Casein reductive methylation

Figure 9. Digestion of casein and chemically modified caseins by bovine a-chy-motrypsin. Amino groups of casein were reductively methylated and the hydrolysis by a-chymotrypsin followed with time., unmodified casein , reductively methylated casein—33% modified , reductively methylated casein—52% modified. The casein concentrations were 0.2 mg/ml in 0.02M borate buffer at pH 8.2 and the a-chymotrypsin was 3.2 fjig/ml. The hydrolysis was followed by the procedure of Lin et al. (62) [Figure from Ref. 56].

The temperature-sensitive precipitation of unmodified and methylated /3-caseins in the presence of calcium was measured also (see Figure 3). Methylation caused an increase of up to 3°C in the precipitation temperature of calcium /3-caseinate. Results from rennet clotting of an asi-K casein micelle system indicated that replacing native asi-casein with the reductively methylated protein had little influence on clotting time, while replacing K-casein with its reductively methylated derivative re-... 

Figure 1. Stability of ols1-k micelles containing 20% reductively methylated (RM) components towards precipitation by Ca2+ O, asl-K micelles A, RMasl-K micelles , asl-RMK micelles A, RMols1-RMk micelles. The asl-casein concentration was 7.4 mg/mL pH 6.6 Ca2+ concentration,...

We chose to prepare 14C-methyl-K-casein (M-k-C) as a tracer because of the important role of K-casein in stabilizing casein micelles (8) and because K-casein is known to participate in heat-induced interactions with whey proteins, thereby influencing the heat stability of milk (9). The reductive methylation radiolabeling procedure used low concentrations of reagents (10) and resulted in M-k-C containing approximately 1 fiinol of 14C-methyl groups for every micromole of protein monomer (about 3 /xCi/mg). When tracer M-k-C was added to skim milk, and trichloroacetic acid was added to a concentration of 2%, about 1% of the radioactivity remained soluble. After clotting of the milk with excess... 

Figure 3. Effect of reductive methylation on the temperature of precipitation of C a-/3-caseinate A, /3-casein , 20% reductively methylated /3-casein O, 60% reductively methylated /3-casein /3-casein concentration, 8.69 mg/mL pH 6.6 (7).

C-Methyl-/3-Casein. 14C-Labeled proteins prepared by reductive methylation have potential as substrates in the study of proteolytic enzymes. A serious limitation is that complete methylation of lysine residues results in inhibition of proteolysis by enzymes with trypsin-like specificity (13). It was interesting to determine whether this problem could be overcome by incomplete methylation which left unaltered most of the lysine residues in more or less random distribution throughout the protein. /3-Casein was selected as a suitable protein for this study since it is cleaved by trypsin-like enzymes to well characterized fragments, the y-caseins, in addition to less well characterized fragments, the proteose peptones. We anticipated that this type of study could provide a basis for a general investigation of milk protein transformation by the native milk proteinase which has a specificity similar to trypsin (14). 

The purified /3-casein was methylated reductively to yield a product in which 10 to 12% of the lysine residues were methylated, with a specific activity of 4.42 /xCi/mg. This was diluted with varying amounts of cold /3-casein before use. 

Practical Coacervate Formation. The composition of the various parts of the coacervate system was determined by using l c-labelled kappa-casein as one component of the coacervates, and then performing a mass balance on the system. The C-kappa-casein was prepared by reductive methylation of kappa-casein with l C-formaldehyde (15), This derivatization of kappa-casein was found not to materially affect its physical properties (16). 

Fig. 1.45. Hydrolysis of a reductively methylated casein by bovine a-chymotrypsin. Modification extents a 0%, b 33%, and c 52%. (according to Galembeck et al., 1977)...

C-Methyl-Labeled c-Casein. The effect of reductive methylatior on the properties of asi-, / -, and c-caseins was investigated in this labora tory (7). The purified proteins were treated to obtain either 20% oi 60% modification of lysine residues. Electrophoretic mobilities at pE 8.5 or 6.5 of the modified proteins were indistinguishable from those oi the native caseins. 

The effect of protein methylation (20% modification) on the stability of casein micelles was evaluated. The stability of the clJk micelle system towards Ca2+ precipitation was decreased when either or both proteins were methylated (see Figure 1). Methylation also resulted in a slight reduction in the stability of the /3-k casein micelle system (see Figure 2). 

Plasmin Hydrolysis of 14C-Methyl-/3-Casein. On the basis of a study by Ottesen and Svensson (2), which described the relative amounts of mono- and dimethyllysinyl derivatives produced by reductive methyla-... 

Fig. 5 - Displacement of the fluorescent probe FHMI by the imidazolinone and non-imidazolinone compounds, (left) Chemical structures of three imidazolinone class compounds and three non-imidazoline agrochemicals used to demonstrate the specificity of the fiber optic sensor, (right) Reduction of steady-state fluorescence by the various analytes. A 25 nM FHMI in casein/PBS solution was perfused to reach a steady state before a solution of 1 jlM compound was added to the FHMI, casein/PBS. Plot 1, imzamethabenz methyl 2, imazapyr 3 imzaaquin 4, chlorimuron ethyl 5, primisulfuron 6, sethoxydim. Reproduced with permission from reference 2, Copyright 1993 American Chemical Society.

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