ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 190, No. 2, October, pp. 657-661, 1978

Cross-Polarization 13C Nuclear Magnetic Resonance Spectroscopy of Collagen

JACOB SCHAEFER,’ E. 0. STEJSKAL,l CURTIS F. BREWER,2* a HAROLD D. KEISER:5 AND H. STERNLICHT’

Monsanto Company, St. Louis, Missouri 63166, Albert Einstein College of Medicine, Bronx, New York 10461, and Case- Western Reserve University School of Medicine, Cleveland, Ohio 44106

Received April 10,1978; revised June 9,1978

Natural abundance ‘% nuclear magnetic resonance (nmr) spectra have been obtained for samples of a variety of native collagens by use of cross-polarization (CP) techniques which permit high resolution natural abundance 13C mnr spectra of solids to be obtained with high sensitivity. The CP ‘% nmr spectra of lyophilized skin and tendon collagens consisted of two broad resonance envelopes spanning a five kHz range. Hydrated tendon collagen gave rise to a CP spectrum very similar to that obtained for the lyophilized sample, indicating that it retains its solid-like properties. In contrast, hydrated skin collagen became denatured under the conditions of the CP experiment and subsequently gave rise to a conventional high-resolution Fourier transform (FT) nmr spectrum. The CP 13C nmr spectrum of ivory was similar to those of lyophiiized skin and tendon collagens, demonstrat- ing the solid-like character of the collagen in dentine, whereas the CP spectrum of bovine nasal cartilage reflected the presence of highly mobile proteoglycan components in addition to relatively rigid collagen molecules. In the case of ivory, the resolution of the CP spectrum was enhanced by “magic angle” spinning to a degree approaching that of conventional FT 13C nmr spectra of denatured collagen in solution, Because of its ability to probe the dynamic properties of solid-like biological molecules, CP 13C nmr spectroscopy should be a valuable investigative tool for future studies.

Conventional high-resolution 13C Fourier transform nuclear magnetic resonance spectroscopy has proven a useful technique for determining the structure, motions, and interactions of relatively small or flexible biological molecules in solution. However, solids or inflexible molecules, such as native

’ Corporate Research Department, Monsanto Com- pany, St. Louis, MO. 63166.

’ Department of Pharmacology and Department of Microbiology and Immunology, Albert Einstein Col- lege of Medicine, Bronx, N. Y. 10461.

’ Research Career Development Awardee, 5-K04 CA 00184, National Cancer Institute.

4 Department of Medicine, Albert Einstein College of Medicine, Bronx. N. Y. 10461.

5 Research Career Development Awclrdee, 5-K04- AM 00024, National Institute of Arthritis, Metabolic and Digestive Diseases.

’ Department of Pharmacology, Case-Western Re- serve University School of Medicine, Cleveland, Ohio 44106.

collagen and DNA, have not yielded con- ventional 13C or ‘H nmr spectra because the static magnetic dipolar interactions be- tween their nuclear spins produce nmr line-widths too broad to be observable. Cross-polarization 13C nmr is a recently in- troduced technique by which the dipolar broadening of 13C resonances by protons is removed by strong proton resonant decou- pling and polarization transfer from protons to carbons is induced via static dipolar in- teractions. This results in strong signal en- hancement of the carbon spin resonances, permitting high resolution natural abun- dance 13C nmr spectra of solids to be ob- tained with high sensitivity (1, 2).

Cross-polarization 13C nmr has been suc- cessfully applied to the study of glassy solid-like organic polymers (3) and to a natural abundance 13C study of lipid meso- phases and selectively labeled carbon sites in bacterial membranes (4). In the present

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0003-9861/78/1902-0657$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

658 SCHAEFER ET AI.

paper, we present the CP7 13C nmr spectra of several types of collagen and two colla- gen-rich tissues, ivory and cartilage; we also indicate the type of information regarding the structural and dynamic properties of collagen which can be made with CP13C nmr in its present state and suggest certain technical refinements which can make CP 13C nmr a highly useful tool for the study of connective tissues.

MATERIALS AND METHODS

Materials. Acid-solubilized calf skin and rat skin collagen? were generous gifts from Dr. Sam Seifter. Bovine achilles tendon collagen was purchased from Calbiochem, , San Diego, Cal. A high quality billiard ball (A. E. Schmidt Co., St. Louis, MO.) was machined into a suitable nmr sample and used as the source of ivory. Bovine nasal septa were obtained fresh from a local slaughter house, stored at -20” and thawed immediately before use. Proteoglycan aggregate frac- tion was prepared as previously described (5-7). Shredded nasal cartilage was depleted of proteoglycan by extraction with 4 M guanadine-HCl, 0.05 M sodium acetate, pH 5.8, at 4’C for 48 hr (5).

Methods. Cross-polarization and dipolar-decoupled FT 13C nmr experiments were performed on a modified Bruker nmr spectrometer operating at 22.6 MHz for carbons and 90 MHz for protons. The spectrometer was equipped with a time-shared external 19F field- frequency stabilization system and a 13C quadrature detector. Dipolar-decoupled CP 13C nmr spectra were obtained at room temperature using single Hartmann- Hahn cross-polarization contact times of 1 msec du- ration (l), a proton spin lock of 6 ms or less and a sequence repetition time of 0.5 s. No provisions were made for cooling to compensate for any radio-fre- quency heating. A short decoupling duty cycle was used to minimize sample heating, but this procedure introduced an intrinsic line broadening of 50 Hz inas- much as the ‘%-free induction decay was recorded for 6 ms or less. Carbon-13 and proton radio-frequency fields of 32 and 8 gauss, respectively, were generated by wide band power amplifiers capable of delivering several hundred watts to a lO-mm dual insert. Data acquisition times varied from a few minutes for dense solids to a few hours for disperse solids containing large quantities of water. Lyophiid collagen samples (50-109 mg) were run in lo-mm nmr tubes. The car- tilage sample was a cylindrical plug, approximately 8 x 30 mm, placed in a IO-mm sample tube.

Magic-angle spinning experiments employed a modification of the rotor geometry introduced by Lowe (8) and further developed by Kessemeier and Norberg (9). The rotor used in these experiments was

’ Abbreviations used: CP, cross-polarization; FT, Fourier transform.

of ivory machined from a piece of a billiard ball. The rotation axis of the rotor was aligned at 54.7” * lo relative to the static magnetic field and the rotor sample was spun at 3 KHz.

Carbon-13 nmr spectra using ‘H scalar decoupling conditions were also obtained on a JEOL PFT-100 spectrometer operating at 24.197 MHz in the pulse FT mode. Sample solutions, 1.3 ml in IO-mm mm tubes, contained 50-100 mg/ml of the appropriate collagen in 1 M NaCl in D20.

RESULTS AND DISCUSSION

The 13C mrrr spectra obtained for tendon, skin and dentine collagens consist of two broad resonance envelopes spanning a 5 KHz range (Fig. 1). By virtue of their chem- ical shift positions, the downfield resonance envelope can be assigned to amino acid carbonyl resonances and the relatively in- tense upfield resonance envelope to ali- phatic amino acid resonances. The similar- ity in line shapes, positions, and intensities of the different collagen samples reflects the similarity of the amino acid composi- tion of these type I collagens and their common solid-state character. These CP nmr spectra resemble the spectra obtained by Torchia and VanderHart for tendon col- lagen and ‘3C-enriched chick calvaria col- lagen using only strong dipolar decoupling conditions and the single spectrum they obtained using cross-polarization signal en- hancement (10).

The dynamic behavior of the collagen in different connective tissues can be differ- entiated by noting whether CP (dipolar- decoupled) or conventional (scalar-decou- pled) FT nmr spectra can be obtained un- der given conditions. Lyophilized skin and tendon collagens were found to yield CP spectra, but not FT spectra, reflecting their solid state character. Upon hydration, ten- don collagen produced a CP spectrum which, except for small changes in intensity, appears to be the same as that obtained prior to hydration (Fig. l), indicating that the hydrated tendon collagen retained its solid-like properties. Hydration of the two skin collagen samples resulted in the loss of their ability to give rise to CP nmr spectra. After completion of the CP nmr experi- ment, the hydrated collagen samples were studied under conventional FT nmr condi- tions. The hydrated tendon collagen sample

CROSS-POLARIZATION 13C NMR OF COLLAGEN 659

FIG. 1. Cross-polarization ‘C mnr spectra of lyophilized bovine tendon, calf skin, and rat skin collagens and of ivory with and without magic-angle spinning; cross-polarization 13C nmr spectrum of hydrated bovine tendon collagen and FT ‘% mnr spectra of similarly hydrated skin collagens

failed to yield an FT nmr spectrum, but the hydrated skin collagen samples gave rise to FT nmr spectra characteristic of denatured collagen (Fig. 1). The CP experiment re- quires strong dipolar decoupling conditions which produce local heat gradients in the samples. Because the probe was not tem- perature regulated, we believe that the hy- drated skin collagen samples were dena- tured by heat in the course of the CP ex- periment, whereas, under the same condi- tions, the tendon collagen retained its over- all solid-like dynamic properties. The fail- ure of tendon collagen to denature under these conditions, together with the persist- ence of a CP spectrum, reflects the added degree of immobilization and stability con- ferred on tendon collagen by its relatively high degree of cross-linkage compared with skin collagen (11).

The 13CP nmr spectra of complex chem- ical systems such as collagen are generally poorly resolved because of complexities in- troduced by overlapping chemical shift an- isotropies. In some cases, chemical shift anisotropy can be removed by rotating a sample around an axis 54.7’ relative to the external magnetic field at a spinning fre- quency somewhat greater than the disper- sion of the chemical shifts of the sample. This procedure averages the chemial shift anisotropic dispersions to their isotropic values (12,13). Ivory or elephant dentine is

composed of 25% conagen (14) and, by com- parison with the CP spectra of lyophilized skin and tendon collagens shown in Fig. 1, it is evident that the CP spectrum of ivory is due to its collagen and that the collagen in the intact tissue has solid-like dynamic properties. High speed rotation of an ivory rotor at the “magic-angle” yielded a CP spectrum of its collagen whose degree of resolution approaches that observed in the conventional FT nmr spectrum of dena- tured collagen in solution. The chemical shifts of about a half-dozen individual res- onances are distinguishable within the up- field aliphatic region and the 2-kHz-wide carbonyl envelope becomes a single reso- nance line only about 200-Hz wide.

Cartilage is an avascular and relatively acellular structural matrix of connective tis- sue which is composed of 75% water and about equal remaining amounts of collagen and proteoglycan (15). The proteoglycan of bovine nasal cartilage consists of subunits, with an average molecular weight of 2.5 x 106, as many as 140 of which are linked noncovalently to hyaluronic acid to form large aggregates. The proteoglycan subunit consists of about 100 chondroitin 4-sulfate chains 30 to 40 disaccharides long, and aboLt 50 relatively short keratin sulfate chains, both covalently linked at one end to a linear core protein (16,17). In terms of its overall composition, bovine nasal cartilage

660 SCHAEFER ET AL.

proteoglycan is 85% chondroitin sulfate, 8% protein, 6% keratin sulfate, and less than 1% hyaluronic acid (18).

Brewer and Keiser (19) and Torchia et. al. (20) found that the high resolution FT 13C nmr spectrum of bovine nasal cartilage reveals only resonances of the chondroitin 4-sulfate chains of its proteoglycan compo- nent. Analysis of the 13C spectra of various proteoglycan subfractions suggested that the chondroitin 4-sulfate chains of proteo- glycan in intact cartilage possess a mobility which approaches that of the free polysac- charide in aqueous solution. The lack of resonances from the collagen component of the cartilage suggested that it was undergo- ing much slower motions and that there was little if any interaction with the much more mobile chondroitin sulfate chains, a finding which has important implications with respect to the structural and func- tional properties of the tissue.

In order to further probe the dynamic properties of the collagen and proteoglycan in cartilage, we obtained the CP 13C nmr spectrum of intact bovine nasal cartilage, lyophilized proteoglycan aggregates, and proteoglycan-depleted cartilage shreds (Fig. 2). For comparative purposes, the fig- ure includes a high resolution FT 13C nmr spectrum of cartilage obtained with suffi- cient instrumental filtering so that the res- onance linewidths of the chondroitin 4-sul- fate chains are approximately the same as in the CP spectra. The CP spectrum of intact cartilage shows the presence of two distinct features: (a) relatively sharp reso- nances associated with the proteoglycan chondroitin 4-sulfate chains (which are the only resonances observed in the high reso- lution FT spectrum); and (b) a broad reso- nance envelope under the sharper peaks. The sharp peaks due to proteoglycan ap- pear in the CP spectrum of cartilage despite the presence of motion because the cross- relaxation transfer rates for these mobile carbons was sufficient to produce a minor signal (3). The intensity distribution of the underlying broad resonance closely resem- ble the CP spectrum of the proteoglycan- depleted cartilage shreds as well as the spectrum of the lyophilized rat skin colla- gen. Considering that most if not all of the

-----4 pmty*n ernaddl shredded nasaL c‘u-c%e

7” ,_. Lyophilized rar-skin couAgen

FIG. 2. Cross-polarization and Fourier transform % nmr spectra of bovine nasal cartilage and cross-polar- ization ‘% nmr spectra of lyophilized proteoglycan aggregate fraction (PGC), proteoglycan-depleted bo- vine nasal cartilage shreds, and lyophilized rat-skin collagen.

proteoglycan in cartilage appears to be in rapid motion (19, 20) and can, therefore, not be expected to give rise to broad reso- nances in the CP spectrum, it is reasonable to conclude that the broad resonance en- velope in the CP spectrum of cartilage is due to its collagen component. The CP spectrum of cartilage thus serves to confirm that the collagen and proteoglycan in car- tilage matrix have independent and very different dynamic properties and do not interact to any great extent, if at all.

The observations reported here indicate that CP 13C nmr techniques can be used to obtain spectra of biologically important rigid polymers such as collagen which yield information even at an elementary level of analysis. The potential for future study is vast. For example, through the incorpora- tion of specific isotopic labels into the col- lagen produced in culture, it should be pos- sible to identify the carbon atoms respon- sible for each of the individual resonances making up the broad envelopes of the CP spectra, permitting characterization of the dynamic behavior of individual atoms un- der various conditions. The dynamic prop- erties of collagens of various types can be compared to determine whether differences in the primary structure of collagens relate to their specific functions. Abnormal colla- gens, such as those present in inherited disorders of connective tissue, may be iden-

CROSS-POLARIZATION 13C NMR OF COLLAGEN 661

tifiable by these techniques and the CP spectra of 13C-enriched collagen produced in vitro by human fibroblasts from biopsies or amniocenteses may be diagnostically useful. Finally, it should be possible to de- velop rotor systems allowing magic-angle spinning of samples which, unlike ivory, cannot be directly machined into rotors. The enhanced degree of resonance resolu- tion achieved by magic-angle spinning will greatly simplify the interpretation of CP spectra and will generally facilitate analysis of CP mrrr spectroscopy in all its potential applications.

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