Studies on serum albumin and hemoglobin: The two principal transport proteins in blood.

摘要

The properties of the two principal transport proteins in human blood, human serum albumin (HSA) and hemoglobin, were studied in this work.;First, the structural changes in human serum albumin upon its binding of fatty acid anions were investigated using three different spectroscopic techniques, UV difference, dansylsarcosine fluorescence and pyridoxal phosphate (PLP) probing spectrophotometry, and isothermal titration calorimetry. Red-shifts in HSA's UV spectrum following the addition of long chain fatty acid (LCFA) anions appear to reflect a major change in its conformation. Similar but smaller changes after the addition of decanoate and other medium chain-length fatty acid (MCFA) anions appear to reflect either a smaller or a less complete conformation change. Dansylsarcosine's fluorescence, when bound to a site near Tyr-411, is sharply reduced by low levels of MCFA anions known to bind to the same site. Similar levels of LCFA anions have only a small effect on its fluorescence but suppress it at higher levels. Prominent, and characteristic, spectral changes between ∼300 and 480 nm accompanying PLP's reaction with fatty acid-free HSA are only slightly affected by four equivalents of LCFA anion but almost completely eliminated by five equivalents. Similar levels of MCFA anions have little affect on PLP's reaction with HSA but gradually suppress it at higher levels. These results appear to reflect a concerted change in HSA's conformation upon the, more or less, simultaneous binding of five LCFA anions. MCFA anions appear to bind in a step-wise manner and to produce a more gradual change in conformation. Isothermal titrations with LCFA and MCFA anions suggest the former bind cooperatively whereas the latter do not.;Second, to understand why the thiol contents of some commercial albumin preparations are so low and HSA isolated form fresh plasma contains only ∼65% reduced thiol, we studied the mechanism of the decrease of the thiol content of HSA in freshly isolated plasma and the inhibition of this process. The thiol content of fresh plasma HSA undergoes a biphasic decrease.;Third, to understand the mechanisms of the reaction of hemoglobin with S-nitrosothiols, the reactions of oxyhemoglobin with various S-nitrosothiols were investigated. The S-nitroso derivatives of glutathione, N-acetyl-penicillamine, L-cysteine, 3-mercaptopropionic acid and 2-mercaptoethanol were shown to react with bovine oxyhemoglobin (HbO2) at different rates and with different kinetics. (Abstract shortened by UMI.).