Evidence of hydrophobic domains in human respiratory mucins. Effect of sodium chloride on hydrophobic binding properties.
Hydrophobic binding properties of purified human respiratory mucins were studied by the fluorescence probe technique using mansylphenylalanine (Mns-Phe) as the fluorescent probe. Mucins were purified from tracheobronchial secretions of cystic fibrosis (CF) and asthmatic patients, as well as from individuals with normal lungs, according to a protocol earlier established in our laboratory. Purified mucins were subjected to reduction-alkylation and Pronase digestion to study the effects of these treatments on the hydrophobic properties of the mucins. In addition, the effects of increased NaCl concentration on the hydrophobic properties of native and reduced-alkylated mucins were also investigated. Native mucins showed evidence of a large number of low-affinity (KD approximately 10(-5) M) binding sites for the hydrophobic ligand Mns-Phe and had between 40 and 50 binding sites/mg of mucin. Reduction of mucin using dithiothreitol in the presence of 6 M guanidine hydrochloride and subsequent alkylation with iodoacetamide apparently caused marked conformational changes in the mucin molecules as revealed by the presence of both high-affinity (KD approximately 10(-6) M) and low-affinity (KD approximately 10(-5) M) binding sites for the probe and an increase in the number of probe binding sites. Pronase digestion of the native and reduced-alkylated mucins almost completely eliminated binding of the fluorescent probe to the mucins, showing that the binding sites are on the nonglycosylated, Pronase-sensitive portion of the mucin molecules. Increasing NaCl concentrations (0.03-1.0 M) did not appreciably alter the native mucin-induced Mns-Phe fluorescence, while that of the reduced-alkylated mucin-induced Mns-Phe fluorescence was progressively increased.(ABSTRACT TRUNCATED AT 250 WORDS)
Shankar
V
V
College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Naziruddin
B
B
Reyes de la Rocha
S
S
Sachdev
G P
GP
D000596:Amino Acids / Q000032:analysis; D001249:Asthma / Q000175:diagnosis; D001665:Binding Sites; D001980:Bronchi / Q000032:analysis / Q000187:drug effects; D002845:Chromatography; D003550:Cystic Fibrosis / Q000175:diagnosis; D005456:Fluorescent Dyes; D006801:Humans; D009077:Mucins / Q000378:metabolism*; D011487:Protein Conformation; D012965:Sodium Chloride / Q000494:pharmacology*; D012995:Solubility; D013183:Sputum / Q000032:analysis*; D014132:Trachea / Q000032:analysis / Q000187:drug effects
D000596:Amino Acids; D005456:Fluorescent Dyes; D009077:Mucins; D012965:Sodium Chloride