Formic Acid → L-Aspartic Acid

HRID 893256Notes

Reaction equation

HRID 893256 reaction equation
NC(N)=O.[CH:5]([OH:7])=[O:6].[NH:8]1CC[CH2:13][C@H:9]1[C:10]([OH:12])=[O:11]>O.Cl.NC(N)=N>[NH2:8][C@H:9]([C:10]([OH:12])=[O:11])[CH2:13][C:5](=[O:7])[OH:6]

Participants

6

Reactant 3

HCID 284Formic Acid2×HCID 1176Urea2×HCID 145742L-Proline

Product 1

HCID 5960L-Aspartic Acid

Reagents, catalysts & solvents 2

HCID 962WaterSolvent2×HCID 5742Guanidine HydrochlorideSolvent

Procedure

An aliquot (30 ul) of the urea soluble fraction was diluted with ten volumes of water, and the insoluble fusion protein was pelleted by centrifugation. The protein was then dissolved in 30 ul of 6M guanidine hydrochloride, and 70 ul 98% formic acid added (Digestion 1). In a parallel experiment, 70 ul 98% formic acid was added to an aliquot (30 ul) of the urea fraction directly (Digestion 2). Following two days incubation at 42° C., ten volumes of water were added, and the insoluble proteins were pelleted by centrifugation. The pellet was resuspended in 1X treatment buffer (100 ul), and 10 ul was used per well on 12.5% SDS-polyacrylamide gel. FIG. 10 shows a sample of the cleaved products (Digestion 1 and Digestion 2) both on a Commassie-stained gel and an immunoblot (using HIVI positive human serum as primary antibody). Only two major bands are visible on the Commassie-stained gel. These represent the products of cleavage at the unique Asp-Pro bond: the CKS portion, MW=26.5 kDa and the p41 portion, MW=23.5 kDa. Peptide sequencing confirmed that the lower molecular weight band was indeed the p41 peptide, and that the amino terminal residue was proline which results from expected cleavage between the Asp and Pro.

Workup

  1. additionwere added

Sources & evidence

ORD record
ord-4a73207270a14545a0db0cd39d6f7112
Source dataset
uspto-grants-1992_06
Patent number
US05124255

Reaction notes

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