Denaturation 3-lactoglobulin

The molar refractions of the amino acids were determined by measurements on their aqueous solutions and the expanded Lorenz-Lorentz equation. The refractive indices of a number of proteins were calculated from their amino acid compositions and the values for the refraction of the amino acid residues. These calculated results are in good agreement with those experimentally determined, demonstrating that refractive index is a unique characteristic of a protein. A comparison of the refractive index of heat denatured /3-lactoglobulin with the native protein demonstrated that changes in structure produced a small change in refractive index, not associated with a change in volume. 

Myoglobin in DjO at pD 6.6 (corrected to 7.0) has an amide I band at 1650 cm, characteristic of a-helical structure. Myoglobin is known to have at least 77% a-helical structure (Kendrew et al., 1960). Timasheff and Susi s (1966) data indicate that in DjO solution the )S-conformation and the a-helical conformation produce amide I bands at the same frequencies as in dry films. Randomly folded proteins (a -casein and denatured ) -lactoglobulin) give rise to a sharp band centering at 1643 cm... 

Slow hydrogen-deuterium exchange (see Chapter 11) of native -lactoglobulin in DjO, pD 1.0 (corrected to 1.4) allowed the amide II band ( 1550 cm ) to remain and be seen in one sample of the lactoglobulin (Fig. 10.18, top curve) which was run immediately after preparation. The presence of this band shows that the native protein must be quite compact. No such bands are observed in denatured lactoglobulin or in a -casein, which have loose randomly folded structures. 

Fig. 10.18. Infrared spectra of native and denatured -lactoglobulin (LG) A, native myoglobin, and a,-casein in D2O solution. The pD values are those read on a pH meter in D2O solution. Consecutive spectra are linearly displaced by 0.1 scale unit Concentration, 20 mg/ml path length, approximately 0.05 mm. (The observed intensities are approximate because of uncertainties in path length. Peak absorptivity values for the amide I band are in the range 3 to 4 liters/g cm, if absorptivity at 1800cm" is taken as reference.) (TimashefT and Susi, 1966.)...

Casein. Milk contains proteins and essential amino acids lacking in many other foods. Casein is the principal protein in the skimmed milk (nonfat) portion of milk (3—4% of the weight). After it is removed from the Hquid portion of milk, whey remains. Whey can be denatured by heat treatment of 85°C for 15 minutes. Various protein fractions are identified as a-, P-, and y-casein, and 5-lactoglobulin and blood—semm albumin, each having specific characteristics for various uses. Table 21 gives the concentration and composition of milk proteins. 

Kauzmann, W. and R.B. Simpson. 1953. Kinetics of protein denaturation. in. Optical rotations of serum albumin, fS-lactoglobulin, and pepsin in urea solutions. J Am Chem 75 5154-5157. 

Chemical reactions Polymerization of casein and whey proteins are due to some kind of chemical reactions. The different proteins as found in the supernatant of milk after precipitation at pH 4.6 are collectively called whey proteins. These globular proteins are more water soluble than caseins and are subject to heat dena-turation. Denaturation increases their water-binding capacity. The principal fractions are P-lactoglobulin, a-lactalbumin, bovine serum albumin (BSA), and immunoglobulins (Ig). 

In the case of cold-induced aggregation and gelation, two different types of gel microstructure, namely filamentous and particulate (Figure 2.1), have been obtained by adding different concentrations of a ferrous salt to solutions of pre-denatured p-lactoglobulin (the major whey protein). This substantial difference in microstructure turns out to have a major impact on the iron delivery, due to the different sensitivities of the structures to the relevant environmental conditions, such as pH and the presence of digestive enzymes. In particular, the filamentous gel micro-... 

Proteins of egg white denature more rapidly than those of whey protein concentrate (13, 34). However, isolated p-lactoglobulin from the whey concentrate was more susceptible to surface denaturation than egg white ovalbumin. These data suggest that whey contains substances that protect the proteins from surface denaturation and may account for the lower stability of whey protein concentrate foams than those of egg white protein. A balance between the disaggregation effect of select pH values and the tendency toward greater aggregation of proteins at higher heating temperatures were correlated closely with maximum foam stability (13, 15). 

Figure 9.14 The denaturation of the total ( ) and individual whey proteins in milk, heated at various temperatures for 30 min /l-lactoglobulin ( ), a-lactalbumin (O). proteose peptone ( ), immunoglobulins (A), and serum albumin ( ) (from Webb and Johnson, 1965).

A light-scattering study of this nature has been carried out on solutions of /J-lactoglobulin A (0-A) dissolved in mixtures of water with 2-chloroethanol in the presence of 0.02M NaCl and 0.01 M HC1. 2-Chloro-ethanol is known to be a structure-forming protein denaturant and can be expected to interact with proteins freshly distilled, its refractive index at 436 m/x is 1.447, different from that of water, 1.340. This results in a large value of dn/dCi. 

The effects of heat treatment of milk on the oxidation-reduction potential have been studied to a considerable extent (Eilers et al 1947 Gould and Sommer 1939 Harland et al 1952 Josephson and Doan 1939). A sharp decrease in the potential coincides with the liberation of sulfhydryl groups by denaturation of the protein, primarily /3-lactoglobulin. Minimum potentials are attainable by deaeration and high-temperature-short-time heat treatments (Higginbottom and Taylor 1960). Such treatments also produce dried milks of superior stability against oxidative flavor deterioration (Harland et al 1952). 

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-... 

In the absence of calcium, /3-lactoglobulin solubility increases as pH is increased from 6.4 to 7. Addition of calcium at any pH causes a decrease in solubility. At any ratio of pH and calcium ion concentration, the increased charge on the protein induced by pH change is balanced by added calcium ions to hold the level of denaturation constant (de Wit 1981). This suggests that calcium-induced precipitation of /3-lactoglobulin occurs by an isoelectric mechanism. However, Hillier et al. (1979) found that an increase in the calcium concentration up to 0.4 mg/ml slowed heat denaturation of /3-lactoglobulin, but additional calcium had little effect. 

Lyster (1970) found denaturation of /3-lactoglobulin to be second order with respect to time. The kinetic constant K2 in log-1 sec-1 is described by the equation... 

Heat denaturation of /3-lactoglobulin is accompanied by alterations in the properties of x-casein (Zittle et al. 1962). x-Casein and /3-lactoglobulin interact through disulfide linkages when heated together or when k-... 

Heat denaturation of protein solutions is normally retarded by concentration. Concentration of milk to total solids levels of 9, 28 and 44% decreases apparent denaturation by 40, 60, and 80% (Whitney 1977). Individual proteins are affected differently by concentration, a-lactal-bumin being denatured more easily as solids are increased and both A and B genetic variants of /3-lactoglobulin being denatured less easily (Hillier et al 1979). 

The variation in lactose concentration in milk is small, but experiments with the adjustment of lactose concentration have shown that lactose plays a part in the stabilization of milk. Addition of lactose increases both denaturation temperatures of /3-lactoglobulin (de Wit 1981). Milk... 

Harwalker, V. R. 1978. Application of differential scanning calorimetry to the study of thermal denaturation of (3-lactoglobulin in solution. J. Dairy Sci. 61 (suppl. 1), 107. 

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