key: cord-0006684-t22a2w1k authors: O'Hern, E. M.; Fishbein, L. title: Studies on histoplasmosis IV. Elution characteristics of the Histo-inhibitory factor (HIF) date: 1968 journal: Mycopathol Mycol Appl DOI: 10.1007/bf02057164 sha: db07dc8be7f36e3f94796f2f0723bdea1493c9eb doc_id: 6684 cord_uid: t22a2w1k The histo-inhibitory factor (HIF) derived from homogenates of liver or kidney from hamsters infected withHistoplasma capsulatum has been fractionated by column chromatography. It shows maximum absorption at 280 mµ, has a molecular weight of 142,000 and can be eluted from DEAE-cellulose or DEAE-Sephadex A—50 with 0.02 M phosphate — 4 M sodium chloride (1 : 1) HIF can be eluted fromHistoplasma yeast cells at pH 10.0, thus a greater number of positive cultures from chronic histoplasmosis could be expected to result from pretreatment of clinical specimens in glycine buffer pH 10.0 prior to culture. cryst.); and hemoglobin (bovine, 2 × cryst.) were all obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio. (a) Sephadex G-150 -The 1.5 × 30 cm column was packed to a bed volume of 20 cm according to the procedure of WHITAKER. (4) (b) DEAE-Sephadex A--50 -The procedure of FLOI)IN (5) was utilized for the preparation of the 1.5 × 30 cm column. (c) DEAE-Cellulose --The 1.5 x 30 cm column was prepared as described by JA~ES & STAS~WORTH (6) . All spectrophotometric measurements were made in silica cells with a 1 cm light path in a Beckman DU-Spectrophotometer and are expressed as optical density (O.D.). Hemoglobin was estimated at 410 m#; gamma globulin, serum albumin and ovalbumin were all estimated at 280 m#. Molecular weight of macromolecular fractions was estimated by gel filtration of a Sephadex G--150 column in accordance with the procedures of WttlTAKER. (4) Compounds used as reference included human gamma globulin, serum albumin (bovine), hemoglobin (bovine) and ovalbumin, Void volume (Vo) was measured utilizing Blue Dextran (MW 2,000,000). One hundred male golden Syrian hamsters (Mesacricetus auratus) weighing 60--80 g were infected by subcutaneous inoculation of 0.05 ml of a saline suspension of 5 × 105 yeast cells from a 3-day culture of H. capsulatum (strain 6650, DR. C. W. EMMONS) grown on brain heart infusion agar containing 5 ~o sheep blood (BHIB agar). Six weeks after infection, the animals' tissues were tested for growth of H. capsulatum and for the presence of the HIF by tile following procedures: Culture on BHIB at 37 ° C These cultures were incubated at 37 ° C, examined daily and held for at least one week if negative. A small sample of spleen, liver and kidney from each animal was fixed in Zenker's formol solution for preparation of tissue sections. Histoplasma yeast cells (approximately 1 × 10*/ml) were mixed with HIF in kidney homogenate, allowed to stand at 4 ° C for two hours, then centrifuged and resuspended in buffer: veronal-acetate buffer pH 4.5--9.0 or glycine -NaOH buffer pH 8.5 -11.0 The sedimented yeast cells were well mixed with buffer and allowed to stand at t° C for 12--24 hours, centrifuged again and resuspended in saline before plating on BHIB agar. The negative control, Histoplasma yeast cells in saline, was processed in the buffers as above. The positive control, Histoplasma yeast cells in the HIF homogenate, was resuspended in saline after the first and second centrifugation. Each buffer supernatant was tested for ttlF by addition of Histoplasma yeast cells to initial concentration, (approximately 1 × 104/ml) at 37 ° C. All tests were run in duplicate. Three bacteria (Escherichia coli, Sfaphylococcus aureus and Bacillus subtilis at approximately 1 × 105/ml) and three yeast-like organisms (Candida albicans, Cryptococcus neo/ormans and Blastomyces dermatitidis at 1 × 10~/ml) were mixed with HIF in a saline-homogenate of liver or kidney or in a 280 m# HIF fraction which had been lyophilized and resuspended in saline. A control test with H. capsulatum was run in parallel. The suspensions were allowed to stand at 4 ° C for two hours, then centrifuged and the supernatants separated from the sediments. The supernatants were boiled (for B. subtilis supernatant autoclaved at 115 ° C) for ten minutes then cooled and H. capsulatum yeast cells were added to a final concentration 1 × 104/ml. Organisms in the sediments were resuspended in saline to the original volume. All suspensions were plated in duplicate on BHIB agar. Cultures were incubated at 37 ° C and checked daily for six days for growth. Kidneys and livers from histoplasma-infected hamsters were separately homogenized with glycine-sodium hydroxide buffer (0.1 M, pH 10.2) in a homogenizer and fractionated initially over a Sephadex G--150 column (prepared as described above). The possible acidic nature of the HIF protein suggested further fractionation over DEAE-cellulose and DEAE-Sephadex anion exchanger. Fig. 1 depicts the elution curves of the histoplasma inhibitory factor (HIF) from kidney as well as a number of protein standards on a Sephadex G--150 column (1.5× 30 cm). HIF, gamma globulin, serum albumin and ovalbumin were estimated at 280 m# and hemogloblin at 410 m#. (Fig. 4) Fig. ¢ depicts the relationship of V/Vo and log molecular weight of the HIF factor (from kidney) as well as a number of standard proteins. Their respective elution volumes molecular and log molecular weights are tabulated in Table I . WHITAKER (4) has shown that a linear relationship exists between V/Vo and the logarithm of the protein molecular weight, wher~ V is the protein retention volume (initial addition of protein sample to the peak of the eluted protein) and Vo is the column void volume. The column void volume was determined utilizing Blue Dextran (MW 2,000,000) and is the volume of buffer required to elute a substance not retained by inclusion within the column packing (the fractionation range for Sephadex G--150 is 5,000--200,000). The molecular weight of HIF (from kidney) utilizing the above procedure is estimated as 142,000. Good correlation has been shown between get-filtration (molecular-sieve chromatography) and the molecular weights of many enzymes and other proteins. (4, (8) (9) (10) (11) (12) Chemical characterization of HIF makes possible a more precise study of the production of the inhibitors during the course of an infection, provides the basis for a meaningful study of the mode of action of HIF and establishes a basis for determining the identity or non-identity of HIF and other known inhibitors found in normalpathological tissues. (1--3, 13--16) Activity of HIF fractions at 280 m# would suggest a compound that is at least in part protein. A molecular weight of 142,000 is within range of known antibody molecules (17) and specificity of the HIF inhibitory activity would also suggest this possibility. Elution of HIF from Hist@lasma at pH 10.2 has shown that the inhibition is not irreversible the organisms are not killed by the inhibitor, at least not within the two hour test period, at 4 ° C. This finding increases the probability of isolating Hist@lasma from infected tissue which has been processed in buffer at approximately pH 10.2, centrifuged and resuspended in saline before culturing. The histo-inhibitory factor (HIF) derived from homogenates of liver or kidney from hamsters infected with Hist@tasma capsulatum has been fractionated by column chromatography. It shows maximum absorption at 280 m/~, has a molecular weight of 142,000 and can be eluted from DEAE-eellulose or DEAE-Sephadex A--50 with 0.02 M phosphate -4 M sodium chloride (1 : 1) HIF can be eluted from Histoplasma yeast cells at pH 10.0, thus a greater number of positive cultures from chronic histoplasmosis could be expected to result from pretreatment of clinical specimens in glycine buffer pH 10.0 prior to culture. Resistance of Hamsters in Infections with Histoplasma capsulatum Studies on Histoplasmosis. II. The Histoplasma Inhibitory Factor in Hamsters Inoculated Intradermally and Subcutaneously Studies in Histoplasmosis. I11. Cross Reactions and Characterization of Histoplasma and Blastomyces Inhibitory Factors Determination of Molecular W'eights of Proteins by Gel Filtration on Sephadex Methodological Aspects of Gel Filtration with Special Reference to Desalting Operations Studies of the Chromatography of Human Serum Proteins of Diethylaminoethyl (DEAE)-Cellulose. 1. The Effect of the Chemical and Physical Nature of the Exchanger Protein Chromatography on Ion Exchange Cellulose Estimation of Molecular Weights of Proteins by Gel Filtration Estimation of the Molecular xNeights of Proteins by Sephadex Gel-Filtration The Chemistry of Xanthine Oxidase. 11. Ultracentrifuge and Gel-Filtration Studies on the Milk Enzyme Zum Molekulargewieht der Laetat-dehydrogenasen. Studien des Verhaltens bei der Gelfiltration Molecular-Sieve Chromatography The Epidermal Chalone: A Preliminary Attempt at Isolation. Exp Inhibition of Growth of Malignant Cells in vitro by a Component of Normal Adult Connective Tissue Humoral Defense Mechanisms in Cryptococcosis: Substances in Normal Serum, Saliva and Cerebralspinal Fluid Affecting the Growth of Cryptococcus neo/ormans Development of Rabies Inhibiting Substance in Skunks Infected with Rabies Virus Experimental Immunochemistry