Denaturation future directions

Given that hydroxylamine reacts rapidly with heme proteins and other oxidants to produce NO [53], the hydrolysis of hydroxyurea to hydroxylamine also provides an alternative mechanism of NO formation from hydroxyurea, potentially compatible with the observed clinical increases in NO metabolites during hydroxyurea therapy. Incubation of hydroxyurea with human blood in the presence of urease results in the formation of HbNO [122]. This reaction also produces metHb and the NO metabolites nitrite and nitrate and time course studies show that the HbNO forms quickly and reaches a peak after 15 min [122]. Consistent with earlier reports, the incubation ofhy-droxyurea (10 mM) and blood in the absence of urease or with heat-denatured urease fails to produce HbNO over 2 h and suggests that HbNO formation occurs through the reactions of hemoglobin and hydroxylamine, formed by the urease-mediated hydrolysis of hydroxyurea [122]. Significantly, these results confirm that the kinetics of HbNO formation from the direct reactions of hydroxyurea with any blood component occur too slowly to account for the observed in vivo increase in HbNO and focus future work on the hydrolytic metabolism of hydroxyurea. 

Some counterbalancing disadvantages must be noted in the method. Very few proteins are naturally fluorescent. Most proteins if they are to be studied by this method must be converted into fluorescent derivatives by attaching to them suitable fluorescent groups, and this must be done in such a way as not to denature the protein, or to alter its size or shape appreciably. Moreover the observed polarization measurements are a function both of the time interval t and of the relaxation time of the protein. The values of t for fluorescent protein molecules have not yet been directly measured and this fact, therefore, places some limitations on the conclusions which can at present be drawn from the method. However, methods are available for the independent determination of r. This source of ambiguity is, therefore, capable of being eliminated in the future. 

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