With respect to the mechanism of chaperone-like activity, we examined the behavior of haptoglobin under heat shock conditions. Secondary structure changes during heat treatment were followed by circular dichroism, Raman and infrared spectroscopy. A model of the haptoglobin tetramer, based on its sequence homology with serine proteases and the CCP modules, has been proposed. Sequence regions responsible for the chaperone-like activity were not fully identical with the region that takes part in formation of the hemoglobin-haptoglobin complex. We can postulate the presence of at least two different chaperone-binding sites on each haptoglobin heavy chain.

Human haptoglobin (Hp) has been shown to have chaperone-like activity in preventing thermally induced aggregation of catalase and γ-crystallin. No differences in the chaperone- like behaviour of genetic types Hp 1-1 and a mixture of types Hp 2-1 and Hp 2-2 (i.e. Hp II) were found. Haptoglobin not only suppresses heat-induced aggregation of proteins but also prevents γ-crystallin from aggregation by oxidative stress. In addition, haptoglobin also provides protection against glycation-induced inactivation of catalase by glyceraldehyde. Chaperone-like activity of haptoglobin decreases in the course of its glycation. Refolding studies have shown that Hp exhibits its chaperone-like activity predominantly on the unfolding and not on the refolding pathway. Although Hp and α-crystallin have no sequence similarities, it seems that their chaperone-like activities are of the same type.

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In order to elucidate the mechanism of complex formation between human haptoglobin, Hp II, and bovine hemoglobin, Hb, measurements of the peroxidase activity and histidine determinations, as well as spectrophtometric and optical rotatroy dispersion titration experiments were performed. Following the raaction by slowly mixing the constituents of the complex in small portinos, the geometry of the complex turns out to depend on the sequence of mixing of the reactants. Adding Hb to Hp, a final complex Hb2→ Hp is formed. Teh inverse sequence of mixing leads to a complex of identical composition (Hp → Hb2), but different arrangement of the constituents, as shown by differences in the accessibility of histidine residues in both types of the complex. As suggested by the comparison of the histidine balance of both complexes, Hb2→ Hp may be described by the combination of αβ‐dimers of Hb with Hp (Hp (αβ)4), while Hp → Hb2 corresponds to the complex with two Hb tetramers (Hp(α2β2)2). Investigating the saturation process of complex formation in greater detail by spectrophtometric titration and optical rotatory dispersion, intermediary complexes are observed which prove the mechanism of complex formation to be a consecutive association process. Both series of complexes, Hp → Hb and Hb → Hp, seem to generate closely related configurations characterized by similar spectral and functional properties.

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