Spectrophotometric determination of low concentrations of proteins at the far-UV region, independently of their amino acid composition, has recently been reported (1). Nucleic acids also absorb strongly at this region. Hence, a study to establish an easy and accurate method of determining nucleic acids and nucleoproteins has been initiated.
Current spectrophotometric methods for determining nucleoproteins are based mainly on the ratio of absorbancies at 260 to 280 mp. The mutual absorption of both components at the above wavelengths, and the dependence of absorbancy rates on the nature of the protein component and the ratio of nucleic acid to protein within the nucleoprotcin. render this method inaccurate. Calorimetric determinations of both components also suffer from inaccuracy either by virtue of their own nature (the Lowry method (2) for proteins), or by interference of one component with the calorimetric determination of the other (many proteins, for example, interfere with the orcinol reaction (3) for RNA).
The present communication reports a method for determining nucleic acids and nucleoproteins that takes advantage of their differential absorbancies at the near-and far-UV regions, by which the amount of both constituents within an individual nucleoprotein can also be determined.
Methods
The following proteins were purchased from Worthington: pancreatic ribonuclease, deoxyribonuclease, and lyophilized trypsin. Bovine serum albumin, torula RNA, polyadenylic acid (poly A), and highly polymerized DNA were obtained from Calbiochem. Alkaline phosphatase (calf intestine) was obtained through Mann Research Laboratories and Escherichia coli tRNA from Schwarz BioResearch Inc. Polyoma DNA was obtained through the court,esy of Professor E. Winocour of the Weizmann Institute of Science.
Tobacco mosaic virus (TMV) was prepared by homogenizing infected tobacco leaves in 0.1 iM EDTA, pH 8.0, squeezing the homogenate through 217