Denaturation serum proteins

Serum Albumin. With a denaturation temperature of 64°C (Table 11.2), bovine serum albumin is denatured almost as easily as a-lactalbumin. Since its denaturation is not as reversible as that of a-lactalbumin, it appears to be the most easily denatured serum protein (de Wit and Klarenbeek 1984). It precipitates between 40° and 50 °C as a result of hydrophobicity-directed unfolding (Lin and Koenig 1976 Macritchie 1973). Some serum albumin remains undenatured even after prolonged heating at 65°C. This may be because already denatured albumin is able to protect native proteins from denaturation (Ter-ada et al. 1980). 

The technological processes used in the dairy industry influence the composition of MFGM. During heating, interactions occur between MFGM components and denatured serum proteins, predominantly p-lactoglobulin. An increase in protein... 

Recio, 1., and Olieman, C., Determination of denatured serum proteins in the casein fraction of heat-treated milk by capillary zone electrophoresis. Electrophoresis, 17, 1228, 1996. 

This enzyme [EC 3.4.21.53], also known as endopepti-dase La, ATP-dependent serine proteinase, and ATP-dependent protease La, catalyzes the hydrolysis of peptide bonds in large proteins (for example, globin, casein, and denaturated serum albumin) in the presence of ATP (which is hydrolyzed to ADP and orthophosphate). Vanadate ion inhibits both reactions. A similar enzyme occurs in animal mitochondria. Protease La belongs to the peptidase family S16. 

About 20% of milk protein is soluble in the aqueous phase of milk. These serum proteins are primarily a mixture of /3-lactoglobulin, a-lactalbumin, bovine serum albumin, and immunoglobulins. Each of these globular proteins has a unique set of characteristics as a result of its amino acid sequence (Swaisgood 1982). As a group, they are more heat sensitive and less calcium sensitive than caseins (Kinsella 1984). Some of these characteristics (Table 11.1) cause large differences in susceptibility to denaturation (de Wit and Klarenbeek 1984). 

Lactoglobulin With a denaturation temperature of 78°C, /3-lacto-globulin is the least denaturable of the serum proteins (Table 11.2). It exhibits a second thermal change near 140°C caused by a breakdown of disulfide bonds and additional unfolding of the molecule (de Wit 1981 Watanabe and Klostermeyer 1976). A change in pH between 6 and 7.5 shifts denaturation between 78° and 140°C, the total denaturation at the two temperatures being nearly constant. pH 6 favors dena-... 

A clinical sample (whole blood, serum, plasma, urine, gastric juice, bile fluid, sweat, etc.) differs from any other analytical sample because of the presence of heterogeneous organic (e.g., proteins) and organic or inorganic components (e.g., urea or sodium ion), sample changes in time (owing to, e.g., denaturation of proteins, escape of C02) and small sample size (even a few tens of microliters). 

Figure 3-3 Time-Temperature Relationships for the Heat Denaturation of Whey Proteins in Skim Milk. Source From H.A. Harland, S.T. Coulter, and R. Jenness, The Effects of Various Steps in the Manufacture on the Extent of Serum Protein Denaturation in Nonfat Dry Milk Solids. / Dairy Sci. 35 363-368, 1952.

Alexander, M., and Dalgleish, D.G. (2005). Interactions between denatured milk serum proteins and easein micelles studied by diffusing wave spectroscopy. Langmuir. 21, 11380... 

For five plates, dissolve 1 g of agarose in 100 ml water, resulting in a 1% (w/v) solution. To buffer, add 50 mM acetic acid, 60 pM ascorbic acid, adjusted to pH 5.0 with sodium hydroxide. Bring slowly to boil (on hot plate stirrer) to completely dissolve the agarose. Cool to 45°C. (Warning Failure to let the mixture completely cool will denature the protein). Add 100 mg bovine serum albumin (BSA) [to 0.1%... 

Denaturation of proteins may result from low concentrations and elevated storage temperatures. These conditions must also be controlled for radioiodinated proteins that are also subject to internal self-irradiation damage, which is more pronounced in I-labeled proteins than I-la-beled proteins owing to the beta radiation emission of I. To minimize such problems, radioiodinated proteins are diluted in protective protein solutions that will not interfere in the subsequent studies to be made, such as 10% normal serum or BSA at 2 mg of protein per milliliter. Aliquots are kept frozen at -20° to -70° and thawed only as needed. 

The alkaline denaturation of serum proteins proved to be of practical use in detecting greater or lesser differences between normal and various pathological sera. For example, the proteins of sera from patients suffering from cancer cannot be denatured with alkali as can those of normal people. 

Specimen Collection and Storage. The preferred specimen is serum. Specimens may be stored at 4 °C for 1 week or at -20 °C for 30 days. Repeated freezing and thawing, mixing using a vortex mixer, or vigorous agitation of serum specimens should be avoided, because such treatment causes denaturation of proteins. The use of hemolyzed or hpemic specimens is also not recommended. 

Increasing pH above the natural pH of milk markedly accelerates the rate of denaturation of p-lactoglobulin. The denatured whey proteins associate with the casein micelles or remain in the serum phase as complexes of denatured whey proteins or denatured whey protein in association with K-casein. 

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