title: Verification of simple hydration/dehydration methods to characterize multiple water compartments on tendon type 1 collagen.
abstract: A molecular model of collagen hydration is used to validate centrifugal dehydration force (CDF) and re-hydration isotherm (RHI) methods to measure and characterize hydration compartments on bovine tendon. The CDF method assesses fluid flow rate from flexor and extensor tendons expressed in (g-water/g-dry mass-minute) and hydration capacity of compartments in (g-water/g-dry mass). Measured water compartment capacities agree with the molecular model of collagen hydration [Fullerton GD, Rahal A. Collagen structure: the molecular source of tendon magic angle effect. J Mag Reson Imag 2007;25:345-361; Fullerton GD, Amurao MR. Evidence that collagen and tendon have monolayer water coverage in the native state. Cell Biol Int 2006;30(1):56-65]. Native tendon hydration has monolayer coverage on collagen h(m)=1.6 g/g which divides into primary hydration on polar surfaces h(pp)=0.8 g/g and secondary hydration h(s)=0.8 g/g bridging over hydrophobic surfaces. Primary hydration is hydrogen bonded to collagen polar side chains h(psc)=0.54 g/g with small free energy or to the protein main chain hydration h(pmc)=0.26 g/g with greater free energy of binding. The CDF method replaces the more time consuming water proton NMR spin-lattice dehydration (NMR titration) method, confirms the presence of three non-bulk water compartments on collagen (h(pmc)=0.26 g/g, h(pp)=0.8 g/g and h(m)=1.6 g/g). This CDF method provides the most reproducible experimental measure of total tissue non-bulk water (TNBW). The re-hydration isotherm method, on the other hand, provides the most accurate measure of the Ramachandran water-bridge capacity h(Ra)=0.0656 g/g. The only equipment needed are: microfilterfuge tubes, a microcentrifuge capable of 14,000 x g or 4MPa, a vacuum drying oven, an accurate balance and curve fitting ability. The newly validated methods should be useful for characterizing multiple water compartments in biological and non-biological materials by allowing direct measurement of water compartment changes induced by pH, co-solute salt, glycation and protein cross-linking.
authors.affiliation: Cellular and Structural Biology Department, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA. cameron@uthscsa.edu
authors.forename: Ivan L
authors.initials: IL
authors.lastname: Cameron
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Short
authors.forename: Gary D
authors.initials: GD
authors.lastname: Fullerton
mesh_terms: D000818:Animals; D002417:Cattle; D002498:Centrifugation; D024042:Collagen Type I / Q000737:chemistry* / Q000378:metabolism; D003890:Desiccation; D008954:Models, Biological; D055672:Static Electricity; D013710:Tendons / Q000737:chemistry* / Q000378:metabolism; D014867:Water / Q000032:analysis* / Q000378:metabolism
chemical_list: D024042:Collagen Type I; D014867:Water