Affinity kinetic data analysis

The inhibition of SuSy by the divalent cations (fCi 15 aM), Zn (fC 25 aM), and Ni (fti 37 aM) was exploited for further purification of SuSyl by immobilized metal affinity chromatography (IMAC). A subsequent gel filtration yielded homogeneous SuSyl suitable for crystallization experiments [24]. The protein chemical characterization revealed a homotetrameric organization of the 93 kDa subunit. Our kinetic data for the cleavage reaction and preliminary immunoblot analysis for phosphoserine suggested that SuSyl may be phosphorylated in the yeast expression system [28]. 

Relaxation kinetic methods provide the most powerful approach to chemical field effects. The quantitative analysis of relaxation kinetic data is appreciably simplified when the z-induced changes are small. A general expression for chemical relaxation conditions may be found in terms of the appropriate chemical reaction affinity according to... 

Cl NMR line width. An analysis analogous to that described in Section 8.1 gave binding constants in good agreement with kinetic data. The following affinity series was obtained CNO > > SCN > I >... 

A comparison of affinity and kinetic data ensures consistency of the analysis. For a 1 1 interaction, affinity is equal to the quotient of dissociation and association rate constants ( d = dissAass)- However, some reactions do not follow a 1 1 interaction, and it must be noted that the apparent affinity of bi- or multivalent reactions can be directly affected by the density of immobilized interaction partners. Nevertheless, the chip surface resembles the situation on a cell surface and possibly reflects the in vivo environment better than an assay free in solution ]17]. 

Most literature on enzyme kinetics is devoted to initial rate data and the analysis of reversible effects on enzyme activity. In many applications and process settings, however, the rate at which the enzyme activity declines is of critical importance. This is especially true when considering its long-term use in continuous reactors. In such situations the economic feasibility of the process may hinge on the useful lifetime of the enzyme biocatalyst. The focus of this section is on the mechanisms and kinetics of loss of enzyme activity. It should also be recognised that the alteration of protein structure is central to the practical manipulation of proteins (e.g. precipitation, affinity and other forms of protein chromatography, and purification in general). 

The kinetic method provides an alternative to equilibrium measurements for the determination of gas-phase thermochemical properties. It has been applied more and more in thermochemical data determination mainly because of its ability to measure very small energy differences and its simplicity. Indeed, it can be executed easily on any tandem mass spectrometer. Furthermore, this method is sensitive and is applicable with impure compounds. Its applications are broad, covering thermochemical properties in the gas phase such as proton affinity [46], electron affinity [47], metal ion affinity [48], ionization energy [49], acidity [50] or basicity [51], In addition to the determination of thermochemical data, the kinetic method has also been applied in structural and chemical analysis such as chiral distinctions. This method is able to distinguish enantiomers and to measure precisely enantiomeric ratios [52],... 

---