A procedure has been developed for the methylation of hemins using trimethyloxonium tetrafluoroborate. The method is rapid and selective for carboxyl groups, and it can be used to methylate polar and nonpolar hemins under neutral and basic conditions. Esterifications of protohemin IX, hematohemin. and heme a have been accomplished in high yield.DEAN, DEFILIPPI AND HULTQUIST

The reactivities of alkaline NH(2)OH and neutral NaHSO(3) with carbonyl and olefinic groups conjugated with the tetrapyrrole nucleus of haems were studied. The reactions were carried out with 2-3nmol of haem a, spirographis haem, isospirographis haem, 2,4-diacetyldeuterohaem and protohaem. Vinyl side chains were found to be insensitive to the chemical action of both alkaline NH(2)OH and neutral NaHSO(3). The formyl-containing haems reacted rapidly with both reagents at room temperature, as evidenced by sizable hypsochromic shifts of the reduced pyridine haemochrome spectrum. In less alkaline solution, the reactions of these formyl-containing haems with NH(2)OH were much slower. 2,4-Diacetyldeuterohaem reacted with alkaline NH(2)OH, but not with neutral NaHSO(3). These rapid, simple and straightforward tests are readily usable in differentiating among formyl, acetyl and other electron-withdrawing side chains conjugated with the tetrapyrrole ring of haems. We applied these observations to an investigation of the two unique prosthetic groups of the bovine erythrocyte green haemoproteins. The prosthetic groups of these two proteins were isolated and spectrally characterized. Under the conditions used, the haems did not react with either NH(2)OH or NaHSO(3), but were altered by dithionite, suggesting that the previous interpretation that a formyl group was present [Hultquist, Dean & Reed (1976) J. Biol. Chem.251, 3927-3932] may have been premature. These studies also provide evidence that the alpha-hydroxyfarnesylethyl side chain of haem a affects the alpha-band maximum, but not the beta- or Soret bands of the reduced pyridine haemochrome spectrum of haem a.

Field desorption mass spectrometry was shown to be a valid technique for determining the molecular weights of hemins and hemin esters, as well as of porphyrins. The observed base peaks of ligand-free protoheme IX, protoheme IX dimethyl ester, and protoporphyrin IX dimethyl ester correspond well to the molecular weights of these compounds and the base peak for hematoporphyrin corresponds to the molecular weight of this porphyrin minus two molecules of water. The technique was employed to confirm the molecular weights of heme a, heme a dimethyl ester, and acetyl heme a dimethyl ester. Heme a dimethyl ester was prepared by reaction of heine a with trimethyloxonium tetrafluoroborate and purified by high-pressure liquid chromatography. The isolated product was converted to the acetylated derivative by reaction with acetic anhydride and was subsequently purified by high-pressure liquid chromatography. A field desorption spectrum of heme a shows a base peak at 582 which is in agreement with previous deductions of the structure of this prosthetic group. Base peaks of the heme a ester and its acetylated derivative demonstrate that the two carboxyl groups have been methylated and the single hydroxyl group has been acetylated without further alteration of the molecule.

Whole cell biocatalysts which enable the concerted use of D‐hydantoinase, D‐carbamoylase, and racemase enzymes are valuable for the production of D‐amino acids. However, Escherichia coli host strains used for this purpose efficiently degrade D‐amino acids. This work demonstrates that D‐amino acid degradation occurs largely through the concerted action of D‐amino acid dehydrogenase, encoded by the dadA gene, and D‐serine dehydratase, encoded by the dsdA gene. Deletion mutants of E. coli which lack these activities were constructed and compared against wild type strains in D‐amino acid degradation. An E. coli dadA mutant reduced the degradation of D‐methionine by one third, D‐phenylalanine by two‐thirds, and D‐2‐aminobutyric acid nearly completely. Even though the dadA mutant had no effect on D‐serine degradation, a dadA dsdA double mutant of E. coli additionally reduced degradation of D‐serine, as well as D‐phenylalanine, almost entirely. These strains are appropriate hosts for whole cell biosynthesis of D‐amino acids using general approaches such as the hydantoinase system.

An analytical and semipreparative procedure has been developed for the isolation of the prosthetic groups of various hemeproteins by use of polyacrylamide gel electrophoresis. Electrophoresis of erythrocyte formylhemeprotein in the presence of 20 mM cyanide and subsequent elution of the sharp hemin band resulted in 87% recovery of hemin and isolation of apoprotein. Removal of the protohemin prosthetic groups from methemoglobin, metmyoglobin, horseradish peroxidase and erythrocyte cytochrome 670