primary drying rates, sample temperature during primary drying, dried product resistance, appearance of the freeze-dried cakes, and protein... 

Pish silage prepared by autolysis of rainbow trout viscera waste was investigated as a substrate for the plastein reaction using pepsin (pH 5.0), papain (pH 6—7), and chymotrypsin (pH 8.0) at 37°C for 24 h (152). Precipitation with ethanol was the preferred recovery method. Concentration of the protein hydrolysate by open-pan evaporation at 60°C gave equivalent yields and color of the final plastein to those of the freeze-dried hydrolysate. 

The coacervation approach uses heating or chemical denaturation and desolvation of natural proteins or carbohydrates. As much as 85% of water-soluble drugs can be entrapped within a protein matrix by freeze-drying the emulsion prepared in this manner. For water-insoluble drugs, a microsuspension-emul-sion procedure has been suggested as a method of choice to achieve high drug payloads. 

Hanafusa [1.36] showed with this method, how the amount of unfrozen water in a 0.57 % solution of ovalbumin reaches practically zero at -20 °C, if 0.01 M sucrose is added (Fig. 1.51). For globular proteins Hanafusa described the freezing process as follows between 0 °C and -20 °C, water molecules from the multilayer hydrate shell are decomposed. Be-... 

For activity assays, proteinase solutions were made fresh daily (10 mg freeze-dried solids in 1 ml pH 10.0 phosphate buffer, 0.1 M). Two ml of the proteinase, 0.5 ml of substrate (azocasein or other proteins in pH 10 buffer), 0.3 ml of 0.1% EDTA, and deionized water were made up to a volume of 3.5 ml. Reaction tubes were incubated in a 40°C water bath for 1 hr, then the reaction was stopped by addition of 1 ml 5% TCA. After removal of the precipitated proteins by centrifugation, absorbance was read at 366 nm (for azocasein) or by the Lowry method (15) for other... 

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