NITRATE REDUCTASE ACTIVITY ( NRA ) AT VEGETATIVE AND REPRODUCTIVE CONDI [ TIONS IN SACCHA RUM CULTIVAR CO
Regulations of flowering and induction of flowering in nonflowering clones of Saccharum has been found possible by the application of extra light (EL) and extra dark (ED) treatments. As to the effect of EL, ED and 'switch over' treatment from EL to ED on NRA relation to flowering NRA was found to be low at vegetative stage and as the plants reached the symptom stage. The activity reached a maximum value and thereafter showing a deckle with progress in flower development. Examination of gemplasm collection for NRA in clones of Saccharum species known to differ in their flowering behaviour was suggested.
MODIFICATION OF ECKERSON METHOD FOR DETERMINING NITRATE REDUCASE.
A satisfactory method for measuring the nitrate reduction by plant extracts should throw some light upon the ability of the plant to assimilate nitrogen in this form. A method for determining the nitrate reducing activity of plant extracts was worked out by ECKERSON.2 A brief statement of her method follows. Extracts of plant tissues are secured by grinding the fresh plant material very finely in a food chopper, and pressing out the juice through cheesecloth. One cubic centimeter of this juice and 1 cc. of 10 per cent. potassium nitrate solution containing 10 mg. of freshly dissolved glucose are made up to 10 cc. with water. This mixture is brought to a pH of 7.2 to 7.4 with sodium hydroxide, using phenol red as an indicator. In the original papers it was contended that there were enough natural buffers present to resist any great changes in hydrogen ion concentration; but, on the other hand, it was pointed out that there was a gradual increase in hydrogen ion concentration during the incubation period. The mixture is incubated under toluene at 350 C. for 17 hours. After incubation the amount of nitrite present is determined by the sulphanilic acid method. The writer and his coworkers found that, although the solutions used in the ECKERSON method were adjusted to the same pH at the beginning of the incubation period in the manner just described, there was an unequal decrease in pH, so that at the end of the period the extracts from different sources showed various degrees of acidity. Some of these extracts probably contained a greater quantity of a natural buffer. The change in pH can be prevented by substituting 4 cc. of 0.2 M phosphate buffer mixture (pH 7.3) for one-half of the water used in making up the original volume. The coloring matter in most plant tissues interferes with a comparison of the color produced by the nitrite reagent. It was found that by removing these colored substances with activated charcoal before treating with the sulphanilic acid reagent, a satisfactory comparison could be made. Upon being removed from the incubator the solutions are treated with 5 gm. of activated charcoal. After being thoroughly mixed and allowed to stand for 15 minutes they are washed through a fine filter paper into 100-cc. volumetric flasks. The flasks are made up to volume and 10-cc. aliquots are used in the determinations. None of the nitrite is absorbed. A standard solution of nitrite treated in the same manner was not affected by the charcoal, since the entire amount of nitrite could be recovered in the filtrate. For solutions which are difficult to clear with charcoal, a second clearing with 2 gm. of
On the fractionation of β-lactoglobulin and α-lactalbumin
Abstract Existing methods for the preparation of α-lactalbumin and β-lactoglobulin are examined critically. It is proposed that the most satisfactory method for the preparation of the whey protein fraction from bovine milk is (NH 4 ) 2 SO 4 fractionation. A number of new gentle procedures for the fractionation of the whey proteins are compared with some existing methods. The distribution of total protein at various stages is determined by total Kjeldahl nitrogen and the kind of protein by starch-gel electrophoresis. The effect of variables, such as pH, salt concentration and temperature on the separation of α-lactalbumin and serum albumin from β-lactoglobulin is studied. Differences in the behavior ( e.g. solubility) of genetic variants of β-lactoglobulin are noted. The solubility of each of the major whey proteins, under similar conditions to those used in the fractionation, is compared with its solubility in the whey protein mixture. The understanding gained of factors important in the fractionation is used in recommending new procedures for α-lactalbumin and β-lactoglobulin isolation.