Insulin titration curve

The titration curve can give no information on the subject of reversible conformational change in insulin because there is extensive pH-dependent association between insulin molecules. The absence of other conformational change is indicated by hydrodynamic measurements of Fredericq (1966). 

Titration curves of insulin have been determined by Tanford and Epstein (1954) and by Fredericq (1954, 1956). As Table XII shows, the count of groups obtained for zinc-free insulin is in agreement with analytical data. This is true in spite of the fact that insulin is insoluble between pH and pH 7. The precipitate is evidently highly hydrated, so that titration of the acidic groups occurs as if they were in solution. 

Tanford and Epstein also determined the titration curve of crystalline zinc insulin, containing one atom of per two insulin molecules. The titration curve of this material differs from that of zinc-free insulin in two ways. (1) Two new groups are titrated for each zinc ion, one near pH 8, the other near pH 12. These presumably represent acidic water molecules attached to Zn... 

Insulin Fig. 1C). Standard inhibition curves are prepared using 10 tubes, each of which contains 0.1 ml of buffer 2 and 0.1 ml of the appropriate dilution of anti-insulin serum. Four pairs of tubes receive 0.1 ml of the insulin standard diluted so as to contain 1.0, 2.5, 5.0, or 10.0 /alU of insulin. The tubes are incubated at 37° for 2 hr, and then 0.1 ml of P I]in-sulin is added to each. After an additional incubation period of 45 min at 37°, 5 ml of Z-gel are added and the tubes are processed as described (see titration curve). 

Understanding of the metal-protein interaction between zinc and insulin has recently been enhanced by studies of titration curves. Crystalline insulin as normally prepared contains a small amount of zinc, and moreover is insoluble between pH 4 and 7, where pH drifts are also observed. Fred-ericq (1954) titrated two fractions of insulin which differed in their solubility at pH 8, and reported that two imidazole groups which dissociate near pH 7 in the soluble form are not evident in the insoluble fraction. 

Fig. 6. Titration curves of insulin, zinc-free (A) and with. 1 mole zinc per 11,500 g. B and C). C is the direct titration of zinc insulin, B the reverse titration with acid or base. A is completely reversible. Data of Tanford and Epstein (1954a, b).

Figure 3 is a graph of the charge on regular insulin as a ftmction of pH. As can be seen from this graph, it is dramatically different from the titration curve assumed in the theoretical calculation. Still, the difference between the assumed titration curve and the actual titration curve would not make any... 

Figure 3. Titration curve of regular insulin. Also shown is the hypothetical titration curve used to show that insulin is theoretically deliverable by iontophoresis. Note that if the actual curve were shifted 5pH units to the right, it would have properties close to ideal for iontophoretic delivery.

Fig. 136. pH Dependence of ADm.t for 0.5% beef zino-insulin relative to a solution at pH 1.60. Measurements in 0.5-om. cells are calculated for 1.0-cm. cells Oi experimental points , points corrected for AD contribution due to light scattering. The curve is the experimental titration curve of Tanford and Epstein (1954) for the COOH ionization range of zinc insulin (Leach and Scheraga, 1960a). 

The titration curves of native and iodinated insulins are compared in Fig. 137. The two curves differ by essentially four groups in the tyrosyl ionization region. A discussion of the departure from four, and the analytical data indicating that the four tyrosyl groups were completely iodinated without any other alteration in the protein, have been presented elsewhere (Gruen et al., 1959a). 

The titration curves of iodinated insulin (in 2 molar KBr to minimize electrostatic effects) are shown in Fig. 138. The first group titrated in the region of positive r is thought to be the e-amino, and the partially titrated second group is the guanidinium group of arginine. The last part of the... 

Fia. 137. Titration curves at 25°C. of zinc-free insulin, , and of iodinated insulin, O, in 0.3 M KCl, showing the difference, Ar, in the number of groups titrated in the region of diiodotyrosyl and tyrosyl ionization. The zero for the r-scale is arbitrary. The protein was partially insoluble in the region to the left of the vertical bar on each curve (Gruen et al., 1959a). 

Fig. 138, Titration curves of iodinated insulin in 2 M KBr at 0, 10, 25, and SS". The filled circles are for the reversed titration the other filled symbols are for duplicate forward titrations. The upper curves are theoretical ones for two independent but overlapping groups with the apparent pK s indicated. The lower curves represent the difference between the experimental points and the theoretical curve (Gruen el al., 1959b).

Iodination of proteins has produced measurable changes in the titration curves. In zein (280), insulin (125), and pepsin (6) the region of the titration curves usually assigned to the phenolic group of tyrosine (pK=10) is displaced in iodinated proteins in the direction of increased acidity by nearly 2 pH units. This is apparently not true for iodinated globin (299). 

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