title: Strategies for labeling proteins with PARACEST agents.
abstract: Reactive surface lysine groups on the monoclonal antibody (3G4) and on human serum albumin (HSA) were labeled with two different PARACEST chelates. Between 7.4 and 10.1 chelates were added per 3G4 molecule and between 5.6 and 5.9 chelates per molecule of HSA, depending upon which conjugation chemistry was used. The immunoreactivity of 3G4 as measured by ELISA assays was highly dependent upon the number of attached chelates: 88% immunoreactivity with 7.4 chelates per antibody versus only 17% immunoreactivity with 10.1 chelates per antibody. Upon conjugation to 3G4, the bound water lifetime of Eu-1 increased only marginally, up from 53μs for the non-conjugated chelate to 65-77μs for conjugated chelates. Conjugation of a chelate Eu-2 to HSA via a single side-chain group also resulted in little or no change in bound water lifetime (73-75μs for both the conjugated and non-conjugated forms). These data indicate that exchange of water molecules protons between the inner-sphere site on covalently attached PARACEST agent and bulk water is largely unaffected by the mode of attachment of the agent to the protein and likely its chemical surroundings on the surface of the protein.
authors.affiliation: Department of Chemistry, University of Texas at Dallas, PO Box 830688, Richardson, TX 75083, USA.
authors.forename: Olga
authors.initials: O
authors.lastname: Vasalatiy
authors.forename: Piyu
authors.initials: P
authors.lastname: Zhao
authors.forename: Mark
authors.initials: M
authors.lastname: Woods
authors.forename: Andrei
authors.initials: A
authors.lastname: Marconescu
authors.forename: Aminta
authors.initials: A
authors.lastname: Castillo-Muzquiz
authors.forename: Philip
authors.initials: P
authors.lastname: Thorpe
authors.forename: Garry E
authors.initials: GE
authors.lastname: Kiefer
authors.forename: A Dean
authors.initials: AD
authors.lastname: Sherry
mesh_terms: D002614:Chelating Agents / Q000138:chemical synthesis / Q000737:chemistry*; D003287:Contrast Media / Q000138:chemical synthesis / Q000737:chemistry*; D000080027:Cyclams; D005063:Europium / Q000737:chemistry; D006571:Heterocyclic Compounds / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D008279:Magnetic Resonance Imaging / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011522:Protons; D015398:Signal Transduction; D014867:Water / Q000737:chemistry; D015018:Ytterbium / Q000737:chemistry
chemical_list: D002614:Chelating Agents; D003287:Contrast Media; D000080027:Cyclams; D006571:Heterocyclic Compounds; D008024:Ligands; D011506:Proteins; D011522:Protons; D014867:Water; D005063:Europium; C038072:cyclen; D015018:Ytterbium