Hemoglobin, tryptic digests

Inserted tip, glass chip EOF-hydraulic pump 200 nL/min ESI-TOF Bovine hemoglobin tryptic digest Mixed off-chip, on-chip tryptic digestion Nil 115... 

Tryptic digest of bovine hemoglobin (4 pM) cytochrome c (0.8 pM) Human hemoglobin (normal and sickle cell, 0.24 pM) Mixed off-chip/on-chip tryptic digestion Nil 807... 

Fig. 5. Elution profiles obtained for the tryptic digest peptides of hemoglobin A using three distinct gradient shapes (number 3,6, and 8 on the Waters M660 solvent programmer). In each case, two /u-Bondapak C, columns were connected in series, a flow of 1.7 ml/min was used, and a 30-min linear gradient was generated from solvent A (0.phosphoric acid, pH 2.2) and solvent B (SOOt acetonitrile-0.1% phosphoric acid, pH 2.2). Reprinted with permission from Bishop et al. (5. ). Copyright by Marcel Dekker, Inc., New York.

Figure 5. Peptide maps of tryptic digests of normal and sickle cell hemoglobins. Adapted from Ref. 73. The single peptide difference between the two hemoglobins is indicated by shying and...

Fig. 1. HPLC of tryptic digest of human globins (a and P)from hemoglobin treated in vitro with [8-14C] styrene-7,8-oxide (a) chromatogram numbers refer to fractions, (b) radiogram annotation refers to tryptic peptide.

The spectrophotometric evidence reviewed above for the binding of a proportion of the phenolic hydroxyl groups of the tyrosine residues of native proteins is supported by work on the action of tyrosinase on proteins. Sizer (1946) found that this enzyme oxidizes the tyrosine residues in native trypsin, pepsin, chymotrypsin, casein, peptone, insulin, and hemoglobin. Native ovalbumin, human and bovine serum albumin, tobacco mosaic virus (nucleoprotein), human y- and bovine /3-globulins, and bovine fibrinogen are not susceptible to tyrosinase, but become so after tryptic digestion. It was shown (Sizer, 1947) that for the proteins which are oxidized by tyrosinase in the native state, the observed reaction does indeed occur with the intact proteins and does not require preliminary degradation to tyrosine peptides or free tyrosine. The kinetics of the oxidation of tyrosine by tyrosinase have been studied spectropho-tometrically (Mason, 1948 etc.). 

FIGURE 47.14 Time-of-flight mass spectra of peptides prepared via tryptic digestion of bovine hemoglobin. (Reprinted with permission from Lazar, I. M., et al., Electrophoresis, 24, 3655, 2003. Copyright 2003 Elsevier B.V.) (a) Peptides separated in the CEC system with monolithic column shown in Figure 47.13, (b) unseparated... 

Guevremont, R., Barnett, D.A., Purves, R.W., Vandermey, J., Analysis of a tryptic digest of pig hemoglobin using ESI-FAIMS-MS. Anal. Chem. 2000, 72, 4577. 

With species closely related to man the hemoglobin concentration in the final preparation could be assayed approximately by the use of spectrophotometric constants established for human hemoglobins. In other species the quantities of material to be used on peptide patterns were estimated roughly from the consumption of sodium hydroxide during tryptic digestion. 

Monoisotopic (mi) and average (av) peptide masses from tryptic digestion of human hemoglobin a chain [11]... 

Fig. 2. Five fragment masses determined from the mass spectrum of the mock unknown protein (human hemoglobin a chain) tryptic digest form the basis of peptide mapping. The mass-to-charge ratio is labeled nih on the horizontal axis.

For example, let us assume that we find three b series fragment ions, miz 908.4, 1021.5, and 1108.5 in the CAD spectrum from the tryptic digest of the human hemoglobin a subunit that belong to the peptide parent ion with m/z 1833.9 (see Fig. 5). This is the peptide between residues 41 and 56 in Table 1. 

The existence of soluble protein-polyelectrolyte complexes makes polyelectrolytes potentially important enzyme carriers, in which the enzyme activity could be controlled. For example, the tryptic digestion of hemoglobin with PAA as a carrier was studied by Morawetz [56] in 1955. The trypsin activity was varied by adjusting pH and polymer concentration. It was found that the carrier could even activate the enzyme at low pH, as shown by Fig. 15.22. As shown in Fig. 15.23, the complex of trypsin with KPVS was found to retain activity in a wide pH range by Kokufuta [57]. Based on the study of trypsin-carboxymethyl-cellulose complexes, Larionova et al. [58] concluded that some protein-polyelectrolyte complexes can provide extra thermal stability at the preservation of high biological activity. 

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