Amino acid sequencing Immobilization

Because the pi of a protein is based on its amino acid sequence, this technique has good resolving power. The resolution can be adjusted further by changing the range of the pH gradient. The use of immobilized pH gradient (IPG) strips has enabled reproducible micropreparative fractionation of protein samples, which is not consistently possible when ampholytes are used in the first dimension (Gorg et al., 2000). 

Bjellqvist, B., Hughes, G. J., Pasquali, C., Paquet, N., Ravier, F., Sanchez, J.-C., Frutiger, S., and Hochstrasser, D. (1993). The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis 14, 1023-1031. 

Combination of two immobilized enzyme columns with HPLC/thermospray MS can be useful for amino acid sequencing and identification. The use of an endopeptidase bioreactor followed by HPLC separation then an exopeptidase column and MS detection can enable sequencing of 3-5 amino acids of each endopeptidase hydrolysis product. The trypsin, hydrolysis/HPLC/ carboxypeptidase A, B, and Y (1 1 1) hydrolysis/ thermospray MS analysis assist in the sequencing of Y-endorphin (Figure 2C,C ). 

For many applications it is important to know what the loading actually is (e.g. to compare activity of different enzymes immobilized to the same loading). Loading determination of an immobilized enzyme is often possible by methods such as quantitative amino acid analysis or active site titration. The latter has the advantage that only active enzyme is quantified. Typically, however, loading is determined indirectly during the immobilization process by measuring enzyme concentration in the aqueous solution before and after incubation with the carrier. The concentration of a pure enzyme in aqueous buffer can be determined from the absorption at 280 nm. The specific absorption can be calculated from the amino acid sequence of the enzyme. For less pure enzyme solutions, the total protein content can be determined with Bradford, BCA or other assays. 

CPA is often used for sequencing protein samples. Whereas there is an effective way to sequence from the amino terminus, such as with Edman degradation with phenyl isothiocyanate, there are few such methods for sequencing from the carboxyl terminus (hyclrazinolysis is one such chemical method). Hence, digestion with CPA is a choice whe e one needs to know the amino acid sequence from the C-terminus. The time course of amino acid release is monitored on the amino acid analyzer. This way to use CPA seems to be made even more effective by immobilizing it on a solid support. Bovine enzyine was... 

An immobilized GL-T-ACA acylase of Pseudomoruis sp. GK16 has been utilized for industrial production of 7-ACA by the chemical and enzymatic method from cephalosporin C via GL 7 ACA by the Asahi Chemical Company (4,21). Several additional cephalosporin acylase genes have been reported (22—28). Enzymatic properties of various cephalosporin acylases are summarized in Table 1, and amino acid sequences of these are shown in Rgure 3. 

The amino acid sequence ROD (Arg-Gly-Asp) is present in many extracellular matrix (ECM) proteins and has been found to play an important role in cellular growth, differentiation, proliferation, and regulation of overall cell function. Immobilized, synthetic, RGD-containing peptides on solid supports such as polymers and silicon oxide surfaces have been reported to mediate specific surface-cell interactions and to promote cell organization. Studies of cell migration suggest the importance of the surface density of RGD sequences for efficient cell-matrix in-teraction. ... 

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