Protein determination, micro-Kjeldahl

Protein content was determined by a semi-automated micro-Kjeldahl method [4], The conversion factor used was 6.25. 

Protein solubilities of soy flour and extrudates in the following solvent systems were determined by the micro-Kjeldahl method ( 9). A portion (0.1 g) of finely-ground sample (No. 60 sieve) was extracted with 9.9 ml of solvent for 1 hr at room temperature followed by centrifugation and filtration. An aliquot of the supernatant was used for nitrogen determination. 

Protein was determined by the method of Lowry (7) after hydrolysis with 0.2N NaOH (100°C, 15 min). Total nitrogen was measured by the micro-Kjeldahl method with sulfuric acid/hydrogen peroxide reagentj the ammonia was detected with Nessler s reagent. Glucose was measured by standard colorimetric assay using dinitrosalicylic acid. Starch was hydrolyzed with concentrated HC1 and then determined as sugar. 

Many physicochemical assays are established to quantify the protein mass. It is determined by exploiting the extinction coefficient in optical density measurements or by colorimetric assays such as the Bradford, Lowry, bicinchoninic (BCA), and biuret assay [13, 14]. Albeit easy to perform, these colorimetric assays suffer from inaccuracies that are due to the use of inappropriate standards like bovine serum albumin. If relevant standards are not available, quantitative amino acid analysis [6], the (micro-)Kjeldahl nitrogen method [14, 15] or gravimetry as very accurate but time-consuming alternatives can be applied. 

Total nitrogen of the AIS was determined by a semi-micro Kjeldahl procedure on a 0.50 g sample and the protein content calculated using the factor 6.25. The ash content was determined on a 0.5 g sample of the AIS ignited In a muffle furnace at 550 C for 16 hours. 

The determination of ammonia after the regular or modified Kjeldahl digestion presents rather less serious problems than those already dis cussed. The advantages of the micro-Kjeldahl distillation (69, 80, 81, 82, 83) as compared with the macro>method, or even the semimicro-method are now generally recognized. A comparative study of the macro-and microscale determination in the analysis of flour, wheat and com for their protein content was made by Robinson and Shellenberger (27). The micro-Kjeldahl method has been used for systematic plasma protein analysis (84, 85), saliva proteins (86), milk proteins (87), and cerebrospinal fluid protein (88),... 

These are the substances in blood, other than proteins, which contain nitrogen. They include urea, creatinine, uric acid, amino acids and ammonia. The non-protein nitrogen fraction can be determined by a micro-Kjeldahl method, followed by Nesslerization. In most laboratories, however, blood urea is determined and the other non-protein nitrogen compounds are measured only where clinically indicated, e.g. uric acid. 

The hydrolysis of 0.06 M BAA by papain is a first-order reaction under the conditions of assay (100). Specific activity (Gi) is expressed as K (first-order rate constant calculated in decimal logarithms) per milligram of protein nitrogen per milliliter of reaction mixture. Protein nitrogen can be estimated by the turbidometric method of Btlcher (36). The nitrogen content of standard papain solutions is determined by micro-Kjeldahl analysis. 

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