We have continued our study of human albumin (HA) at pH 7.0 by investigating the stabilizing effects of Cl- on thermal denaturation. At low ionic strength (i.e in water), the protein shows 2 major thermal transitions by differential scanning calorimetry and by changes in intrinsic trp fluorescence and circular dichroism (CD), i.e. HA denatures in a non-cooperative manner. The denaturation temperatures (Td's) measured by these techniques correlate well. In view of the presence of a single trp in HA, the fluorescence experiments suggest that with increase in temperature an unfolding of secondary structure near the trp initially occurs followed by unfolding of the region containing the trp at a higher temperature. With increasing [NaCl], Td(1) and Td(2) increase but Td(1) more rapidly such that between 75 and 150 mM NaCl the 2 transitions coalesce. At 150 mM NaCl, HA unfolds in an almost ideal 2-state manner, i.e. highly cooperatively. NaI appears to promote cooperativity more efficiently than NaCl thereby suggesting that halide anion binding is the driving force. CD spectra of HA in water and 150 mM NaCl are essentially the same thereby indicating a comparable secondary structure content of the 2 protein forms. We have attempted to measure the trp to free SH distance by fluorescence resonance energy transfer (FRET) to determine whether HA is more relaxed in water than 150 mM NaCl. However, these groups appear to be too close for such a determination. A fragment, T45, comprised of domains II and III, was prepared by tryptic digest. A small amount of pure although nicked T45 was obtained. Thermal denaturation of this T45 in water by CD revealed a single transition with a Td intermediate to those observed with the intact HA. This suggests that removal of domain I causes an opening of the fragment about the """"""""hinge"""""""" region between domains II and III thereby making T45 a poor model for the intact HA. FRET is being used to investigate the proximity of CD32 and CD45 receptors on human neutrophils. Occurrence of FRET between labeled CD32 and CD45 receptors establishes the feasibility of regulation by CD45 of CD32 Fc receptor mediated mobilization and tyr phosphorylation. Preliminary results of the thermal unfolding of alpha-1 proteinase inhibitor, a serpin (i.e. serine protease inhibitor), in 150 mM NaCl at pH 7.0 shows by CD, upon the first heating, a single transition with a Td of ca. 61 degrees C.
