Protein/ligand molar ratio

In an ideal pure preparation of Na,K-ATPase from outer renal medulla, the al subunit forms 65 70% of the total protein and the molar ratio of a to is 1 1, corresponding to a mass ratio of about 3 1 [1,5]. Functionally the preparation should be fully active in the sense that each a/ unit binds ATP, Pj, cations and the inhibitors vanadate and ouabain. The molecular activity should be close to a maximum value of 7 000-8 000 Pj/min. The highest reported binding capacities for ATP and phosphate are in the range 5-6 nmol/mg protein and close to one ligand per otjS unit [29], when fractions with maximum specific activities of Na,K-ATPase [40 50 pmo Pj/min mg protein) are selected for assay. 

Derrick studied the interaction of L-tryptophan and ibuprofen with human serum albumin (HSA),74 which is an abundant transport blood protein capable of binding efficiently several species.75 They acquired 1H NMR spectra of L-Tryptophan-HSA system for different ligand protein molar ratios, that is 3 1, 5 1, 7 1 and 10 1. The aromatic resonances of L-Tryptophan are difficult to be observed due to the overlap with HSA signals, even at 10 1 molar ratio, so that the spectral subtraction was performed. D values of L-Tryptophan were calculated by integration of the subtracted spectra and were in good agreement with those predicted by computer simulations. In the case of ibuprofen, only for 140 1 molar ratio, the resonances of ibuprofen are clearly visible also in this case, the... 

Fig. 3.3 The raw data output of ITC is transformed to show the heat exchange at each injection (kcal mol of injectant), obtained by integration of the area of each spike in the raw data output, as a function of the molar ratio of the protein-ligand binding interaction. The curve is then computer-generated as the best fit to either a one-site or multi-site binding model.

Figure 20. Titration of bovine serum albumin by 2-naphthol-3,6-disulfonate. Titration was carried out with 14.7 xiW protein (pH = 6.0) and increasing ligand concentration. The fluorescence emission ratio of the dissociated vs. undissociated emitter is drawn with respect to the molar ratio of protein to ligand. The emission ratio of free ligand is indicated. Note discontinuity of abcissa and ordinate. (Insert). Sketchard plot of the titration.

PKR, Particulate Protein Kinase rf, ligand/nucleotide molar ratio... 

Except for high-affinily ligands, the protein-ligand complex usually coexists in solution in an equilibrium with significant amounts of free protein and/or free ligand, depending on their relative molar ratios. The dissociation constant can be expressed as the ratio between off- and on-rate (governing dissociation and formation of the complex, respectively) ... 

ApoD is a member of the 2 -microglobulin superfamily of proteins (also known as lipocalins Flower, 1994). The physiological role of plasma apoD is not known. Although several, putative ligands have been suggested by in vitro studies and theoretical considerations (Milne, Rassart Marcel, 1993), our studies identified, for the first time, an in vivo ligand for apoD as it appears in apocrine secretion. We also determined that there is a 2 1 molar ratio of 3M2H to apocrine apoD. 

Add 5 XL of an ethanolic solution of 0.88 mM RME to the spectrophotometer cuvet containing 7 5 jiM apo-CRBP in 1 mL phosphate buffer, pH 7 3 (see Note 2) The excess of CRBP relative to RME (the CRBP/RME molar ratio m the system is approx 1 7) ensures that nearly all the ligand is bound to the protein (a correction for the contribution of free ligand to the spectra of the RME-CRBP complex IS, therefore, not needed). Stir gently and let RME bind to CRBP in the dark at 20°C for 15-20 min (see Note 3). Use this solution to record subsequently both absorption and fluorescence spectra... 

The result from a titration is a plot of the recorded power, dQ/dt, vs. time, as shown in the right upper panel of Fig. 1. Each peak represents the thermal effect associated with an injection. The right lower panel shows the integrated areas as a function of the ligand/protein molar ratio. 

For evaluation of efficiency of medical sorbents, the issue of their deliganding properties, i.e. an ability to withdraw toxic protein bound compounds (ligands) is of great importance. If a sorbent possesses strong deliganding capability as, for example, some types of modem carbon hemosorbents do [11,12], then after contact with such an adsorbent, the ratio of molar concentrations of ligand - protein carrier (M /Mp) decreases, i.e. the transport protein transforms into a more purified state than it was initially (Table 21.3). 

Table3 Further hydration and volume data (Si, V) for anhydrous and hydrated proteins as obtained by simple calculation procedures, together with some secondary parameters (V/M, Ny,/S, Vw(2rw) /5 ). Calculation procedures hydration Si according to Kuntz (K) volumes V according to Traube (T) or Cohn and Edsall (CE) these calculations were performed without ligands, using the molar masses M from the SWISS-PROT database (Table 1). Volumes V obtained by the programs SIMS (Table 1) or HYDCRYST (HC) and/or HYD-MODEL (HM) (Table 2) are inclusive of the contributions of ligands for calculating the V/M ratios, the molar masses from the crystal structure (Mcryst, Table 2) were used if these were different from the SWISS-PROT data...

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