Recovery of bone and serum proteins from human skeletal tissue • 53 



Allowing an electrophoresed total protein extract to par- 

 tially destain after Coomassie Blue staining produced dis- 

 crete bands of protein in 12 out of the 13 skeletal fragments 

 analyzed. The number and the molecular weight of these 

 bands varied among bone fragments, but generally, the most 

 common band observed was at approximately 68 Ka. Several 

 bands were also observed at 60 Ka, 45 Ka, and 42 Ka. The 

 ability to see bands of protein as the collagen degradation 

 products destain is due to the fact that collagen is a relatively 

 poor Coomassie binder. Therefore difi'erent bands could be 

 observed at varying stages of the destaining process, and any 

 protein that binds Coomassie Blue equally or less well than 

 collagen would not be seen at all. Another problem with the 

 extensive collagen degradation products is the interference in 

 transferring the fossil protein to nitrocellulose paper for im- 

 munological detection. Because a percentage of the protein 

 moves from the gel to the paper with time, it is difficult to 

 transfer enough of the putative intact protein in a reasonable 

 amount of time. 



The total protein extracts were digested with bacterial col- 

 lagenase. Bacterial collagenase is an enzyme that degrades 

 any string of amino acids, Gly-X-Pro or Hypro, to tripep- 

 tides. Since only collagen has repeats of Gly-X-Pro or 

 Hypro, this is the only protein that will be affected by the 

 collagenase treatment. All of the skeletal fragments pro- 

 duced protein that was partially degradable by bacterial col- 

 lagenase, and all digested fossil bone protein extracts ex- 

 hibited at least one Coomassie stainable band upon 

 postdigestion electrophoresis (Figure 2). 



The ability to digest the background smear on the gels with 

 bacterial collagenase is proof that the staining was caused by 

 collagen breakdown products. The existence of Coomassie 

 stainable bands after collagenase treatment is proof of the 

 preservation of several noncollagenous proteins in these fos- 

 sil bones. 



While preliminary identification of these bands can be 

 made based on their molecular weight, immunological iden- 

 tification by reacting the transferred bands to purified anti- 

 bodies is the basic criterion upon which protein identification 

 should be made. This immunodetection technique (com- 

 monly called Western blotting) requires that one and only one 

 band at the appropriate molecular weight recognize the anti- 

 body being used. 



In preliminary analysis of the collagenase digested elec- 

 trotransfers, the proteins albumin, osteonectin, and IgG were 

 identified from these skeletal remains. Albumin was by far 

 the most prevalent among the preserved noncollagenous pro- 

 teins in the samples. The identification of IgG in some of the 

 collagenase extracts demonstrates the ability to detect intact 

 proteins from fossil extracts even in the absence of a clear 

 Coomassie stained band on the gel. 



The demonstration of preservation of noncollagenous pro- 

 teins in fossil bones that have been washed, treated and 

 stored in a museum collection is an important addition to the 



COLLAGENASE 



B. 



oi 



c 



£ 



X 



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lO lO lO 

 CO CO CO 



lo" in lo" 



CVJ C\J CM 

 CO CO CO 



Figure 2. Bacterial collagenase digestion of bone protein 

 from three Mobridge site individuals on an SDS 4-20% 

 polyacrylamide gel stained with Coomassie Brilliant Blue. 

 This is a fully stained (Coomassie) gel of protein bands re- 

 maining after bacterial collagenase digestion of I mg of bone 

 protein extract. The smear of degradation products from Fig- 

 ure 1 is removed, and intact, collagenase-resistant proteins 

 can be seen. A band just above the 66 Ka marker was positive 

 to the rabbit antihuman albumin antibody upon electro- 

 transfer, and two of the three individuals (325357 and 

 325358) had bands that recognized rabbit antihuman os- 

 teonectin antibody. 



growing evidence that fossil mineralized tissue contains mo- 

 lecular information of importance to the paleopathologist. 

 Digestion of fossil bone protein extracts with bacterial col- 

 lagenase will increase our ability to screen these samples for 

 molecular preservation. The preservation of immuno- 

 globulins is particularly intriguing, and opens up the possi- 

 bility that an independent record of disease states remains in 

 the bones of many individuals. 



Zagreb Paleopathology Symp 19SS 



