Antisera directed against the cytoplasmic portion of human erythrocyte Band 3 were used to follow the degradation of the band 3 molecule. Small amounts of Band 3 were degraded when well-washed red cell membrane ghosts were incubated in the cold; this process was greatly accelerated by incubating ghosts were incubated in the cold; this process was greatly accelerated by incubating ghosts at 37 degrees C. Band 3 labeled with pyridoxal-phosphate was digested at comparable rates. Band 3 digestion also took place when alkali-extracted ghost membranes were incubated at 37 degrees for prolonged periods. These results suggest that human erythrocytes contain tightly bound, membrane-associated proteolytic activity.

We examined chemical reactions in mouse lysozyme after incubation under physiological conditions (pH 7 and 37 degrees C). After incubation for 8 weeks, racemization was observed specifically at Asn127 among the 19 Asp/Asn residues in mouse lysozyme. Furthermore, analysis of the primary structure showed that the racemized residue was not Asp, but Asn, which demonstrates that deamidation and isomerization did not occur. These results mean that this racemization occurs without forming a succinimide intermediate. This is the first example of D-asparaginyl formation in a protein occurring during the racemization process under physiological conditions.

Stored, depleted RBC were rejuvenated with respect to their levels of adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG), and P50 by acid-citrate-dextrose perservatives containing phosphoenolpyruvate (PEP) without sucrose. The restorations of P50 and 2,3-DPG were dependent on the phosphoenolpyruvate concentration. Erythrocyte P50 and 2,3-DPG, even after treatment with these preservatives, decreased with increasing storage period, but the P50 and 2,3-DPG of five-week-old blood were still higher than the corresponding values of fresh blood. ATP concentration was also increased by treating stored blood with preservatives containing phosphoenolpyruvate, but the elevated ATP of five-week-old blood was only about 50 percent of fresh blood. The ATP level could not be raised further by increasing phosphoenolpyruvate concentration but was improved by supplementation with adenine and nucleosides. Incubation of stored blood with 15 mM phosphoenolpyruvate was sufficient to restore ATP, 2,3-DPG and P50 of three-week-old blood to nearly normal. The results of these studies indicate that sucrose is not necessary for PEP to be effective as a preservative additive.

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