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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Meat Safety and Quality » Research » Publications at this Location » Publication #112671
Title: EFFECT OF EXTRACTION BUFFER ON ESTIMATING CALPAIN AND CALPASTATIN ACTIVITY IN POSTMORTEM OVINE MUSCLE

Author
item VEISETH, EVA - AGRIC. UNIV. OF NORWAY
item Koohmaraie, Mohammad

Submitted to: Journal of Meat Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/18/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary: Tenderness is the most important factor related to consumers' acceptability of meat, and it is known to improve during postmortem storage. Tenderization is caused by breakdown of key muscle proteins, and it is caused by the calpain enzyme system. Calpains have been a subject of intense research in meat science during the last two decades. Some proteins, such as the calpains, will start to precipitate when the pH approaches their isoelectric point (pI). To prevent loss of calpains during extraction, it is important that the homogenate pH is kept well above their pI. Different groups have used different buffers and extraction volumes to study the calpain system in postmortem muscle. There seems to be an agreement with regard to effect of postmortem storage on u-calpain and calpastatin but not for the effect of postmortem storage on m-calpain. Some studies have indicated that m-calpain also loses its activity during postmortem storage. However, we have failed to observe any change in the activity of m-calpain. We hypothesized that m-calpain discrepancy is caused by use of different extraction buffers. In this study, we show that it is crucial to keep homogenate pH above 6.2 (either by using an appropriate combination of buffering capacity and buffer volume or by adjusting the pH of the muscle homogenate prior to the first centrifugation), for accurate quantification of calpain and calpastatin activity in postmortem muscle.

Technical Abstract: The effect of extraction buffer on extractable calpain and calpastatin activity in postmortem muscles was examined. Muscles were removed from ovine carcasses 24 h after slaughter and extracted with 3 volumes of two extraction buffers containing 20 (pH 7.5) and 100 (pH 8.3) mM Tris base. There was a significant difference in pH of the muscle homogenates, having a pH of 5.84 and 7.58 for 20 and 100 mM Tris base, respectively. Calpastatin, u-calpain, and m-calpain all had significantly reduced activity in extracts made with 20 mM Tris base buffer compared to 100 mM, showing a loss of, respectively, 30, 57, and 37%. These results indicate the impact of choice of buffer on the extractable calpains and calpastatin activity from postmortem muscle. To avoid loss of calpains due to isoelectric precipitation, the pH of the muscle homogenate (after homogenization and prior to the first centrifugation) must be above 6.2.