The mechanism of heme uptake and release by cell membranes and proteins was investigated using a model system of carbon monoxide heme, human globin, and phosphatidyl choline single bilayer vesicles. The combination of heme with globin involves the rapid (t(, 1/2) << 1 ms) formation of an intermediate complex described by the equation:
K(,d) k(,3)
heme + globin heme-globin hemoglobin
From stopped flow measurements, observed rates were obtained and absolute spectra were constructed for intermediate reaction mixtures. At pH 7.2, 10(DEGREES)C, the K(,d) is 22 (mu)M and k(,3) equals 700 s('-1). The effects of pH, glycerol, organic phosphate, and substituted heme were examined. The results suggested that heme is rapidly adsorbed onto hydrophobic areas of the protein to form a complex exhibiting spectral characteristics of heme dissolved in liposomes. The proper orientation of heme induces a rate limiting conformational change in globin, followed by rapid iron-histidine binding.
The reaction of heme with phospholipid vesicles was analyzed as a simple binding phenomenon by calculating equilibrium fractions of bound and unbound heme. The K(,d) is 0.7 (mu)M with 7.8 lipid molecules/binding site. The observed binding rate was defined as k(,obs) = k(,1)(lipid phosphate) + k(,2), where the association rate, k(,1), equals 1.36 (mu)M P('-1)s('-1) and the dissociation rate, k(,2), equals 2-5 s('-1). Multilamellar vesicles and liposomes containing stearylamine exhibited predictable deviations.
Fluorescence measurements using pyrene labelled phospholipid indicate that rates of heme transfer between and release from liposomes are similar. The combination of globin with membrane bound heme is much slower (t(, 1/2) (DBLTURN) 200 ms) than with free heme (t(, 1/2) (DBLTURN) 1 ms) due to partitioning between the various phases. A theoretical expression for k(,obs) was derived and evaluated using constants obtained from independent experiments.
Our results demonstrate the rapid exchange of heme between liposomes and extraction by apoproteins which occurs by a rate limiting first order step of heme dissociation into the aqueous phase. A carrier protein is not necessary for heme transport but may be required for compartmentalization.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/15720 |
| Date | January 1982 |
| Creators | ROSE, MELANIE YARBROUGH |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | application/pdf |
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