Investigation of molecular motion of proteoglycans in cartilage by 13C magnetic resonance.

D A Torchia,M A Hasson,V C Hascall

doi:10.1016/s0021-9258(17)40297-3

Abstract

13C nmr spectral parameters were measured for intact bovine nasal cartilage tissue, the purified proteoglycan aggregate, and chondroitin 4-sulfate. A comparison of integrated intensities obtained for four different samples of fresh tissue with an ethylene glycol standard indicated that at least 80% of the total glycosaminoglycan carbons in the tissue contributed to the spectrum. This result was confirmed by intensity measurements obtained at 56 degrees on fresh tissue and at 37 degrees after extensive papain digestion of fresh tissue. Spin lattice relaxation times and nuclear Overhauser enhancements were analyzed in terms of the following models of molecular motion: (a) single correlation time; (b) log X2 distribution of correlation times; and (c) anisotropic motion. The analysis indicates that the segmental motions of glycosaminoglycan chains are characterized by a broad distribution of correlation times centered at about 50 ns. Slow motion contributions to glycosaminoglycan line widths were reduced by dipolar decoupling (gammaH2/2pi = 65 kHz). Collagen intensity was observed in dipolar decoupled spectra, but not in scalar decoupled spectra of intact tissue, showing that the type II collagen in cartilage undergoes anisotropic motion like the type I collagen in tendon. Only glycosaminoglycan resonances were observed in spectra of a solution of proteoglycan aggregate before and after chondroitinase digestion. After subsequent digestion with papain, protein resonances were observed. These results suggest that the protein portions of the proteoglycan aggregate structure, in contrast with the glycosaminoglycan chains, have restricted backbone mobility and consequently a defined backbone structure.

Highlights

The fact that the line widths observed in the tissue are 5100 Hz implies that the vast majority of correlation times characterizing reorientation of the C-H vectors in the glycosaminoglycan are less than 1 ps [13]
It is interesting that collagen spectral intensity is observed upon dipolar decoupling since this shows that anisotropic motion is present even in the highly organized collagen fibrils
That a secondary structure exists and does not depend upon the presence of the glycosaminoglycan chains is indicated by the absence of protein resonances in the spectrum of the chondroitinase-treated aggregate

Summary

Introduction

A comparison of integrated intensities obtained for four different samples of fresh tissue with an ethylene glycol standard indicated that at least 80% of the total glycosaminoglycan carbons in the tissue contributed to the spectrum. This result was confirmed by intensity measurements obtained at 56” on fresh tissue and at 37” after extensive papain digestion of fresh tissue. Collagen intensity was observed in dipolar decoupled spectra, but not in scalar decoupled spectra of intact tissue, showing that the type II collagen in cartilage undergoes anisotropic motion like the type I collagen in tendon

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Conclusion