Drabkin method

Concentrations, expressed per heme, were determined by the Drabkin method and were all close to 18 mM. Concentrations of.CO--heme were also determined breaking the cells in distilled water and using known extinction coefficient of CO-hemoglobin results of both methods were invariably found to be equal in the experimental error. Magnetic volume susceptibility measurements were performed with our superconducting susceptometer (25) as described elsewhere (23). The overall accuracy of the method for protein aqueous solutions referred to calibrations with pure deoxygenated water is 7x10" cgs units, or 1/1000 of the volume susceptibility of water. 

Samples of whole blood were obtained by heart puncture using and EDTA-treated syringe. The whole blood was immediately assayed for hematocrit (percent packed cell volume) and hemoglobin by the method of Drabkin and Austin (ll). 

For blood heparinized venous blood is centrifuged and the upper layer is removed. Wash the erythrocyte sediment three times with 0.9% (w/v) NaCl solution. Haemolyse the washed red cells by adding 4 parts (v/v) of distilled water per volume of packed cells to give a stock haemolysate solution (approx. 5% (w/v)). For assay, dilute the stock solution 1 500 with the phosphate buffer immediately before the assay is to be carried out and determine the haemoglobin content of the solution by the method of Drabkin. The catalase activity is expressed per unit of haemoglobin. 

Samples of blood were obtained from sickle cell anemic individuals who had not been transfused within three months prior to the time of sampling. Stroma-free eoneentrated solutions of human adult hemoglobin were prepared by the method used by Drabkin (5). These solutions were diluted just before use with the... 

Inasmuch as cytochrome c is a relatively small protein easily extractable and easily recognized by spectro-photometric methods, it is quite natural that its biosynthesis was investigated in vivo in a variety of tissues. Much of the in vivo work comes from Drabkin s laboratory [116]. These workers used labeled glycine as a precursor and followed its incorporation in both the heme and the protein moiety of cytochrome c. The following is a summary of their findings (1) the isotope is incorporated in both the porphyrin and the protein portion of the molecule (2) the incorporation is more rapid in the heme than in the protein fraction (3) practically all tissues are capable of independent cytochrome c synthesis (4) studies in regenerating liver also demonstrate that the rate of cytochrome c synthesis outweighs the active increase in the cellular population and (5) new cytochrome c is made in the endoplasmic reticulum and transferred by an unknown mechanism to mitochondria. 

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