Casein amino acid analysis

Groves, M. L. and Gordon, W. G. 1969. Evidence from amino acid analysis for a relationship in the biosynthesis of 7- and 0-caseins. Biochim. Biophys. Acta 194, 421-432. 

Figure 1. Amino acid analysis of a hydrolysate of casein heated in water...

Figure 1 summarizes the potential pathways involved in the formation of dehydroalanine. It appears that dehydroalanine can be formed in a variety of amino acids protein, suggesting that any or all of the routes in Figure 1 could be involved in dehydroalanine formation. Table 1 contains results of partial amino acid analysis of several alkaline treated proteins. The results support the suggestion that both serine and cystine or their derivatives can be sources of dehydroalanine and subsequently the lysinoalanine measured in the proteins. In casein there is substantial LAL formation with a measurable loss in serine. In isolated soy protein and lactalbumin it can be seen that cystine shows the most significant losses. It should be noted that a significant portion of the serine in casein is present as phosphoserine. The relatively rapid 6-elimination of phosphoserine (15) accounts for the formation of considerable quantities of dehydroalanine and subsequently the substantial levels of LAL found in casein. In addition, as mentioned above, the presence of calcium would accelerate dehydroalanine formation from the phosphoserine present in the casein. The variability of... 

Protein adsorption to PS latex has also been determined by sedimentation field flow fractionation (SdFFF). The maximum surface coverages of P-casein and p-lactoglobulin on negatively charged PS latex calculated using this method were similar at around Img/m [19]. This figure, which was confirmed by amino acid analysis of the material irreversibly bound to the surface, was significantly lower... 

Although the results are consistent with a B-elimination reaction leading to formation of dehydroalanine, the conclusions are based on the assumption that the absorbance at 241 nm is associated with dehydroalanine side chains derived from cystine residues. This assumption may not always be justified for the following reasons. First, alkali treatment of casein which has very few or no disulfide bonds also yields significant amounts of dehydroalanine residues (52). These presumably arise from serine side chains. Second, Nashef et al. (41) cite evidence that other functionalities may contribute to the 241 nm absorption. These considerations suggest that there is a need to directly measure dehydroalanine in proteins. This is now possible with our method (52), whereby the dehydroalanine residues are first transformed to S-pyridylethyl side chains by reaction with 2-mercaptopyridine (Figure 12). Amino acid analysis of the acid-hydrolyzed protein permits estimation of the dehydroalanine content as S-B-(2-pyridylethyl)-L-cysteine along with the other amino acids. 

Amino acid analysis is commonly used for the identification of proteins in paint layers, where contamination is low, the conservation state is generally good, and the range of possible proteins is very limited (mainly egg, collagen, or casein). However, the identification of archaeological proteinaceous residues is by no means an easy task and requires peptide mapping using HPLC/ MS or MALDI techniques (see Section 36.5). With the increased availability of advanced instrumentation, pro-teomics may become the favored approach for protein determination also in paint layers [21]. 

Figure 9.2 PCA score plot of amino acidic profiles obtained in the GC/MS analysis of samples from the collection of paint reference materials of Opificio delle Pietre Dure (+), containing egg, casein and animal glue as binders, and of samples from the OL17bis series (x ) from the Leonetto Tintori Collection [ 10]...

The electronic property of the amino acid on the C-terminus also has an effect on antioxidant activity (Li et al., 2011), that is, the larger the electronic property, the higher is the activity. The C-terminus is a polar position that is thus affected by its electrostatic potential, to some extent therefore, the amino acids Trp, Glu, Leu, lie, Met, Val, Tyr, etc. are suitable at the C-terminus. Some researchers have speculated that the identity of the amino acid on the C-terminus would play an important role in its activity. Suetsuna (2000) separated and identified a radical scavenging peptide, Tyr-Phe-Tyr-Pro-Glu-Leu, from casein hydrolysate, and it was confirmed that the Glu-Leu on the C-terminus mainly contributed to its antioxidant activity. Kim et al. (2009) speculated that the hydrophobic property of the amino acid on the C-terminus, for example, Val and Leu, had a distinct effect on the activity, as determined from the analysis of antioxidative peptides derived from venison hydrolysate. 

Fluorimetric methods for the determination of amino acids are generally more sensitive than colorimetric methods. Fluorescamine (4-phenyl-spiro[furan-2(3H),l -phthalan]-3,3 -dione) and o-phthaldialdehyde (OPA) substances are used for protein analysis. Fluorescamine reacts with amino groups to form fluorophores that excite at 390 nm and emit at 475 nm (Weigele et al., 1972). Applications of fluorescamine include monitoring the hydrolysis of K-casein (Beeby, 1980 Pearce, 1979) and quantification of proteins, peptides, amino acids in extracts (Creamer et al., 1985). OPA produces fluorescence on reaction with 2-mercaptoethanol and primary amines, with strong absorption at 340 nm. Lemieux et al. (1990) claimed that this method was more accurate, convenient, and simple for estimating free amino acids than the TNBS, ninhydrin, or fluorescamine methods. 

It appears that the heterogeneity of the reaction products results from the heterogeneity of the K-casein rather than from a random attack on the K-casein by rennin (2). This suggests that perhaps a single peptide bond might be split by rennin. If this is the case, then new C-terminal and N-terminal amino acid residues should be detectable after rennin action. The same C-terminal amino acids were found in K-casein and in the macropeptide indicating that the macropeptide occupied the C-terminal position of the K-casein (164, 165). Thus a new C-terminal residue should be exposed in p-K-easein and a new N-terminal amino acid in the macropeptide. Carboxypeptidase analysis of p-K-easein indicated a C-terminal phenylalanine or leucine (165). Reduction of... 

Casamino acids, commercial acid-hydrolyzed casein. Hydrolysis is carried out until all the nitrogen in the casein is converted to amino acids or other compounds of relative chemical simplicity. Prepn Mueller, Miller, J. Immunol 40, 21 (1941) Mueller, Johnson, ibid. 33. Typical analysis N 10%, NaCI 14%, ash 20%, phosphorus as P04 2%, Fe 15 mg/3 g. 

The principal component analysis also clusters related functional measures. We selected three representative activities based on the functional clustering shown in Figure 6 for further analysis activity towards AAPL-p-NA at pH 7.0, absolute activity towards AAPL-p-NA following 5 minutes at 65°C and activity towards casein. For each of these activities we constructed PLSR models similar to that shown in Figure 3, and calculated the regression coefficients for each amino acid variation as shown for thermal tolerance in Figure 4. The changes calculated to contribute positively to each property are shown in Table 1. 

Focuses on the role of protein intake on performance with analysis of whey, soy, and casein proteins, and creatine, glutamine, branched-chain, and other amino acids... 

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